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Highlights • SAR-CoV-2 infection lead to sympathetic overactivity. • People with co morbid condition due to pre-sympathetic overactivity, there will be always possible of worse outcome. • FDA-approved drug clonidine reduces sympathetic activity during COVID-19 infection and prevent complication and death. • Clonidine should be considered early in incremental fashion to mitigate SAR-CoV-2 related complication. • This is the first case series demonstrating the effectiveness of early use of clonidine in COVID-19., Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a life-threating viral infection that is highly transmissible and be lethal. Although many patients with mild symptoms recover, an acute form of the infection is characterized by rapidly evolving respiratory failure, an acute inflammatory response, organ failure, and death. Herein, we describe the use of clonidine to modulate the acute inflammatory consequences of this infection in three cases. The patients were three men between 40–50 years from Kathmandu valley, during the peak of COVID-19 (September 2020- January 2021). All three patients presented with typical COVID-19 symptoms (daily fever, loss of smell and taste, excessive fatigue, cough) and had pneumonia with typical finding in CT Scan of chest. Patient 1was able to maintain adequate oxygenation despite having pneumonia, managed at home by regular self-monitoring of vitals and treatment with oral clonidine whereas patient 2 and 3 developed significant pneumonia and had difficult in maintaining oxygenation hence admitted in hospital and treated with clonidine and supplemental oxygen. All three patients recovered completely. In this limited report, we proposed several mechanisms by which clonidine may be useful in managing rapidly evolving SARS-CoV-2 infection based on the rationale that early clonidine administration can intervene in the catecholaminergic response that characterizes rapid clinical deterioration including presumptive cytokine storm that occurs in COVID-19 infection in vulnerable populations.

Summary We developed a spatially-tracked single neuron transcriptomics map of an intrinsic cardiac ganglion, the right atrial ganglionic plexus (RAGP) that is a critical mediator of sinoatrial node (SAN) activity. This 3D representation of RAGP used neuronal tracing to extensively map the spatial distribution of the subset of neurons that project to the SAN. RNA-seq of laser capture microdissected neurons revealed a distinct composition of RAGP neurons compared to the central nervous system and a surprising finding that cholinergic and catecholaminergic markers are coexpressed, suggesting multipotential phenotypes that can drive neuroplasticity within RAGP. High-throughput qPCR of hundreds of laser capture microdissected single neurons confirmed these findings and revealed a high dimensionality of neuromodulatory factors that contribute to dynamic control of the heart. Neuropeptide-receptor coexpression analysis revealed a combinatorial paracrine neuromodulatory network within RAGP informing follow-on studies on the vagal control of RAGP to regulate cardiac function in health and disease., Graphical abstract, Highlights • Spatially-tracked single neuron transcriptomics map of intrinsic cardiac ganglia • Distinct composition of intrinsic cardiac neurons compared to the central neurons • Correlation of cholinergic and catecholaminergic markers suggesting neuroplasticity • Adaptive and dynamic system driven by putative paracrine neuromodulatory networks, Cardiovascular medicine; Molecular physiology; Systems neuroscience; Transcriptomics

Highlights • Medullary autonomic dysfunction may contribute to the cascade leading to SUDEP. • Polyglucosan bodies (PGB) identified primarily in the medullary catecholaminergic neurones has not previously been reported. • Deposition of PGB in the medulla could compromise brainstem function in the agonal peri-ictal period., Polyglucosan bodies have been reported in the context of hypoxic-ischaemic perinatal brain injury, mainly in the pallidum but with rare reports in brainstem neurons. We report a case of a five-year-old boy with cerebral palsy and complex neurological features including epilepsy who experienced sudden nocturnal death. At post-mortem long-standing bilateral necrosis of basal ganglia and hippocampal atrophy was identified in keeping with hypoxic-ischaemic perinatal injury. In addition numerous polyglucosan bodies, which were PAS, p62 and ubiquitin positive, were noted in brainstem neurones and dendrites, primarily involving the ventrolateral and dorsomedial medulla. Immunohistochemistry confirmed relative preservation of medullary neuronal populations in the reticular formation, including catecholaminergic (tyrosine hydroxylase, TH), serotonergic (tryptophan hydroxylase) and neurokinin1 receptor/somatostatin positive neurones. The polyglucosan bodies predominated in catecholaminergic neurones which could indicate their selective vulnerability and a functional deficiency, which during a critical peri-ictal period contributed to the sudden unexpected death in epilepsy.

Parkinson’s disease is a common degenerative disorder of the brain for which the cellular basis is in large part the loss of dopaminergic neurons from the midbrain, making it an attractive target for cell replacement therapy. Available medical and surgical treatments have significant drawbacks, further encouraging such efforts. Attempts to achieve this with various sources of dopaminergic and catecholaminergic tissue have been undertaken over several decades, and the use of human fetal midbrain, although associated with numerous problems, has suggested the possibility of significant benefit from cell replacement therapy. iPSC technology has now provided a possible solution to many of these problems. We present the history of cell replacement therapy for Parkinson’s disease and the various current methods and strategies to create, differentiate and develop iPSC as a clinically applicable therapeutic option for both autologous and allogeneic use.

