Large-scale momentary brain co-activation patterns are associated with hyperalgesia and mediate focal neurochemistry and cross-network functional connectivity in fibromyalgia.

ABSTRACT: Fibromyalgia has been characterized by augmented cross-network functional communication between the brain's sensorimotor, default mode, and attentional (salience/ventral and dorsal) networks. However, the underlying mechanisms of these aberrant communication patterns are unknown. In this study, we sought to understand large-scale topographic patterns at instantaneous timepoints, known as co-activation patterns (CAPs). We found that a sustained pressure pain challenge temporally modulated the occurrence of CAPs. Using proton magnetic resonance spectroscopy, we found that greater basal excitatory over inhibitory neurotransmitter levels within the anterior insula orchestrated higher cross-network connectivity between the anterior insula and the default mode network through lower occurrence of a CAP encompassing the attentional networks during sustained pain. Moreover, we found that hyperalgesia in fibromyalgia was mediated through increased occurrence of a CAP encompassing the sensorimotor network during sustained pain. In conclusion, this study elucidates the role of momentary large-scale topographic brain patterns in shaping noxious information in patients with fibromyalgia, while laying the groundwork for using precise spatiotemporal dynamics of the brain for the potential development of therapeutics.

Systemic inflammation and cortical neurochemistry in never-medicated first episode-psychosis individuals.

Studies of cellular and cytokine profiles have contributed to the inflammation hypothesis of schizophrenia; however, precise markers of inflammatory dysfunction remain elusive. A number of proton magnetic resonance spectroscopy (1H-MRS) studies in patients with first-episode psychosis (FEP) have shown higher brain levels of metabolites such as glutamate, myo-inositol (mI) and choline-containing compounds (tCho), suggesting neuroinflammation. Here, we present peripheral inflammatory profiles in antipsychotic-naive FEP patients and age-and-sex matched healthy controls, as well as cortical glutamate, mI and tCho levels using 1H-MRS. Inflammatory profiles were analyzed using cytokine production by peripheral blood mononuclear cells, that were either spontaneous or stimulated, in 48 FEP patients and 23 controls. 1H-MRS of the medial prefrontal cortex was obtained in 29 FEP patients and 18 controls. Finally, 16 FEP patients were rescanned after 4 weeks of treatment (open-label) with Risperidone. FEP patients showed a higher proportion of proinflammatory Th1/Th17 subset, and an increased spontaneous production of Interleukin (IL)-6, IL-2 and IL-4 compared with the control group. Results obtained from 1H-MRS showed no significant difference in either glutamate, mI or tCho between FEP and control groups. At baseline, CD8% showed a negative correlation with glutamate in FEP patients; after 4 weeks of risperidone treatment, the FEP group exhibited a decrease in glutamate levels which positively correlated with CD4 + T cells. Nevertheless, these correlations did not survive correction for multiple comparisons. FEP patients show evidence of immune dysregulation, affecting both the innate and adaptive immune response, with a predominantly Th2 signature. These findings, along with the changes produced by antipsychotic treatment, could be associated with both systemic and central inflammatory processes in schizophrenia.

Effect of standardized Eucalyptus globulus leaf extract on brain oxidative stress and aberrant neurochemistry of fructose-streptozotocin-induced diabetic rats.

The neuro-pharmacological effect of Eucalyptus globulus ethanol leaf extract in fructose-streptozotocin-induced diabetic rats was evaluated in this study. The phytochemical analysis of the extract was carried out using HPLC-DAD. Diabetes was induced in rats with 10% fructose in drinking water and a single intraperitoneal injection of 40 mg/kg streptozotocin (STZ). Diabetic animals were orally treated with 100-400 mg/kg of the extract for 21 days with glibenclamide as the reference drug. Blood and brain tissue were processed for the determination of serum electrolyte levels, hematological indices, and biochemical estimations. Ergosterol, pinitol, catechin, quercetin, robinetinidol, and other polyphenols were identified in the extract. Diabetic animals showed decreased serum potassium and sodium ion levels and decreased hematocrit, hemoglobin, red blood cells, white blood cells and lymphocytes but increased neutrophils. The brains of animals in the untreated diabetic group with increased blood glucose level showed oxidative stress (increased level of MDA and myeloperoxidase but decreased level of reduced glutathione and superoxide dismutase) and disturbed neurochemistry (increased level of acetylcholinesterase and monoamine oxidase but decreased level of Na+K+ATPase, tyrosine hydroxylase and dopamine). Administration of the Eucalyptus globulus leaf extract remarkably ameliorated the observed hyperglycemia, electrolyte, and hematological imbalances in animals. In addition, the administration of the extract attenuated the brain redox imbalance, and neurochemical disturbances in the rats. These results show that Eucalyptus globulus leaves contain antioxidant and neurotransmitter modulating phytochemicals with the potential to be developed as therapeutic agents for the management of diabetic cerebrovascular problems and related complications.

