Catecholaminergic Neurons Research Articles

Midbrain catecholaminergic neurons co-express α-synuclein and tau in progressive supranuclear palsy.

Objective: To analyze the frequency and distribution of α-synuclein deposits in progressive supranuclear palsy (PSP).Methods: The brains of 25 cases of pathologically confirmed PSP were evaluated with immunohistochemistry for α-synuclein and tau. Multiple immunofluorescent stains were applied to analyze the expression of tau and α-synuclein aggregates in catecholaminergic neurons. Patients’ clinical symptoms were retrospectively recorded.Results: Deposits α-synuclein in the form of typical Lewy bodies (LBs) were only found in two PSP cases (8%) that fulfilled the clinical subtype of PSP known as Richardson’s syndrome (RS). LBs were present in the locus ceruleus (LC), substantia nigra pars compacta (SNc), basal forebrain, amygdala and cingulated cortex in a distribution mimicking that of Parkinson’s disease (PD). Triple-immunolabeling revealed co-expression of α-synuclein and tau proteins in some tyrosine hydroxilase (TH)-positive neurons of the LC and SNc.Conclusions: There is no apparent clinical correlation between the presence of LBs in PSP. Tau protein co-aggregate with α-synuclein in catecholaminergic neurons of PSP brains suggesting a synergistic interaction between the two proteins. This is in keeping with the current view of neurodegenerative disorders as “misfolded protein diseases”.

Impact of subanesthetic doses of ketamine on AMPA-mediated responses in rats: An in vivo electrophysiological study on monoaminergic and glutamatergic neurons.

The rapid antidepressant action of a subanesthetic dose of ketamine in treatment-resistant patients represents the most striking recent breakthrough in the understanding of the antidepressant response. Evidence demonstrates tight interactions between the glutamatergic and monoaminergic systems. It is thus hypothesized that monoamine systems may play a role in the immediate/rapid effects of ketamine. In vivo electrophysiological recordings were carried in male rats following ketamine administration (10 and 25 mg/kg, i.p.) to first assess its effects on monoaminergic neuron firing. In a second series of experiments, the effects of ketamine administration on α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)- and N-methyl-D-aspartate receptor (NMDA)-evoked responses in hippocampus CA3 pyramidal neurons were also investigated using micro-iontophoretic applications. Although acute (~2 hours) ketamine administration did not affect the mean firing activity of dorsal raphe serotonin and ventral tegmental area dopamine neurons, it did increase that of locus coeruleus norepinephrine neurons. In the latter brain region, while ketamine also enhanced bursting activity, it did increase population activity of dopamine neurons in the ventral tegmental area. These effects of ketamine were prevented by the prior administration of the AMPA receptor antagonist 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide. An increase in AMPA-evoked response of CA3 pyramidal neurons was also observed 30 minutes following acute ketamine administration. The present findings suggest that acute ketamine administration produces a rapid enhancement of catecholaminergic neurons firing activity through an amplification of AMPA transmission. These effects may play a crucial role in the antidepressant effects of ketamine observed shortly following its infusion in depressed patients.

Valproic acid enhances neuronal differentiation of sympathoadrenal progenitor cells.

The antiepileptic drug valproic acid (VPA) has been shown to influence the neural differentiation and neurite outgrowth of neural stem cells. Sympathoadrenal progenitor cells share properties with neural stem cells and are considered a potential cell source in the treatment of neurodegenerative diseases. The present study therefore aims at modulating the neural differentiation potential of these cells by treatment with the histone deacetylase inhibitor VPA. We studied the epigenetic effects of VPA in two culture conditions: suspension conditions aimed to expand adrenomedullary sympathoadrenal progenitors within free-floating chromospheres and adherent cell cultures optimized to derive neurons. Treatment of chromospheres with VPA may launch neuronal differentiation mechanisms and improve their neurogenic potential upon transplantation. However, also transplantation of differentiated functional neurons could be beneficial. Treating chromospheres for 7 days with clinically relevant concentrations of VPA (2 mm) revealed a decrease of neural progenitor markers Nestin, Notch2 and Sox10. Furthermore, VPA initiated catecholaminergic neuronal differentiation indicated by upregulation of the neuronal marker β-III-tubulin, the dopaminergic transcription factor Pitx3 and the catecholaminergic enzymes TH and GTPCH. In adherent neural differentiation conditions, VPA treatment improved the differentiation of sympathoadrenal progenitor cells into catecholaminergic neurons with significantly elevated levels of nor- and epinephrine. In conclusion, similar to neural stem cells, VPA launches differentiation mechanisms in sympathoadrenal progenitor cells that result in increased generation of functional neurons. Thus, data from this study will be relevant to the potential use of chromaffin progenitors in transplantation therapies of neurodegenerative diseases.