The bed nucleus of the stria terminalis (BNST) is a complex integrative centre in the\ud forebrain, composed of multiple sub-nuclei, each with discrete populations of neurons.\ud Progress in understanding BNST function, both in the adult and during postnatal\ud maturation, is dependent upon a more complete characterization of neuronal\ud phenotypes in the BNST. The aim of the current study was to define the molecular\ud phenotype of one postnatal BNST neuronal population, in order to identify molecular\ud factors that may underlie both (protein marker-related) immaturity, and secondly, the\ud transience of this phenotype. This BNST population was originally identified by high, but\ud transient expression of the EGR1 transcription factor (TF) in postnatal rat lateral\ud intermediate BNST (BNSTLI). The current results confirm a high level of Egr1 activation\ud in postnatal day 10 (PN10) male BNSTLI that is lost at PN40, and now demonstrate a\ud similar pattern of transient activation in female brains. Apparent cellular immaturity in this\ud population, as indicated by low levels of the adult neuronal marker NeuN/RBFOX3, was\ud found to be uncorrelated with both key neuronal regulator protein expression (SOX2 and\ud REST), and also RBFOX2 protein levels. The BNSTLI neurons have a partial\ud catecholaminergic phenotype (tyrosine hydroxylase-positive/dopa decarboxylasenegative;\ud TH+ve/DDC-ve) that is lost at PN40. In contrast, the co-expressed\ud neuropeptide, somatostatin, is maintained, albeit at lower levels, at PN40. The\ud transcriptional basis of the transient and partial catecholaminergic phenotype was\ud investigated by analysing TFs known to maintain adult dopaminergic (TH+ve/DDC+ve)\ud neuronal phenotypes. The BNSTLI neurons were shown to lack forkhead TFs including\ud FOXA1, FOXA2 and FOXO1. In addition, the BNSTLI neurons had low, primarily\ud cytoplasmic, expression of NR4A2/ NURR1, an orphan nuclear receptor that is critical\ud for adult maintenance of midbrain dopamine neurons. These results detail the molecular\ud 3\ud features of an immature neuronal phenotype, and reveal TF deficiencies that may\ud underlie postnatal transience of the phenotype.

We investigated whether the descending noradrenergic system regulates allergic itch. Mosquito allergy of the hind paw elicited biting, an itch-related response, in sensitized mice. The biting was inhibited by intrathecal clonidine and reversed by yohimbine, an α2-adrenoceptor antagonist. The biting was increased by intrathecal pretreatment with the catecholaminergic neurotoxin 6-hydroxydopamine and the α-adrenoceptor antagonist phentolamine but not the serotonergic neurotoxin 5,7-dihydroxytryptamine. We propose that α2-adrenoceptors are involved in the inhibition of allergic itch in the spinal cord and that the descending noradrenergic system exerts a tonic inhibition on the itch signaling. The serotonergic system may not be involved. Keywords:: allergic itch, clonidine, descending noradrenergic system

Here, we provide a detailed account of how to denervate white and brown adipose tissue (WAT and BAT) and how to measure sympathetic nervous system (SNS) activity to these and other tissues neurochemically. The brain controls many of the functions of WAT and BAT via the SNS innervation of the tissues, especially lipolysis and thermogenesis, respectively. There is no clearly demonstrated parasympathetic innervation of WAT or the major interscapular BAT (IBAT) depot. WAT and BAT communicate with the brain neurally via sensory nerves. We detail the surgical denervation (eliminating both innervations) of several WAT pads and IBAT. We also detail more selective chemical denervation of the SNS innervation via intra-WAT/IBAT 6-hydroxy-dopamine (a catecholaminergic neurotoxin) injections and selective chemical sensory denervation via intra-WAT/IBAT capsaicin (a sensory nerve neurotoxin) injections. Verifications of the denervations are provided (HPLC-EC detection for SNS, ELIA for calcitonin gene-related peptide (proven sensory nerve marker)). Finally, assessment of the SNS drive to WAT/BAT or other tissues is described using the alpha-methyl-para-tyrosine method combined with HPLC-EC, a direct neurochemical measure of SNS activity. These methods have proven useful for us and for other investigators interested in innervation of adipose tissues. The chemical denervation approach has been extended to nonadipose tissues as well.

Based on previous work describing the distribution of somatostatin-28 (1-12) in the male alpaca (Lama pacos) diencephalon, and owing to the well known interactions between this peptide and the catecholaminergic system, the aims of this work are (1) to describe the distribution of putative catecholaminergic cell groups in the alpaca diencephalon and (2) to study the possible morphological basis of the interactions between these substances in the diencephalon of the alpaca by using double immunohistochemistry methods. Thus, the distribution of catecholaminergic cell groups in the alpaca diencephalon agrees with that previously described in the diencephalon of other mammalian species of the same order: the A11, A12, A13, A14 and A15d cell groups have been identified; however, we have observed an additional hitherto undescribed cell group containing tyrosine hydroxylase in the medial habenula. In addition, double-labelling procedures did not reveal neurons containing tyrosine hydroxylase and somatostatin, suggesting that the hypothalamic interactions between catecholamines and somatostatin at intra-cellular level must be carried out by a somatostatin molecule other than fragment (1-12). Otherwise, the overlapping distribution patterns of these substances would suggest some interconnections between groups of chemospecific neurons. These results could be the starting point for future studies on hypothalamic functions in alpacas, for example those concerning reproductive control, since other physiological studies have suggested that this species could have different regulatory mechanisms from other mammalian species. Our results support the Manger hypothesis that the same nuclear complement of neural systems exists in the brain of species of the same order.

The locus coeruleus (LC) has been implicated in a variety of physiological functions including sleep/wakefulness, cognition/memory, stress/emotion, and pain. Marked loss of LC-noradrenergic (NAergic) neurons is observed in autopsy specimens of patients with Alzheimer’s disease and Parkinson’s disease (PD), and part of the clinical symptoms of these diseases may be related to dysfunction of the LC. Neurotoxins have been utilized to ablate LC-NAergic neurons in experimental animals for elucidating the pathophysiological implication of the loss of LC, but there are methodological drawbacks in previously utilized methods. We employed immunotoxin-mediated neuronal targeting to overcome these problems. Following complete disruption of the LC-NAergic neurons by immunotoxin, mice showed behavioral changes, which resembled the nonmotor symptoms of PD. The LC-NAergic neurons did not regenerate following ablation, so the immunotoxin-mediated neuronal targeting may be useful especially for studying the long-term effects of the loss of LC-NAergic neurons on brain functions.

Compared to neurons that communicate using synapses, some neuroendocrine neurons release relatively large quantities of peptide into the vasculature to control neuroendocrine function. Maintaining adequate amounts of peptide for release through controlled biosynthesis is therefore critical for their function. But how neuroendocrine-or in fact, any neuropeptide-neurons link appropriate levels of peptide biosynthesis with the action potentials that drive peptide release is unknown. Here, we review possible mechanisms in paraventricular hypothalamic CRH neuroendocrine neurons to coordinate these processes in response to catecholaminergic inputs. We show that CRH synthesis and release mechanisms are not invariably linked as CRH neurons are activated. Instead, coupling mechanisms exist in the premotor network that provides their synaptic inputs and in their intracellular signal transduction mechanisms, where transmitter-regulated phosphorylation of p44/42 mitogen-activated protein kinases (ERK1/2) may play a prominent role. These versatile and dynamic coupling mechanisms provide a way to link peptide biosynthesis and release.