Altered prefrontal neurochemistry in the DJ-1 knockout mouse model of Parkinson's disease: complementary semi-quantitative analyses with in vivo magnetic resonance spectroscopy and MALDI-MSI.

In vivo proton magnetic resonance spectroscopy (1H-MRS) and matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) are two semi-quantitative analytical methods commonly used in neurochemical research. In this study, the two methods were used complementarily, in parallel, to investigate neurochemical perturbations in the medial prefrontal cortex (mPFC) of 9-month-old DJ-1 knockout mice, a well-established transgenic model for Parkinson's diseases. Convergingly, the results obtained with the two methods demonstrated that, compared with the wild-type (WT) mice, the DJ-1 knockout mice had significantly increased glutathione (GSH) level and GSH/glutamate (Glu) ratio in the mPFC, which likely presented an astrocytic compensatory mechanism in response to elevated regional oxidative stress induced by the loss of DJ-1 function. The results from this study also highlighted (1) the need to be cautious when interpreting the in vivo 1H-MRS results obtained from aged transgenic animals, in which the concentration of internal reference, being whether water or total creatine, could no longer be assumed to be the same as that in the age-matched WT animals, and (2) the necessity and importance of complementary analyses with more than one method under such circumstances.

SRF in Neurochemistry: Overview of Recent Advances in Research on the Nervous System.

Serum response factor (SRF) is a representative transcription factor that plays crucial roles in various biological phenomena by regulating immediate early genes (IEGs) and genes related to cell morphology and motility, among others. Over the years, the signal transduction pathways activating SRF have been clarified and SRF-target genes have been identified. In this overview, we initially briefly summarize the basic biology of SRF and its cofactors, ternary complex factor (TCF) and megakaryoblastic leukemia (MKL)/myocardin-related transcription factor (MRTF). Progress in the generation of nervous system-specific knockout (KO) or genetically modified mice as well as genetic analyses over the last few decades has not only identified novel SRF-target genes but also highlighted the neurochemical importance of SRF and its cofactors. Therefore, here we next present the phenotypes of mice with nervous system-specific KO of SRF or its cofactors by depicting recent findings associated with brain development, plasticity, epilepsy, stress response, and drug addiction, all of which result from function or dysfunction of the SRF axis. Last, we develop a hypothesis regarding the possible involvement of SRF and its cofactors in human neurological disorders including neurodegenerative, psychiatric, and neurodevelopmental diseases. This overview should deepen our understanding, highlight promising future directions for developing novel therapeutic strategies, and lead to illumination of the mechanisms underlying higher brain functions based on neuronal structure and function.

Sleep-Wake Neurochemistry.

Behavioral states naturally alternate between wakefulness and the sleep phases rapid eye movement and nonrapid eye movement sleep. Waking and sleep states are complex processes that are elegantly orchestrated by spatially fine-tuned neurochemical changes of neurotransmitters and neuromodulators including glutamate, acetylcholine, γ-aminobutyric acid, norepinephrine, dopamine, serotonin, histamine, hypocretin, melanin concentrating hormone, adenosine, and melatonin. However, as highlighted in this brief overview, no single neurotransmitter or neuromodulator, but rather their complex interactions within organized neuronal ensembles, regulate waking and sleep states. The neurochemical pathways presented here are aimed to provide a conceptual framework for the understanding of the effects of currently used sleep medications.