Localization of α-synuclein in teleost central nervous system: immunohistochemical and Western blot evidence by 3D5 monoclonal antibody in the common carp, Cyprinus carpio.

Alpha synuclein (α-syn) is a 140 amino acid vertebrate-specific protein, highly expressed in the human nervous system and abnormally accumulated in Parkinson's disease and other neurodegenerative disorders, known as synucleinopathies. The common occurrence of α-syn aggregates suggested a role for α-syn in these disorders, although its biological activity remains poorly understood. Given the high degree of sequence similarity between vertebrate α-syns, we investigated this proteins in the central nervous system (CNS) of the common carp, Cyprinus carpio, with the aim of comparing its anatomical and cellular distribution with that of mammalian α-syn. The distribution of α-syn was analyzed by semiquantitative western blot, immunohistochemistry, and immunofluorescence by a novel monoclonal antibody (3D5) against a fully conserved epitope between carp and human α-syn. The distribution of 3D5 immunoreactivity was also compared with that of choline acetyltransferase (ChAT), tyrosine hydroxylase (TH), and serotonin (5HT) by double immunolabelings. The results showed that a α-syn-like protein of about 17 kDa is expressed to different levels in several brain regions and in the spinal cord. Immunoreactive materials were localized in neuronal perikarya and varicose fibers but not in the nucleus. The present findings indicate that α-syn-like proteins may be expressed in a few subpopulations of catecholaminergic and serotoninergic neurons in the carp brain. However, evidence of cellular colocalization 3D5/TH or 3D5/5HT was rare. Differently, the same proteins appear to be coexpressed with ChAT by cholinergic neurons in several motor and reticular nuclei. These results sustain the functional conservation of the α-syn expression in cholinergic systems and suggest that α-syn modulates similar molecular pathways in phylogenetically distant vertebrates.

Plasma biomarkers of decreased vesicular storage distinguish Parkinson disease with orthostatic hypotension from the parkinsonian form of multiple system atrophy.

Parkinson disease with orthostatic hypotension (PD + OH) and the parkinsonian form of multiple system atrophy (MSA-P) can be difficult to distinguish clinically. Recent studies indicate that PD entails a vesicular storage defect in catecholaminergic neurons. Although cardiac sympathetic neuroimaging by (18)F-dopamine positron emission tomography can identify decreased vesicular storage, this testing is not generally available. We assessed whether plasma biomarkers of a vesicular storage defect can separate PD + OH from MSA-P. We conceptualized that after F-dopamine injection, augmented production of F-dihydroxyphenylacetic acid (F-DOPAC) indicates decreased vesicular storage, and we therefore predicted that arterial plasma F-DOPAC would be elevated in PD + OH but not in MSA-P. We measured arterial plasma F-DOPAC after (18)F-dopamine administration (infused i.v. over 3 min) in patients with PD + OH (N = 12) or MSA-P (N = 21) and in healthy control subjects (N = 26). Peak F-DOPAC:dihydroxyphenylglycol (DHPG) was also calculated to adjust for effects of denervation on F-DOPAC production. Plasma F-DOPAC accumulated rapidly after initiation of (18)F-dopamine infusion. Peak F-DOPAC (5-10 min) in PD + OH averaged three times that in MSA-P (P < 0.0001). Among MSA-P patients, none had peak F-DOPAC > 300 nCi-kg/cc-mCi, in contrast with 7 of 12 PD + OH patients (χ(2) = 16.6, P < 0.0001). DHPG was lower in PD + OH (3.83 ± 0.36 nmol/L) than in MSA-P (5.20 ± 0.29 nmol/L, P = 0.007). All MSA-P patients had peak F-DOPAC:DHPG < 60, in contrast with 9 of 12 PD + OH patients (χ(2) = 17.5, P < 0.0001). Adjustment of peak F-DOPAC for DHPG increased test sensitivity from 58 to 81% at similar high specificity. After F-dopamine injection, plasma F-DOPAC and F-DOPAC:DHPG distinguish PD + OH from MSA-P.