In addition to catecholaminergic neurons possessing all the enzymes of catecholamine synthesis and the specific membrane transporters, neurons partly expressing the catecholaminergic phenotype have been found a quarter of a century ago. Most of them express individual enzymes of dopamine (DA) synthesis, tyrosine hydroxylase (TH), or aromatic l -amino acid decarboxylase (AADC), lacking the DA membrane transporter and the vesicular monoamine transporter, type 2. These so-called monoenzymatic neurons are widely distributed throughout the brain in ontogenesis and adulthood being in some brain regions even more numerous than dopaminergic (DA-ergic) neurons. Individual enzymes of DA synthesis are expressed in these neurons continuously or transiently in norm and pathology. It has been proven that monoenzymatic TH neurons and AADC neurons are capable of producing DA in cooperation. It means that l -3,4-dihydroxyphenylalanine ( l -DOPA) synthesized from l -tyrosine in monoenzymatic TH neurons is transported to monoenzymatic AADC neurons for DA synthesis. Such cooperative synthesis of DA is considered as a compensatory reaction under a failure of DA-ergic neurons, for example, in neurodegenerative diseases like hyperprolactinemia and Parkinson's disease. Moreover, l -DOPA, produced in monoenzymatic TH neurons, is assumed to play a role of a neurotransmitter or neuromodulator affecting the target neurons via catecholamine receptors. Thus, numerous widespread neurons expressing individual complementary enzymes of DA synthesis serve to produce DA in cooperation that is a compensatory reaction at failure of DA-ergic neurons.

The sympathetic nervous system not only regulates cardiovascular and metabolic responses to stress but also is altered by stress. The sympathoneural and sympathoadrenomedullary systems are modified by different metabolic pathways and have different responses to short- and to long-term stressors. Stress also induces nonneuronal catecholamine enzymes, primarily through corticosteroids. Catecholamine synthetic enzymes are induced by different pathways in response to short- and long-term acting stressors, like cold exposure or immobilization, and differently in the sympathetic ganglia and the adrenal medulla. However, a long-term exposure to one stressor can increase the response to a second, different stressor. Tyrosine hydroxylase gene transcription increases after only 5min of immobilization through phosphorylation of CREB, but this response is short lived. However, repeated stress gives a longer-lived response utilizing transcription factors such as Egr-1 and Fra-2. Glucocorticoids and ACTH also induce sympathoneural enzymes leading to distinct patterns of short-term and long-lived activation of the sympathetic nervous system. Nonneuronal phenylethanolamine N-methyltransferase (PNMT) develops early in the heart and then diminishes. However, intrinsic cardiac adrenergic cells remain and nonneuronal PNMT is present in many cells of the adult organism and increases in response to glucocorticoids. Both stress-induced and administered glucocorticoids induce fetal PNMT and hypertension. Human stressors such as caring for an ill spouse or sleep apnea cause a persistent increase in blood norepinephrine, increased blood pressure, and downregulated catecholamine receptors. Hypertension is associated with a loss of slow-wave sleep, when sympathetic nerve activity is lowest. These findings indicate that stress-induced alteration of the sympathetic nervous system occurs in man as in experimental animals.

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the biosynthesis of the catecholamines dopamine, noradrenaline, and adrenaline. In response to short-term stimuli, TH activity is regulated by feedback inhibition by the catecholamines and relief of that inhibition by phosphorylation. This chapter examines the current understanding of these regulatory mechanisms and the roles that they play in different catecholaminergic cells. This chapter also examines hierarchical phosphorylation in TH and how it provides a mechanism for the differential regulation of the major human TH isoforms.

The interaction between the endocannabinoid system and catecholaminergic circuits has gained increasing attention as it is recognized that the development of synthetic cannabinoid receptor agonists/antagonists or compounds targeting endocannabinoid synthesis/metabolism may hold some therapeutic potential for the treatment of psychiatric disorders. The noradrenergic system plays a critical role in the modulation of emotional state, primarily related to anxiety, arousal, and stress. Recent evidence suggests that the endocannabinoid system mediates stress responses and emotional homeostasis, in part, by targeting noradrenergic circuits. This review summarizes our current knowledge regarding the anatomical substrates underlying regulation of noradrenergic circuitry by the endocannabinoid system. It then presents biochemical evidence showing an important effect of cannabinoid modulation on adrenergic receptor signaling. Finally, new evidence from behavioral pharmacology studies is provided demonstrating that norepinephrine is a critical determinant of cannabinoid-induced aversion, adding another dimension to how central noradrenergic circuitry is regulated by the cannabinoid system, Grant support: NIDA DA020129.

The distribution of tyrosine hydroxylase (TH) in the brainstem of alpaca (Lama pacos) has been analysed using immunohistochemical methods. The following catecholaminergic cell nuclei have been detected: A1, C1, A2, C2 and area postrema in the medulla oblongata; A5, A6d, A7sc and A7d in the pons; as have several mesencephalic groups: A8, A9l, A9m, A9v, A9pc, A10, A10c, A10d and A10dc. This nuclear parcellation differs from that found in rodents, but agrees with the results reported in other members of the Artiodactyla order, such as giraffe or pig, and with the catecholaminergic distribution detected in species of other mammalian orders. Thus, these findings support the hypothesis that the animals included in the same order show the same nuclear complement in the neuromodulatory systems. In addition, it seems that other species share the same catecholaminergic groups as the alpaca, suggesting that a specific nuclear disposition was important and worth maintaining throughout evolution. Moreover, the distribution of TH has been compared with that of CGRP by double immunohistochemistry. Double-labelled neurons were very isolated and observed only in a few catecholaminergic groups: A1 and C2 in the medulla oblongata, A6d, A7sc and A7d in the pons, and A9l in the mesencephalon. However, interaction between TH and CGRP may be possible in more brainstem regions, particularly the area postrema. This interaction may prove important in the regulation of the specific cardiovascular control of alpacas given their morphological characteristics.