Developmental exposure to the A6-pesticide causes changes in tyrosine hydroxylase gene expression, neurochemistry, and locomotors behavior in larval zebrafish.

Purpose: In recent years, the increase in the biopesticides synthesis for alternative agricultural uses has required their impacts study. Among these compounds, several of them are known to exert endocrinedisrupting (EDs) effects causing deregulation of physiological functions affecting cell signaling pathways involved in neural cell differentiation leading to developmental neurotoxicity. The objective of our study was to determine the impact of the biopesticide A6 structurally related to estrogenic EDs on zebrafish larvae, to define its toxicity, the mechanisms responsible, and to monitor the locomotors activity at nanomolar concentrations (0. 0.5, 5 and 50 nM).Materials and methods: Using imaging analysis tools, immunohistochemistry, quantitative PCR, and an automated behavior recording system (Zebrabox) we were able to assess these effects.Results: We have shown through its blue fluorescence properties that it accumulates in different parts of the body such as the intestine, adipose tissue, muscles, yolk sac and head. A6 also disrupted swimming behavior by affecting the expression of tyrosine hydroxylase (TH) in dopaminergic neurons.Conclusions: In conclusion, our study provided a mechanistic understanding of the A6 neurotoxic effect which could be the result of its binding to the estrogen receptor.

Integral methods for automatic quantification of fast-scan-cyclic-voltammetry detected neurotransmitters.

Modern techniques for estimating basal levels of electroactive neurotransmitters rely on the measurement of oxidative charges. This requires time integration of oxidation currents at certain intervals. Unfortunately, the selection of integration intervals relies on ad-hoc visual identification of peaks on the oxidation currents, which introduces sources of error and precludes the development of automated procedures necessary for analysis and quantification of neurotransmitter levels in large data sets. In an effort to improve charge quantification techniques, here we present novel methods for automatic selection of integration boundaries. Our results show that these methods allow quantification of oxidation reactions both in vitro and in vivo and of multiple analytes in vitro.

Exposure to DMSO during infancy alters neurochemistry, social interactions, and brain morphology in long-evans rats.

INTRODUCTION: Dimethyl sulfoxide (DMSO) is a widely used solvent to dissolve hydrophobic substances for clinical uses and experimental in vivo purposes. While usually regarded safe, our prior studies suggest changes to behavior following DMSO exposure. We therefore evaluated the effects of a five-day, short-term exposure to DMSO on postnatal infant rats (P6-10). METHODS: DMSO was intraperitoneally injected for five days at 0.2, 2.0, and 4.0 ml/kg body mass. One cohort of animals was sacrificed 24 hr after DMSO exposure to analyze the neurometabolic changes in four brain regions (cortex, hippocampus, basal ganglia, and cerebellum) by hydrophilic interaction liquid chromatography. A second cohort of animals was used to analyze chronic alterations to behavior and pathological changes to glia and neuronal cells later in life (P21-P40). RESULTS: 164 metabolites, including key regulatory molecules (retinoic acid, orotic acid, adrenic acid, and hypotaurine), were found significantly altered by DMSO exposure in at least one of the brain regions at P11 (p < .05). Behavioral tests showed significant hypoactive behavior and decreased social habits to the 2.0 and 4.0 ml DMSO/kg groups (p < .01). Significant increases in number of microglia and astrocytes at P40 were observed in the 4.0 ml DMSO/kg group (at p < .015.) CONCLUSIONS: Despite short-term exposure at low, putatively nontoxic concentrations, DMSO led to changes in behavior and social preferences, chronic alterations in glial cells, and changes in essential regulatory brain metabolites. The chronic neurological effects of DMSO exposure reported here raise concerns about its neurotoxicity and consequent safety in human medical applications and clinical trials.

Effects of infliximab on brain neurochemistry of adults with bipolar depression.