Distribution of the creatine transporter throughout the human brain reveals a spectrum of creatine transporter immunoreactivity

ABSTRACTCreatine is a molecule that supports energy metabolism in cells. It is carried across the plasma membrane by the creatine transporter. There has been recent interest in creatine for its neuroprotective effects in neurodegenerative diseases and its potential as a therapeutic agent. This study represents the first systematic investigation of the distribution of the creatine transporter in the human brain. We have used immunohistochemical techniques to map out its location and the intensity of staining. The transporter was found to be strongly expressed, especially in the large projection neurons of the brain and spinal cord. These include the pyramidal neurons in the cerebral cortex, Purkinje cells in the cerebellar cortex, and motor neurons of the somatic motor and visceromotor cranial nerve nuclei and the ventral horn of the spinal cord. Many other neurons in the brain also had some degree of creatine transporter immunoreactivity. By contrast, the medium spiny neurons of the striatum and the catecholaminergic neurons of the substantia nigra and locus coeruleus, which are implicated in neurodegenerative diseases, showed a very low to almost absent level of immunoreactivity for the transporter. We propose that the distribution may reflect the energy consumption by different cell types and that the extent of creatine transporter expression is proportional to the cell's energy requirements. Furthermore, the distribution indicates that supplemented creatine would be widely taken up by brain cells, although possibly less by those cells that degenerate in Huntington's and Parkinson's diseases. J. Comp. Neurol. 523:699–725, 2015. © 2014 Wiley Periodicals, Inc.

Neuropeptide Y in the noradrenergic neurones induces obesity and inhibits sympathetic tone in mice.

Neuropeptide Y (NPY) co-localized with noradrenaline in central and sympathetic nervous systems seems to play a role in the control of energy metabolism. In this study, the aim was to elucidate the effects and pathophysiological mechanisms of increased NPY in catecholaminergic neurones on accumulation of body adiposity. Transgenic mice overexpressing NPY under the dopamine-beta-hydroxylase promoter (OE-NPY(DβH) ) and wild-type control mice were followed for body weight gain and body fat content. Food intake, energy expenditure, physical activity, body temperature, serum lipid content and markers of glucose homoeostasis were monitored. Thermogenic and lipolytic responses in adipose tissues, and urine catecholamine and tissue catecholamine synthesizing enzyme levels were analysed as indices of sympathetic tone. Homozygous OE-NPY(DβH) mice showed significant obesity accompanied with impaired glucose tolerance and insulin resistance. Increased adiposity was explained by neither increased food intake or fat absorption nor by decreased total energy expenditure or physical activity. Adipocyte hypertrophy and decreased circulating lipid levels suggested decreased lipolysis and increased lipid uptake. Brown adipose tissue thermogenic capacity was decreased and brown adipocytes filled with lipids. Enhanced response to adrenergic stimuli, downregulation of catecholamine synthesizing enzyme expressions in the brainstem and lower adrenaline excretion supported the notion of low basal catecholaminergic activity. Increased NPY in catecholaminergic neurones induces obesity that seems to be a result of preferential fat storage. These results support the role of NPY as a direct effector in peripheral tissues and an inhibitor of sympathetic activity in the pathogenesis of obesity.

Optogenetic release of norepinephrine from cardiac sympathetic neurons alters mechanical and electrical function.

Release of norepinephrine (NE) from sympathetic neurons enhances heart rate (HR) and developed force through activation of β-adrenergic receptors, and this sympathoexcitation is a key risk for the generation of cardiac arrhythmias. Studies of β-adrenergic modulation of cardiac function typically involve the administration of exogenous β-adrenergic receptor agonists to directly elicit global β-adrenergic receptor activation by bypassing the involvement of sympathetic nerve terminals. In this work, we use a novel method to activate sympathetic fibres within the myocardium of Langendorff-perfused hearts while measuring changes in electrical and mechanical function. The light-activated optogenetic protein channelrhodopsin-2 (ChR2) was expressed in murine catecholaminergic sympathetic neurons. Sympathetic fibres were then photoactivated to examine changes in contractile force, HR, and cardiac electrical activity. Incidence of arrhythmia was measured with and without exposure to photoactivation of sympathetic fibres, and hearts were optically mapped to detect changes in action potential durations and conduction velocities. Results demonstrate facilitation of both developed force and HR after photostimulated release of NE, with increases in contractile force and HR of 34.5 ± 5.5 and 25.0 ± 9.3%, respectively. Photostimulation of sympathetic fibres also made hearts more susceptible to arrhythmia, with greater incidence and severity. In addition, optically mapped action potentials displayed a small but significant shortening of the plateau phase (-5.5 ± 1.0 ms) after photostimulation. This study characterizes a powerful and clinically relevant new model for studies of cardiac arrhythmias generated by increasing the activity of sympathetic nerve terminals and the resulting activation of myocyte β-adrenergic receptors.