Publisher Summary This chapter presents a study to examine endogenous defenses that protect dopamine neurons. The study involves using 6-hydroxydopamine (6-OHDA) models of Parkinson's disease (PD). 6-OHDA has been useful in evaluating the therapeutic potential of compounds such as GDNF in high throughput and inexpensive manner and has been useful in answering some fundamental questions about cell biology. As with sublethal ischemic episodes, oxidative stress produced by 6-OHDA has the capacity to elicit self-protective responses. This study suggests that many individuals do not develop PD because they have a strong defensive stress response, which can be defined as a protective reaction to what would otherwise be toxic cellular stress. It is hypothesized that in people who do develop the illness, this defensive stress response—while not adequate to prevent the disease—serves to keep the neurodegenerative process at bay for many years. It is also suggested that although a response to minor stress may be defensive and thereby serve to “precondition” an individual against further insults, a more intense stress might actually sensitize an individual to a potential toxin. The chapter reviews endogenous defenses and then focus upon stress responses initiated in common models of PD, those involving cells and animals exposed to the catecholaminergic neurotoxin 6-OHDA, while also highlighting some of the advantages and limitations of 6-OHDA.

Stress is the response of the cell or the organism to challengers of homeostasis (a dynamic state of balance between the stress response and the stressors). Homeostasis is essential for the preservation of life. In humans and other higher organisms, components of the stress system include various neurotransmitters and hormones, which in most species constitute the effectors of the hypothalamic-pituitary-adrenal (HPA) axis. Thus, the cholinergic and catecholaminergic systems, corticotropin releasing hormone (CRH), arginine vasopressin, adrenocorticotropic hormone (ACTH), and the glucocorticosteroids are all essential parts of the stress system in higher organisms.

The catecholamines dopamine (DA) and norepinephrine (noradrenaline, NA) exert a strong modulatory control over the “motive circuit”. This neuronal circuit comprises cortical, striatal and thalamic areas and underlies the behavioural expression of motivated behaviour, including classical and operant conditioning. Conditioned and unconditioned stimuli with a strong motivational salience have rapid access to the catecholaminergic systems and activation by such stimuli may result in the modulation of a wide array of neuronal processes in the brain. Microdialysis is the universally applicable method to determine the transmitter efflux resulting from this activation. In this chapter, the evidence that DA and NA efflux is increased by conditioned approach and avoidance behaviour and during instrumental actions is reviewed and data obtained using microdialysis are compared with results from in vivo electrophysiological and electrochemical studies. Special attention is paid to the suggestions that the catecholamines support flexibility in operant responding and may have a unique role in reward-related learning.

Publisher Summary This chapter discusses the sleep and autonomic nervous system (ANS) dysfunction. The autonomic nervous system, through its complex central and peripheral circuits, controls vital involuntary functions of the body, such as circulation, respiration, thermoregulation, neuro endocrine secretion, gastrointestinal, and genitourinary functions. The autonomic nervous system and sleep are closely related along anatomical, physiological, and neurochemical lines. The central autonomic network (CAN), through its ascending and descending connections between the hypothalamic–limbic region and the nucleus tractus solitarius (NTS) in the medulla, orchestrates the sympathetic and parasympathetic divisions of the ANS. Sleep-promoting neurons, which are scattered in the vicinity of the central autonomic network (CAN) and its connections (e.g. preoptic–anterior hypothalamic or POAH region and NTS) along with cholinergic REM-on and catecholaminergic REM-off cells in the ponto-mesencephalic junction and pons, control non-rapid eye movement (NREM) and rapid eye movement (REM) sleep cycles. Sleep induces profound changes in the functions of the ANS, and disorders of the ANS adversely affect vital functions during sleep, including circulation and respiration.

Publisher Summary This chapter describes the strategy of immunotoxin-mediated cell targeting (IMCT) and its use to generate a transgenic mouse model for autonomic neuropathy (AN) derived from disruption of the peripheral catecholaminergic cell types. In the strategy of IMCT, transgenic mice are generated that express human interleukin-2 receptor α-subunit (IL-2Rα) under the control of a cell type-specific gene promoter. IMCT was used to generate an animal model that exhibits autonomic failure caused by ablation of the peripheral catecholaminergic cell types. To express human IL-2Rα in these cell types, the dopamine (DA) β-hydroxylase gene promoter was used. In the transgenic mice, IL-2Rα was expressed in the adrenal medulla chromaffin cells and the sympathetic ganglion principal neurons. The histopathologic examination indicated no apparent damages in other cell types in the treated transgenic mice but displayed progressive phenotypic abnormalities, including cardiac dysfunction and hypothermia. These abnormalities reflected deficits in autonomic regulation of heart function and cardiovascular system because of ablation of the peripheral catecholaminergic cell types. IMCT provides a technique for conditionally ablating specific neuronal types involved in autonomic functions. IMCT will be useful for elucidating the role of different neuronal types and their interactions in the development and in the symptoms of autonomic disorders.

Publisher Summary This chapter discusses a carotid chemodenervation approach to study oxygen sensing in brain stem catecholaminergic cells. The physiological conditions of hypoxia known as “moderate” or “tolerable” hypoxia allows a long-term exposure lasting for weeks. The exposure to tolerable hypoxia enables the definition of the morphofunctional mechanisms involved in the ventilatory acclimatization of intact or carotid chemodenervated animals. Exposure to moderate hypoxia induces the activation of brain stem catecholaminergic cells located in cardiorespiratory areas and/or involved in the modulation of cardiorespiratory output. The chapter describes the in vivo and in vitro preparations used to test the effect of a moderate hypoxia on brain stem respiratory areas and then the methods used to evaluate the consequences of hypoxia on ventilation, catecholamine neurochemistry, and gene expression. Methods used to test the physiological effect of hypoxia are discussed.