OBJECTIVES: To explore the relationship between inflammation and neuronal metabolism in bipolar disorder (BD) by evaluating the neurochemical effects of the tumor necrosis factor-α (TNF-α) antagonist infliximab among individuals with bipolar depression METHODS: This is a post-hoc, exploratory analysis from a 12-week, randomized, double-blind, placebo-controlled trial with infliximab for adults with bipolar depression. We assessed the effects of infliximab on concentration of metabolites in the prefrontal cortex, using proton-magnetic resonance spectroscopy (1H-MRS), as well as its association with clinical outcomes (i.e. depressive symptom severity and cognitive function). RESULTS: Eighteen participants in the placebo and 15 in the infliximab group were included in this analysis. In the pre-specified primary outcome, there were no significant effects of treatment on prefrontal concentrations of N-acetylaspartate (NAA; p = 0.712). In the secondary analyses, there was a significant treatment by time interaction for glutamate (Glx; p = 0.018), indicating that Glx levels decreased in infliximab-treated patients, relative to placebo. Treatment group significantly moderated the association between changes in Glx levels and changes in a neurocognitive test (i.e. Digit Symbol Substitution Test; p = 0.014), indicating that in infliximab-treated participants reductions in Glx were associated with cognitive improvement. CONCLUSIONS: Treatment with infliximab did not affect prefrontal NAA concentration in adults with BD. Exploratory analysis suggested a potential effect of treatment on the glutamate system, a finding that should be confirmed and validated by additional studies.

Effect of short-term intake of high- and low-concentrations of sucrose solution on the neurochemistry of male and female mice.

The effect of short-term intake of high- and low-concentrations of sucrose solution on the neurochemistry of male and female mice was studied. The body weight, feed intake, sucrose solution consumption and brain monoamine neurotransmitters were determined after 34 days' intake of 1% and 8% sucrose solutions. The gene expression and protein levels related to dopamine and opioids were also determined. The results showed that the intake of 1% and 8% sucrose solution for 34 days did not cause significant changes in the weight development of both male and female mice. The preference for sucrose varies with sex. Both males and females had greater preference for the high concentration sucrose solution than the low concentration sucrose solution. The continuous intake of sucrose stimulated the release of monoamine neurotransmitters (DA, 5-HT, NE) in the brains of mice, and the reward effect of 8% sucrose solution is significantly higher than that of 1% sucrose solution. The sex of mice did not affect the release of neurotransmitters. The gene expressions of D1 and D2 were up-regulated in the 1% sucrose group of male mice, while the OPRM1 gene expression was down-regulated. The expression of these three genes in the 8% sucrose group of male mice was all down-regulated, while the gene expressions of D1 and D2 in the 1% and 8% sucrose group (p < 0.05) of female mice were both up-regulated.

Behavioral and Neurochemical Effects of Extra Virgin Olive Oil Total Phenolic Content and Sideritis Extract in Female Mice.

The aim of this study was to determine the cognitive and behavioral effects of extra virgin olive oil total phenolic content (TPC) and Sideritis (SID) extracts in female mice, and identify the associated neurochemical changes in the hippocampus and the prefrontal cortex. All animals received intraperitoneal low or high doses of TPC, SID or vehicle treatment for 7 days and were subjected to the Open Field (OF), Novel Object Recognition (NOR) and Tail Suspension Test (TST). The prefrontal cortex and hippocampus were dissected for analysis of neurotransmitters and aminoacids with high performance liquid chromatography with electrochemical detection (HPLC-ED). Both TPC doses enhanced vertical activity and center entries in the OF, which could indicate an anxiolytic-like effect. In addition, TPC enhanced non-spatial working memory and, in high doses, exerted antidepressant effects. On the other hand, high SID doses remarkably decreased the animals' overall activity. Locomotor and exploratory activities were closely associated with cortical increases in serotonin turnover induced by both treatments. Cognitive performance was linked to glutamate level changes. Furthermore, TPC reduced cortical taurine levels, while SID reduced cortical aspartate levels. TPC seems to have promising cognitive, anxiolytic and antidepressant effects, whereas SID has sedative effects in high doses. Both extracts act in the brain, but their specific actions and properties merit further exploration.