Nucleotide sequence conservation of novel and established cis-regulatory sites within the tyrosine hydroxylase gene promoter.

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine biosynthesis and its gene proximal promoter ( < 1 kb upstream from the transcription start site) is essential for regulating transcription in both the developing and adult nervous systems. Several putative regulatory elements within the TH proximal promoter have been reported, but evolutionary conservation of these elements has not been thoroughly investigated. Since many vertebrate species are used to model development, function and disorders of human catecholaminergic neurons, identifying evolutionarily conserved transcription regulatory mechanisms is a high priority. In this study, we align TH proximal promoter nucleotide sequences from several vertebrate species to identify evolutionarily conserved motifs. This analysis identified three elements (a TATA box, cyclic AMP response element (CRE) and a 5'-GGTGG-3' site) that constitute the core of an ancient vertebrate TH promoter. Focusing on only eutherian mammals, two regions of high conservation within the proximal promoter were identified: a ∼250 bp region adjacent to the transcription start site and a ∼85 bp region located approximately 350 bp further upstream. Within both regions, conservation of previously reported cis-regulatory motifs and human single nucleotide variants was evaluated. Transcription reporter assays in a TH -expressing cell line demonstrated the functionality of highly conserved motifs in the proximal promoter regions and electromobility shift assays showed that brain-region specific complexes assemble on these motifs. These studies also identified a non-canonical CRE binding (CREB) protein recognition element in the proximal promoter. Together, these studies provide a detailed analysis of evolutionary conservation within the TH promoter and identify potential cis-regulatory motifs that underlie a core set of regulatory mechanisms in mammals.

Another chapter in the understanding of angiotensin-catecholamine interactions relevant to blood pressure control.

Another chapter in the understanding of angiotensin-catecholamine interactions relevant to blood pressure control.

Catecholaminergic C3 neurons are sympathoexcitatory and involved in glucose homeostasis.

Brainstem catecholaminergic neurons play key roles in the autonomic, neuroendocrine, and behavioral responses to glucoprivation, yet the functions of the individual groups are not fully understood. Adrenergic C3 neurons project widely throughout the brain, including densely to sympathetic preganglionic neurons in the spinal cord, yet their function is completely unknown. Here we demonstrate in rats that optogenetic stimulation of C3 neurons induces sympathoexcitatory, cardiovasomotor functions. These neurons are activated by glucoprivation, but unlike the C1 cell group, not by hypotension. The cardiovascular activation induced by C3 neurons is less than that induced by optogenetic stimulation of C1 neurons; however, combined stimulation produces additive sympathoexcitatory and cardiovascular effects. The varicose axons of C3 neurons largely overlap with those of C1 neurons in the region of sympathetic preganglionic neurons in the spinal cord; however, regional differences point to effects on different sympathetic outflows. These studies definitively demonstrate the first known function of C3 neurons as unique cardiovasomotor stimulatory cells, embedded in the brainstem networks regulating cardiorespiratory activity and the response to glucoprivation.

Angiotensin type 1A receptor expression in C1 neurons of the rostral ventrolateral medulla contributes to the development of angiotensin‐dependent hypertension

Chronic low-dose systemic infusion of angiotensinII induces hypertension via activation of the angiotensinII type1A receptor (AT1AR). Previously, we have demonstrated that expression of the AT1AR on catecholaminergic neurons is necessary for the full development of angiotensin-dependent hypertension. In the present study, we examined the mechanism by which selective deletion of the AT1AR from these cells affects the development of hypertension. We also tested the hypothesis that AT1ARs expressed by catecholaminergic C1 neurons in the rostral ventrolateral medulla play an important role in angiotensin-induced hypertension. A Cre-lox approach was used to delete the AT1AR from all catecholaminergic cells or from C1 neurons selectively. Subcutaneous administration of angiotensinII induced hypertension in all mice, with delayed onset and reduced maximal response in the global AT1AR catecholaminergic knockout mice. The AT1AR catecholaminergic knockout mice had decreased renal fluid and electrolyte retention and urinary noradrenaline excretion. The blood pressure response was reduced only during the second week of angiotensinII infusion in the mice with selective C1 AT1AR deletion, demonstrating that AT1AR expression by C1 neurons plays a moderate role in angiotensin-induced hypertension. The difference in the time course of development of hypertension between the mice with global AT1AR knockout from catecholaminergic cells and the mice with C1 AT1AR deletion suggests that other catecholaminergic neurons are important.