Age-related changes of the catecholaminergic nerve fibers of the trachea, bronchial smooth muscle, lung capillaries and bronchus-associated lymphoid tissue (BALT) were studied in male Wistar rats aged 3 months (young), 12 months (adult) and 24 months (old/aged). Catecholamine histo- and immuno-fluorescence techniques were used, associated with image analysis and high pressure liquid chromatography with electrochemical detection of nor-epinephrine (nor-adrenaline). In young rats, blue-green fluorescent nerve fibers supply the trachea-bronchial smooth muscle and tracheal and bronchial glands. These structures are innervated by a delicate network of nerve fibers, being rich in varicosities. Pulmonary capillaries are sparsely innervated. The highest nor-epinephrine concentration was found in the trachea and bronchi, followed by BALT. The density and the pattern of noradrenergic nerve fibers of the trachea-bronchial tree or of the pulmonary vessels were similar in young and adult rats. In aged rats, a loss of noradrenergic nerve fibers, involving primarily the supply to the smooth muscle of the trachea-bronchial tree, was observed. Fluorescence microscopic techniques demonstrated a higher sensitivity than nor-epinephrine assay in detecting changes of the sympathetic nerve supply of the trachea-bronchial tree, pulmonary vessels and BALT. The possible significance of reduced noradrenergic nerve supply of the trachea-bronchial-pulmonary tree in aged rats is discussed.

The topographical distribution of catecholaminergic nerve fibres and their anatomical relationship to cholinergic elements in the rat globus pallidus were studied. Peroxidase-antiperoxidase and two-colour immunoperoxidase staining procedures were used to demonstrate tyrosine hydroxylase (TH), dopamine β-hydroxylase (DBH), phenylethanolamine N-methyltransferase (PNMT) and choline acetyltransferase (ChAT) immunoreactivities, combined with acetylcholinesterase (AChE) pharmacohistochemistry. TH immunoreactive nerve fibres were seen to enter the globus pallidus from the medial forebrain bundle. The greatest density of such fibres was found in the ventral region of the globus pallidus, which was also characterized by the greatest density of ChAT immunoreactive neurons. TH immunoreactive nerve fibres showed varicose arborizations and sparse boutons, which were occasionally seen in close opposition to cholinergic structures. In all regions of the globus pallidus, there were also larger, smooth TH immunoreactive nerve fibres of passage to the caudate putamen. A smaller number of DBH immunoreactive nerve fibres and terminal arborizations were found in the substantia innominata, internal capsule and in the globus pallidus bordering these structures. A few PNMT immunoreactive nerve fibres in the substantia innominata and internal capsule did not enter the globus pallidus. Electron microscopy revealed TH immunoreactive synaptic profiles in the ventromedial area of the globus pallidus corresponding to the nucleus basalis magnocellularis of Meynert (nBM). These made mainly symmetrical and only a few asymmetrical synaptic contacts with dendrites containing AChE reaction product. The results indicate that cholinergic structures in the nBM are innervated by dopaminergic fibres and terminals, with only a very small input from noradrenergic fibres.

Publisher Summary Both biosynthesis and release functions linked in that changes in release must be balanced by changes in synthesis, so it is appropriate that they are considered together. The regulation of synthesis relative to release is, however, influenced importantly by functions of catecholaminergic systems that affect or contribute to transmitter turnover independently of transmitter release. This chapter covers the processes of catecholamine biosynthesis and release. These processes are considered in relation to the catecholamine transporter. As the first and rate-limiting enzyme in catecholamine biosynthesis, tyrosine hydroxylase (TH) is subject to multiple controls. Short-term regulation includes activation of TH in response to increased nerve traffic and negative feedback control through end-product inhibition. Long-term regulation involving production of new TH enzyme occurs at the levels of transcription and translation. Although several cis-acting DNA motifs in the upstream sequence of the TH gene have been characterized to control TH gene expression, some controversial results remain. Regulation of other catecholamine synthesizing-enzymes, such as DBH and PNMT, is controlled importantly at the transcriptional level. Researchers have described increased binding of AP1-like factors to a composite regulatory element in the promoter containing two adjacent cAMP regulatory elements (CREI, CRE2) in nuclear extracts from repeatedly immobilized animals. The first direct evidence of quanta1 release was obtained by amperometric recording of the evoked catecholamine oxidation current spikes at a small carbon fiber electrode at the surface of a chromaffin cell, in combination with membrane capacitance measurements. The results indicated that chromaffin cells sometimes release all, sometimes only part, of the catecholamine content of single vesicles. Progress in this area is important both in its own right and because it may help to elucidate the control of transmitter release in other peripheral or central neurons with terminals of similar morphology, regardless of the nature of the transmitter.

Publisher Summary In this chapter several studies have been summarized to reveal the pattern of expression of several candidate markers in the embryonic adrenal gland of wild-type mice. It has been suggested that Phox 2 is a marker for both differentiated and presumptive catecholaminergic adrenal chromaffin cells prior to reaching detectable levels of tyrosine hydroxylase (TH) immunoreactivity. In addition to TH, Phox 2, peanut agglutinin (PNA) and neuropeptide Y (NPY) are detectable in all postnatal chromaffin cells. Noradrenergic chromaffin cells possess the full repertoire of chromaffin cell functions except the capacity to synthesize adrenaline. Adult noradrenergic chromaffin cells exhibit at least three markers that they do not share with adrenergic chromaffin cells, GAP43, the adhesion molecule L1, and vesicular monoamine transporter-2 (VMAT2). The data presented in the chapter provide a useful basis for further analyzing the phenotypes of adrenal chromaffin cells in glucocorticoid-receptor knockout mice. Furthermore, the results suggest that one marker found predominantly or exclusively in mature adrenergic chromaffin cells in mice, chromogranin B can be detected in chromaffin progenitor cells prior to the expression of other key markers for the adrenergic phenotype, phenylethano1amine-Nmethyltransferase (PNMT), and secretogranin II. This suggests that differentiative properties of adrenergic chromaffin cells can be regulated sequentially and are probably, in part, independent from glucocorticoid signaling.