V Simpósio Internacional de Neuroquímica e Fisiopatologia da Célula Glial / V International Symposium of Neurochemistry and Pathophysiology of the Glial Cell

O 5º Simpósio Internacional de Neuroquímica e Fisiopatologia da Célula Glial aconteceu em conjunto com o 10º Simpósio de Atualização em Farmacologia da Universidade Federal da Bahia, de 09 a 12 de outubro de 2019 no Pavilhão Felipe Serpa (PAF I) no Campus Ondina, em Salvador-Bahia. Além de pesquisadores locais, o evento contou com a participação de renomados palestrantes do Brasil, das Américas e da Europa e Oceania, que apresentaram e discutiram avanços no entendimento de aspectos da neuroquímica, imunologia, fisiologia e fisiopatologia de doenças que afetam o sistema nervoso e sobre o entendimento de alvos moleculares para descoberta de drogas.

An Update on the Neurochemistry of Essential Tremor.

BACKGROUND: The pathophysiology and neurochemical mechanisms of essential tremor (ET) are not fully understood, because only a few post-mortem studies have been reported, and there is a lack of good experimental model for this disease. OBJECTIVE: The main aim of this review is to update data regarding the neurochemical features of ET. Alterations of certain catecholamine systems, the dopaminergic, serotonergic, GABAergic, noradrenergic, and adrenergic systems have been described, and are the object of this revision. METHODS: For this purpose, we performed a literature review on alterations of the neurotransmitter or neuromodulator systems (catecholamines, gammaaminobutyric acid or GABA, excitatory amino acids, adenosine, T-type calcium channels) in ET patients (both post-mortem or in vivo) or in experimental models resembling ET. RESULTS AND CONCLUSION: The most consistent data regarding neurochemistry of ET are related with the GABAergic and glutamatergic systems, with a lesser contribution of adenosine and dopaminergic and adrenergic systems, while there is not enough evidence of a definite role of other neurotransmitter systems in ET. The improvement of harmaline-induced tremor in rodent models achieved with T-type calcium channel antagonists, cannabinoid 1 receptor, sphingosine-1-phosphate receptor agonists, and gap-junction blockers, suggests a potential role of these structures in the pathogenesis of ET.

Neurochemical insights into the radiation protection of astronauts: Distinction between low- and moderate-LET radiation components.

Radiation protection of astronauts remains an ongoing challenge in preparation of deep space exploratory missions. Exposure to space radiation consisting of multiple radiation components is associated with a significant risk of experiencing central nervous system (CNS) detriments, potentially influencing the crew operational decisions. Developing of countermeasures protecting CNS from the deleterious exposure requires understanding the mechanistic nature of cognitive impairments induced by different components of space radiation. The current study was designed to identify differences in neurochemical modifications caused by exposure to low- and moderate-LET radiations and to elucidate a distinction between the observed outcomes. We exposed rats to accelerated protons (170 MeV; 0.5 keV/µm) or to carbon ions (12C; 500 MeV/u; 10.5 keV/µm) delivered at the same dose of 1 Gy. Neurochemical alterations were evaluated 1, 30, and 90 days after exposure via indices of the monoamine metabolism measured in five brain structures, including prefrontal cortex, hypothalamus, nucleus accumbens, hippocampus and striatum. We obtained the detailed patterns of neurochemical modifications after exposure to the mentioned radiation modalities. Our data show that the enhancement in the radiation LET from relatively low to moderate values leads to different neurochemical outcomes and that a particular effect depends on the irradiated brain structure. We also hypothesized that exposure to the moderate-LET radiations can induce a hyperactivation of feedback neurochemical mechanisms, which blur metabolic deviations and lead to the delayed impairments in brain functions. Based on our findings we discuss possible contribution of the observed changes to behavioural impairments.

Using positron emission tomography to investigate hormone-mediated neurochemical changes across the female lifespan: implications for depression.