GDNF signaling implemented by GM1 ganglioside; failure in Parkinson's disease and GM1-deficient murine model

GDNF is indispensible for adult catecholaminergic neuron survival, and failure of GDNF signaling has been linked to loss of dopaminergic neurons in Parkinson's disease (PD). This study demonstrates attenuated GDNF signaling in neurons deficient in ganglio-series gangliosides, and restoration of such signaling with LIGA20, a membrane permeable analog of GM1. GM1 is shown to associate in situ with GFRα1 and RET, the protein components of the GDNF receptor, this being necessary for assembly of the tripartite receptor complex. Mice wholly or partially deficient in GM1 due to disruption of the B4galnt1 gene developed PD symptoms based on behavioral and neuropathological criteria which were largely ameliorated by gene therapy with AAV2-GDNF and also with LIGA20 treatment. The nigral neurons of PD subjects that were severely deficient in GM1 showed subnormal levels of tyrosine phosphorylated RET. Also in PD brain, GM1 levels in the occipital cortex, a region of limited PD pathology, were significantly below age-matched controls, suggesting the possibility of systemic GM1 deficiency as a risk factor in PD. This would accord with our finding that mice with partial GM1 deficiency represent a faithful recapitulation of the human disease. Together with the previously demonstrated age-related decline of GM1 in human brain, this points to gradual development of subthreshold levels of GM1 in the brain of PD subjects below that required for effective GDNF signaling. This hypothesis offers a dramatically different explanation for the etiology of sporadic PD as a manifestation of acquired resistance to GDNF.

Prenatal sodium arsenite affects early development of serotonergic neurons in the fetal rat brain

Prenatal arsenite exposure has been associated with developmental disorders in children, including reduced IQ and language abnormalities. Animal experiments have also shown that exposure to arsenite during development induced developmental neurotoxicity after birth. However, the evidence is not enough, and the mechanism is poorly understood, especially on the exposure during early brain development. This study assessed effects of sodium (meta) arsenite shortly after exposure on early developing fetal rat brains.Pregnant rats were administered 50mg/L arsenite in their drinking water or 20mg/kg arsenite orally using a gastric tube, on gestational days (GD) 9–15. Fetal brains were examined on GD16.Pregnant rats administered 20mg/kg arsenite showed reductions in maternal body weight gain and food consumption during treatment, but not with 50mg/L arsenite. Arsenite did not affect fetal development, as determined by body weight, mortality and brain size. Arsenite also did not induce excessive cell death or affect neural cell division in any region of the fetal neuroepithelium. Thyrosine hydroxylase immunohistochemistry revealed no difference in the distribution of catecholaminergic neurons between fetuses of arsenite treated and control rats. However, reductions in the number of serotonin positive cells in the fetal median and dorsal raphe nuclei were observed following maternal treatment with 20mg/kg arsenite. Image analysis showed that the serotonin positive areas decreased in all fetal mid- and hind-brain areas without altering distribution patterns. Maternal stress induced by arsenite toxicity did not alter fetal development. These results suggest that arsenite-induced neurodevelopmental toxicity involves defects in the early development of the serotonin nervous system.

Distribution of the creatine transporter throughout the human brain reveals a spectrum of creatine transporter immunoreactivity.