Publisher Summary This chapter summarizes several studies on novel catecholaminergic systems. The researches are related to catecholestrogens, nonneuronal biosynthesis of catecholamines, L-dopa as a neurotransmitter, and dopamine (DA) as a renal autocrineparacrine substance. Production of 2-hydroxylated catecholestrogens (2-OH-CEs) constitutes a major pathway for hepatic metabolism of estrone (E 1 ) and estradiol (E 2 ). Aromatic hydroxylation of E 2 by estradiol-2/4 hydroxylase yields the catecholestrogens 2-OH-E 2 and 4-OH-E 2 . In human breast cancer microsomes, 4-OH-E 2 constitutes a major catechol metabolite, consistent with a role of CEs in production of estrogen-dependent tumors. CEs also appear to play an important role in blastocyst implantation. Investigators have viewed DA as only an intermediary in the biosynthesis of norepinephrine (NE) in sympathetic nerves and of epinephrine (Epi) in adrenomedullary cells. Recent evidence has supported a physiological role for DA as an autocrine-paracrine hormone that influences Na + disposition in the periphery. L-Dopa undergoes conversion to DA in many types of cells, including neurons, and DA acts as a classical central neurotransmitter. In the nucleus of the solitary tract (NTS), L-dopa may modulate the arterial baroreflex. The renal DOPA-DA system constitutes the most well studied nonneuronal catecholaminergic system. L-dopa in the tubular filtrate undergoes Na + - dependent uptake into proximal tubular cells. Classical endocrine systems may contribute to regulation of the renal DOPA-DA natriuretic system. The studies support the view that the renal DOPA-DA system mediates some hormonal influences on renal sodium handling.

Publisher Summary In this chapter the identification of cell interactions involved in the induction of a specific neurotransmitter phenotype and characterization of the growth factors that are mediating these inductive interactions have been summarized. The analysis of the noradrenergic differentiation of postganglionic sympathetic neurons has been accelerated by the availability of markers indicating catecholamine biosynthesis and storage in embryonic tissue. In the chick embryo, tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, is expressed after the neural crest-derived precursors of the sympathetic neurons have gathered at the dorsal aorta to form the primary sympathetic ganglia. In addition to the dorsal aorta, several embryonic tissues have been demonstrated to promote catecholaminergic differentiation in developing sympathetic neurons or their precursors. In particular, ventral neural tube and notochord are necessary for catecholaminergic differentiation of sympathetic neurons in vivo . The search of identified growth factors that mediate the induction of noradrenergic differentiation in sympathetic neurons revealed that bone morphogenetic proteins (BMPs) are effective in inducing TH expression in vitro and in vivo . This observation indicates that BMPs are able to promote catecholaminergic differentiation in neural crest-derived sympathetic precursors in vitro as well as in vivo . Significantly, BMP-4 and -7 are expressed in the dorsal aorta before and during noradrenergic differentiation of sympathetic neurons. Markers of the cholinergic neurotransmitter phenotype, such as the enzymes choline acetyltransferase (CHAT), acetylcholine esterase (AChE), and the neuropeptide vasoactive intestinal peptide (VIP), are detectable only after sweat gland innervation. Whereas BMPs have been shown to be prime candidates for the growth factors mediating the induction of noradrenergic differentiation of sympathetic neurons in the primary sympathetic ganglia, the factors responsible for the induction of CHAT expression are unknown.

Publisher Summary There is a close correlation between forearm venous plasma norepinephrine (NE) and muscle sympathetic nerve activity, as measured by microneurography. This has been observed not only at rest, but also during mental stress, hypoglycemia, and after raising and lowering arterial blood pressure, as well as during several pathophysiological states. Other than forearm organ release of NE can be calculated from the venous-arterial difference of NE corrected for the extraction of NE across the organ and from the blood flow to the organ. Studies in animals have indicated that the overflow is proportional to the impulse activity in sympathetic nerves to the organ, although this may not be true during all situations. The catecholamines may be derived from internalization of β2-adrenoceptors because there is a close correlation between cyclic adenosine monophosphate and the level of catecholamines in lymphocytes. Further studies of lymphocyte NE have demonstrated a marked difference in the NE content of lymphocytes from elderly smokers as compared with nonsmokers. It is well established that plasma NE increases with age. This phenomena is most pronounced in long-term smokers but also may be observed in nonsmokers. In lymphocytes, NE concentration was found to be increased in elderly nonsmokers but was markedly reduced in smokers. Further studies have shown that β2-adrenoceptor mRNA concentration is not reduced in long-term smokers, suggesting that the decrease in NE reflects a defective membrane function, especially of NK-cells, in long-term smokers.

Publisher Summary Responses of central catecholaminergic systems as well as the Hypothalamic-Pituitary-Adrenocortical (HPA) axis vary during exposure to different stressors. Extracellular norepinephrine (NE) levels in the paraventricular nucleus (PVN) increase markedly with immobilization (IMMO) or with formalin (FORM)-induced pain and relatively little with insulin (INS)-induced hypoglycemia, cold (COLD) stress, or hemorrhage (HEM). Levels of 3,4-dihydroxyphenylacetic acid, a metabolite of dopamine, increase in the locus ceruleus with IMMO or ether but are unchanged during INS-induced hypoglycemia. These and other findings have led to the hypothesis of the existence of stressor-specific central noradrenergic pathways participating in regulation of the HPA axis. In the study described in this chapter, conscious rats have been exposed to one of several stressors-HEM, i.v. INS, s.c. FORM, COLD, or IMMO. Fos immunoreactivity of the immediate early gene c-fos was used to investigate changes in the activity of brain stem neurons. The results were correlated with previous findings about stress-induced central noradrenergic activation in the PVN using in vivo microdialysis and simultaneous measurements of plasma adrenocorticotropic hormone (ACTH) levels. The present results indicate that the magnitude of activation of c-fos in brain catecholaminergic regions and of PVN corticotrophin releasing hormone (CRH) neurons varies widely across stressors. The findings support the notion of stressor-specificity of responses of central catecholaminergic systems and the HPA axis and indicate that different central pathways regulate HPA reactivity.