Ovarian hormones, particularly oestrogen and progesterone, undergo major fluctuations across the female lifespan. These hormone transition periods, such as the transition from pregnancy to postpartum, as well as the transition into menopause (perimenopause), are also known to be times of elevated susceptibility to depression. This study reviews how these transition periods likely influence neurochemical changes in the brain that result in disease vulnerability. While there are known associations between oestrogen/progesterone and different monoaminergic systems, the interactions and their potential implications for mood disorders are relatively unknown. Positron Emission Tomography (PET) allows for the in-vivo quantification of such neurochemical changes, and, thus, can provide valuable insight into how both subtle and dramatic shifts in hormones contribute to the elevated rates of depression during pre-menstrual, post-partum, and perimenopausal periods in a woman's life. As one better understands how to address the challenges of PET studies involving highly vulnerable populations, such as women who have recently given birth, one will gain the insight necessary to design and individualize treatment and therapy. Understanding the precise time-line in younger women when dramatic fluctuations in the hormonal milieu may contribute to brain changes may present a powerful opportunity to intervene before a vulnerable state develops into a diseased state in later life.

Interaction Between the Trace Amine-Associated Receptor 1 and the Dopamine D2 Receptor Controls Cocaine's Neurochemical Actions.

Recent evidence suggests that the trace amine-associated receptor 1 (TAAR1) plays a pivotal role in the regulation of dopamine (DA) transmission and cocaine's actions. However, the underlying mechanisms through which TAAR1 activation mediates these effects have not yet been elucidated. Here, we used fast-scan cyclic voltammetry to measure DA dynamics and explore such mechanisms. We show, first, that the full TAAR1 agonist, RO5256390, dose-dependently blocked cocaine-induced inhibition of DA clearance in slices of the nucleus accumbens. Second, subthreshold inhibition of PKA or PKC phosphorylation did not prevent TAAR1 suppression of cocaine effects whereas subeffective doses of the DA D2 receptor antagonist, L-741,626, rescued cocaine's ability to produce changes in DA uptake in the presence of full TAAR1 activation, thus indicating that TAAR1 modulation of cocaine effects requires simultaneous DA D2 receptor activation. Predictably, inhibition of glycogen synthase kinase-3 (GSK-3), which results from activation of D2/TAAR1 heterodimers, fully reproduced the inhibitory effects of TAAR1 activation on cocaine-induced changes in DA transmission. Collectively, the present observations reveal that the ability of TAAR1 to regulate cocaine effects is linked to cooperative interactions with D2 autoreceptors and associated downstream molecular targets converging on GSK-3 and suggest a new mechanism to disrupt cocaine neurochemical actions.

Neurochemical and neuropharmacological characterization of ASP2905, a novel potent selective inhibitor of the potassium channel KCNH3.

KCNH3 (BEC1) is a member of the ether-à-go-go (KCNH) family of voltage-gated K+ channels. The aim of this study was to determine the pharmacological profiles in vitro and in vivo of a KCNH3 inhibitor N-(4-fluorophenyl)-N'-phenyl-N''-(pyrimidin-2-ylmethyl)-1,3,5-triazine-2,4,6-triamine (ASP2905). We analyzed the effects of ASP2905 on channel activity in vitro and its neuropharmacological properties in young and aged rats as well as in mice. ASP2905 potently inhibited potassium currents in CHO cells expressing KCNH3 (IC50 = 9.0nM). In contrast, ASP2905 (≤ 10µM) minimally bound with low affinities to 55 transmembrane proteins. ASP2905 (0.1µM, 1µM) decreased the frequency of spontaneous inhibitory postsynaptic currents in cultured rat hippocampal neurons. In mice, ASP2905 reversed the disruption of spontaneous alternation behavior induced by MK-801 and scopolamine (minimum effective dose of ASP2905: 0.0625mg/kg, po). ASP2905 ameliorated the cognitive deficits of aged rats in step-through passive avoidance (0.0313 and 0.0625mg/kg, po) and Morris water-maze tasks (0.01mg/kg, po) and effectively penetrated the brain. The mean plasma and brain concentrations of ASP2905 reached their maxima (Cmax = 0.399ng/ml and 1.77ng/g, respectively) 1h after a single oral administration and then decreased (t1/2 = 1.5-1.6h) (brain plasma ratio = 2.7-4.9). The present study suggests that ASP2905 is a selective, orally administered inhibitor of KCNH3, which can enhance cognitive performance.

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.