Creatine is a molecule that supports energy metabolism in cells. It is carried across the plasma membrane by the creatine transporter. There has been recent interest in creatine for its neuroprotective effects in neurodegenerative diseases and its potential as a therapeutic agent. This study represents the first systematic investigation of the distribution of the creatine transporter in the human brain. We have used immunohistochemical techniques to map out its location and the intensity of staining. The transporter was found to be strongly expressed, especially in the large projection neurons of the brain and spinal cord. These include the pyramidal neurons in the cerebral cortex, Purkinje cells in the cerebellar cortex, and motor neurons of the somatic motor and visceromotor cranial nerve nuclei and the ventral horn of the spinal cord. Many other neurons in the brain also had some degree of creatine transporter immunoreactivity. By contrast, the medium spiny neurons of the striatum and the catecholaminergic neurons of the substantia nigra and locus coeruleus, which are implicated in neurodegenerative diseases, showed a very low to almost absent level of immunoreactivity for the transporter. We propose that the distribution may reflect the energy consumption by different cell types and that the extent of creatine transporter expression is proportional to the cell's energy requirements. Furthermore, the distribution indicates that supplemented creatine would be widely taken up by brain cells, although possibly less by those cells that degenerate in Huntington's and Parkinson's diseases.

Over-expression of the Sirt3 sirtuin Protects neuronally differentiated PC12 Cells from degeneration induced by oxidative stress and trophic withdrawal

Sirt3 is a mitochondrial sirtuin whose deacetylase activity regulates facets of oxidative metabolic efficiency, anti-oxidative capacity, and intra-mitochondrial signaling. In this study, we tested whether the over-expression of a human Sirt3-myc transgene in differentiated PC12 cells, a model of sympathetic catecholaminergic neurons, would affect the sensitivity of these cells to oxidative stress or trophic withdrawal insults. Expression analysis revealed the Sirt3-myc product was expressed as a 45kDa pro-form, which localized primarily within the cytosol, and a 30kDa processed form that localized predominantly within mitochondria. When subjected to acute glucose deprivation or acute oxygen–glucose deprivation, differentiated PC12 cells over-expressing Sirt3-myc displayed significantly lower levels of cytotoxicity, both at the end of the insult, and at different times following media reperfusion, than cells transfected with a control plasmid. Further, Sirt3-myc over-expression also protected differentiated PC12 cells from apoptosis induced by trophic withdrawal. Collectively, these data indicate that an elevation of Sirt3 is sufficient to protect neuronal PC12 cells from cytotoxic insults, and add to the growing evidence that Sirt3 could be targeted for neuroprotective intervention.

Catecholaminergic neurons of mammalian brain and neuromelanin

Brain catecholaminergic neurons belong to the most extensively studied populations of nerve cells. Presence of a pigment neuromelanin in their cytoplasm is a specific morphological feature of these neurons in many mammalian species. Elucidation of the role of neuromelanin is of importance for comparative neurobiology, as it is absent in neurons of another neurotransmitter systems and, moreover, even in catecholaminergic neurons of some laboratory animals, which limits the possibility of experimental verification of existing hypotheses of its functions under physiological and pathological conditions. For recent years, neuromelanin is an object of particular interest in the scientific community involved in research of neurotoxicity and modeling the Parkinson's disease. The present review summarizes and analyzes new data on the structure and functions of neuromelanin and its probable role in pathogenesis of Parkinson's disease is discussed.

Abstract 201: Overexpression of Calcium/Calmodulin-Dependent Protein Kinase II (CaMKII) Potentiates Angiotensin II (AngII)-Mediated Signaling in Neurons

AngII signaling in the brain regulates systemic cardiovascular function. AngII modulates neuronal ion channel activity and neuronal firing via reactive oxygen species (ROS) and redox-sensitive proteins. Our recent studies indicate that in neurons AngII increases activity (i.e. phosphorylation) of CaMKII, a signaling intermediate known to inhibit potassium (K+) channel current. However, the potential cross-talk between AngII, ROS, and CaMKII in modulating neuronal K+ channel activity and the in vivo central AngII-induced pressor response remains unclear. Here, we tested the hypothesis that CaMKII overexpression exacerbates the AngII-dependent inhibition of K+ channel current and the acute central AngII-induced increase in blood pressure. The neuron specific isoform of CaMKII (CaMKIIα) was cloned, and CaMKIIα adenovirus was generated (AdCaMKIIα). Adenovirus-mediated overexpression of total and active CaMKII protein were assessed in differentiated mouse catecholaminergic (CATH.a) neurons by measuring total and phosphorylated CaMKII protein levels, respectively, via Western blot analysis. To investigate the effect of CaMKIIα overexpression on K+ channel activity, CATH.a neurons were transduced with AdCaMKIIα or control adenovirus, AdEmpty, and voltage-gated K+ current (IKV) was measured by the whole cell patch-clamp technique. Baseline IKV was measured followed by 5 mins of AngII (100 nM) superfusion. AngII lowered steady-state current (ISS) and peak current (Ipeak) in AdEmpty-transduced neurons (ISS by 20 ± 4%; Ipeak by 19 ± 3%), and this inhibitory response was significantly potentiated in AdCaMKIIα-transduced neurons (ISS by 40 ± 7%; Ipeak by 41 ± 5%; p&lt; 0.05). Preliminary in vivo studies suggest that the central AngII-induced pressor response is exacerbated in mice intracerebroventricularly injected with AdCaMKIIα as compared to control virus-injected mice. These data indicate that CaMKIIα overexpression potentiates AngII-mediated inhibition of IKv and the central AngII-induced pressor response. Future studies will investigate the ROS-mediated post-translational modifications of CaMKIIα in AngII-stimulated neurons and the contribution of these modifications to ion channel activity and blood pressure regulation.