Publisher Summary Recent research related to catecholamines in the periphery has dealt with assessment of peripheral catecholaminergic function, catecholamines and stress, catecholamines and pain, catecholamines and neuroimmunology, adrenomedullary secretion and cosecretion, neurocardiology, catecholamines and metabolism, and roles of catecholamines in the brain in regulation of the cardiovascular system. All of these are discussed in this chapter. Peripheral catecholaminergic systems probably play a role in many neurocardiologic disorders, either secondarily as homeostatic effectors or primarily as etiologic factors. The hemodynamic hallmark of neurocardiogenic syncope is systemic vasodilation, with or without bradycardia. The vasodilation results from sudden, marked decreases in sympathoneural outflows. In contrast, plasma epinephrine (Epi) levels generally increase in this setting. Studies show a close association between lymphocyte concentrations of catecholamines and of cyclic adenosine monophosphate (cAMP). The usefulness of lymphocyte norepinephrine (NE) concentrations in evaluating sympathoneural function remains unknown. Studies using microdialysis, specific activity of [ 3 H]normetanephrine, and NE concentrations in lymph is likely to enable estimates of NE concentrations in interstitial fluid and, thereby, estimates of release of NE from sympathetic nerves. More convincing, consistent evidence is required to understand the role of catecholaminergic systems in components of immune responses. Increased use of microdialysis in adipose tissue and skeletal muscle is likely to enhance understanding of catecholaminergic systems in patients with a variety of metabolic disorders, including insulin-dependent diabetes mellitus (IDDM) and obesity.

This chapter provides an overview of catecholamines and their receptors, and possible catecholaminergic mechanisms by which central structures can contribute to the regulation of the cerebral circulation. The possibility of catecholamine-induced changes in the cerebral circulation was first addressed concomitant with the role of perivascular sympathetic innervation. However, there is multiplicity of catecholaminergic neurotransmitters, their various origins, and mechanisms of action. This chapter also discusses dopamine that is primarily located in three midbrain nuclei. These nuclei give rise to two major dopaminergic systems: first, the mesocortical pathway that arises from the substantia nigra and densely innervates the striatum and additionally sends axons to the frontal cortex; second, the mesolimbic pathway which arises from the ventral tegmental area and innervates both cortical and subcortical structures that comprise the limbic system. These systems are principally involved in extrapyramidal motor control; furthermore, their dysfunction underlies various human pathologies such as Parkinson's disease as well as certain aspects of schizophrenia and drug abuse. The chapter describes the general anatomical and functional data of the central catecholaminergic systems, and discusses circulating catecholamines and activation of the reticular formation.

Publisher Summary Stress has been defined as a multihormonal response pattern organized in a rather specific or selective manner, depending on the particular stimulus. The weight of evidence supports the view that different neuroendocrine systems are activated specifically by different stressors. Some studies have shown that even components of the same system might be activated differentially. For instance, the two branches of the sympathoadrenal system (SAS)—the adrenal medulla and the sympathetic nerves—can be activated independently by different stressors. The studies have been based on determinations of plasma epinephrine (Epi) and norepinephrine (NE) levels. Measurements of plasma levels of the catecholamine (CA) precursor dihydroxyphenylalanine (DOPA) and of catecholamine metabolites give better information about mechanism of sympathoadrenal activation. The aim of the present chapter is to examine activation of the two components of the SAS by simultaneous measuring the adrenomedullary (AM) and sympathoneural responses during exposure to different intensities of various stressors. Plasma levels of NE, Epi, dopamine (DA), their precursor DOPA, and the metabolites dihydroxyphenylglycol (DHPG) and dihydroxyphenylacetic acid (DOPAC) are assessed, as well as gene expression of the CA biosynthetic enzyme tyrosine hydroxylase (TH) in the adrenal medulla.

Publisher Summary The mixed-type dopamine D1-D2-receptor agonist apomorphine has had an impressive career as a replacement for L-dopa in the therapy for Parkinson's disease in the late stage of the disease. Earlier attempts to introduce apomorphine as a dopamine replacement had failed because of its short duration of action and gastrointestinal side effects. Presently, the pharmacokinetic problems have been overcome with sophisticated drug delivery devices, as portable infusion pumps or insulin pens. Coadministration of the peripherally acting dopamine receptor antagonist domperidone prevents the undesired noncentral effects of apomorphine, which has become the medication of choice for patients suffering from on-off fluctuations after years of L-dopa administration. The discovery that catecholamines can be cytotoxic has raised the question of the long-term effects of the treatment in Parkinson's disease with drugs like L-dopa or apomorphine on the progression of the disease. Catecholaminergic drugs can act as reducing agents and iron chelators. They can interfere with cellular redox reactions and promote the formation of free radicals, which are thought to be mediators of neurodegeneration. Damage is caused close to the site where free radicals are formed. Catecholamines can act as radical scavengers and slow down the progression of neurodegenerative diseases. In this chapter it has been demonstrated that apomorphine can act as a potent free radical scavenger in vitro and in cell culture. The balance between catecholamine toxicity and possible beneficial effects because of antioxidation was looked into in PC12 cell culture, a system to study apoptotic and necrotic cell death. Because the dopamine prodrug L-dopa and apomorphine are used in the treatment of Parkinson's disease, it is of interest to investigate the influence of these agents on the biochemical processes involved in the progression of neurodegeneration. It was found that apomorphine is a representative of a catecholamine with pronounced antioxidant effects.

Publisher Summary In the study discussed in this chapter, immunohistochemical, in situ hybridization histochemistry, tract-tracing, and in vivo microdialysis techniques have been used to investigate the participation of the central catecholaminergic (CA) systems in the organization of responses to an acute neurogenic (pain) stress: subcutaneous injection of 0.5 ml of 4% formalin into the hind paw of rats. This single injection produces a local pain that develops immediately after injection and results in four- to fivefold increase in corticosterone and adrenocorticotropic hormone (ACTH) levels in the plasma and a similar increase in norepinephrine levels in the paraventricular nucleus (PVN). Formalin also induced a rapid expression of c-fos and corticotropin-releasing hormones (CRHs) mRNA in the PVN. The possible crossover of the nociceptive spinal afferents to the medullary CA neurons and the crossover of the ascending CA neurons to the PVN were investigated after spinal and medullary surgical hemisections. Hemisection between the spinal cord and the medulla oblongata did not influence formalin-induced elevation of plasma corticosterone and ACTH levels, and strong c-fos immunoreactivity was observed in the medullary CA cell groups and in the PVN bilaterally, as in the sham-operated animals. Hemisection between the medullary CA cell groups and the PVN (unilateral knife cut at the pons-medulla oblongata border) decreased CRH immunoreactivity and CRH gene expression in the PVN ipsilateral to the transection but remained unchanged contralateral to the knife cut. Although, unilateral pontomedullary hemisection reduce TH and CRH immunoreactivity and CRH gene expression in the PVN, it does not influence the high plasma corticosterone and ACTH levels in response to formalin stress. However, acute immobilization stress in hemisected animals is able to induce CRH expression. These findings suggest that in brain stem-hemisected animals, the remaining intact ascending pathways are sufficient to allow normal, or almost normal, activation of the hypothalamopituitary-adrenal axis during stress.