The rate of fall of blood glucose determines the necessity of forebrain-projecting catecholaminergic neurons for male rat sympathoadrenal responses.

Different onset rates of insulin-induced hypoglycemia use distinct glucosensors to activate sympathoadrenal counterregulatory responses (CRRs). Glucosensory elements in the portal-mesenteric veins are dispensable with faster rates when brain elements predominate, but are essential for responses to the slower-onset hypoglycemia that is common with insulin therapy. Whether a similar rate-associated divergence exists within more expansive brain networks is unknown. Hindbrain catecholamine neurons distribute glycemia-related information throughout the forebrain. We tested in male rats whether catecholaminergic neurons that project to the medial and ventromedial hypothalamus are required for sympathoadrenal CRRs to rapid- and slow-onset hypoglycemia and whether these neurons are differentially engaged as onset rates change. Using a catecholamine-specific neurotoxin and hyperinsulinemic-hypoglycemic clamps, we found that sympathoadrenal CRRs to slow- but not rapid-onset hypoglycemia require hypothalamus-projecting catecholaminergic neurons, the majority of which originate in the ventrolateral medulla. As determined with Fos, these neurons are differentially activated by the two onset rates. We conclude that 1) catecholaminergic projections to the hypothalamus provide essential information for activating sympathoadrenal CRRs to slow- but not rapid-onset hypoglycemia, 2) hypoglycemia onset rates have a major impact on the hypothalamic mechanisms that enable sympathoadrenal CRRs, and 3) hypoglycemia-related sensory information activates hindbrain catecholaminergic neurons in a rate-dependent manner.

The orexinergic neurons receive synaptic input from C1 cells in rats.

The C1 cells, located in the rostral ventrolateral medulla (RVLM), are activated by pain, hypoxia, hypoglycemia, infection, and hypotension and elicit cardiorespiratory stimulation, adrenaline and adrenocorticotropic hormone (ACTH) release, and arousal. The orexin neurons contribute to the autonomic responses to acute psychological stress. Here, using an anatomical approach, we consider whether the orexin neurons could also be contributing to the autonomic effects elicited by C1 neuron activation. Phenylethanolamine N-methyl transferase-immunoreactive (PNMT-ir) axons were detected among orexin-ir somata, and close appositions between PNMT-ir axonal varicosities and orexin-ir profiles were observed. The existence of synapses between PNMT-ir boutons labeled with diaminobenzidine and orexinergic neurons labeled with immunogold was confirmed by electron microscopy. We labeled RVLM neurons with a lentiviral vector that expresses the fusion protein ChR2-mCherry under the control of the catecholaminergic neuron-selective promoter PRSx8 and obtained light and ultrastructural evidence that these neurons innervate the orexin cells. By using a Cre-dependent adeno-associated vector and TH-Cre rats, we confirmed that the projection from RVLM catecholaminergic neurons to the orexinergic neurons originates predominantly from PNMT-ir catecholaminergic (i.e., C1 cells). The C1 neurons were found to establish predominantly asymmetric synapses with orexin-ir cell bodies or dendrites. These synapses were packed with small clear vesicles and also contained dense-core vesicles. In summary, the orexin neurons are among the hypothalamic neurons contacted and presumably excited by the C1 cells. The C1-orexin neuronal connection is probably one of several suprabulbar pathways through which the C1 neurons activate breathing and the circulation, raise blood glucose, and facilitate arousal from sleep.