Publisher Summary Sweat glands are innervated exclusively by cholinergic sympathetic neurons. Several studies summarized in this chapter indicate that the cholinergic differentiation factor produced by sweat glands is a novel member of the neuropoietic cytokine family. The changes in neurotransmitter properties that occur during normal development of the sweat gland innervation can be induced in cultured sympathetic neurons by members of the neuropoietic cytokine family, including leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), and cardiotrophin-1 (CT-1) but not by other cytokines and growth factors. Further, monoclonal antibodies that block activation of LIFR β , one of the receptor subunits utilized by members of the neuropoietic cytokine family, block the induction of cholinergic function in sweat glandhympathetic neuron cocultures. Although, the studies on adult mice deficient in LIF, CNTF, or LIF and CNTF have revealed that the sweat gland innervation possessed cholinergic and peptidergic properties. Similarly, the induction of cholinergic and peptidergic properties in sympathetic neurons cocultured with sweat gland cells is not prevented by the presence of antibodies that block CT-1 function. Thus, the known members of the neuropoietic cytokine family do not appear to account for the cholinergic induction by sweat glands. In addition, culture studies suggest that the catecholamines produced by the early innervation are important in its regulation. Recently the periosteum or connective tissue covering of the bone has been identified a new cholinergic sympathetic target. Like the developing sweat gland innervation, periosteal innervation initially expresses noradrenergic properties that are suppressed as cholinergic and peptidergic ones appear, and this change is regulated by interactions with the target. In contrast to the developing sweat gland innervation, a significant proportion of the sympathetic axons that reach the sternum have already expressed immunoreactivity for the vesicular acetylcholine transporter (VAChT) and vasoactive intestinal peptide (VIP) in addition to catecholaminergic properties.

Publisher Summary This chapter provides an overview of adrenocorticosteroids and cardiovascular regulation, and describes the methods for surgery and blood pressure measurements. The chapter also describes the chronic intracerebroventricular infusion methods used to complement the receptor and lesioning studies. The design of such a study must consider the acute sensitivity of the hypothalamic–pituitary–adrenal axis and the catecholaminergic system to stress from any source. In ideal situation, the administration of test agents and the measurement of blood pressure should not alter the blood pressure. Blood pressure or behavioral changes are terminal effects that can be modified not only by extraneous variables, including method of measurement, but also by unforeseen changes secondary to experimental manipulations. Because the perfect experimental design is rare or nonexistent, it is crucial that several approaches with different potential problems be used to examine the central nervous system with its myriad interactions and complexities. There are several models of mineralocorticoid hypertension in rats, such as systemic mineralocorticoid-salt excess, of which DOCA-salt is the prototype, central mineralocorticoid excess, and adrenal regeneration hypertension. Doses of corticosteroids, their antagonists, and other agents to be studied are chosen so that they do not cause overt clinical illness or distress that could independently alter blood pressure and the humoral environment, particularly that of the adrenocorticosteroids and catecholamines.

Publisher Summary The basal forebrain cholinergic projection system (BFC) has been the focus of considerable attention as a result of evidence implicating it in a number of behavioral functions, including arousal, sensory processing, motivation, emotion, learning, and memory. Cholinergic projection neurons are distributed across a number of classically defined regions of the basal forebrain and collectively project to the entire cortical mantle, including allocortical areas, such as the hippocampus, the amygdala, and the olfactory bulb. In spite of detailed information, concerning the efferent projections of these neurons, as well as significant recent progress in our understanding of the mechanisms of acetylcholine action in the cortex, relatively little is known about the afferents, which drive BFC neurons. This chapter discusses the recent efforts to identify catecholaminergic inputs to BFC neurons in the rat using this approach. Additional biochemical studies test the effect of specific removal of ascending brainstem catecholaminergic fibers on forebrain choline acetyltransferase (ChAT) activity.

Publisher Summary This chapter examines an experimental situation based on the effect of a series of 10 electroconvulsive shocks. The question is could an induction of tyrosine hydroxylase (TH) gene expression be found in catecholaminergic cells 24 hr after the last shock. For this purpose, the messenger RNA coding for TH, the TH protein amount, and the TH activity were simultaneously evaluated in three nuclei, namely, the substantia nigra (SN), the ventrotegmental area (VTA), and the locus coeruleus (LC), in an experiment described in the chapter. These structures were chosen because the main ascending catecholamine pathways of the central nervous system innervating the forebrain originate within them. The TH mRNA and TH activity were measured using the same samples with in vitro assays realized on dissected tissues. In separate experiments, the amount of TH protein was measured in situ with two different methods, allowing a better regional localization. In the three regions of interest, the enzymatic activity was enhanced but the LC was the only one in which this increase was correlated with a rise in the amount of protein and TH mRNA, suggesting that it was the only region in which an increased expression of TH gene could occur.

The dorsolateral pontine tegmentum of the cat is known to contain a large population of catecholaminergic neurons. Additionally, several studies have also shown the presence of other neurochemicals (acetylcholine, enkephalin, neuropeptide Y, serotonin, somatostatin and substance P). In this study, we have employed retrograde transport of horseradish peroxidase in combination with immunocytochemistry to determine the locations of pontospinal neurons which contain catecholamine, enkephalin, neuropeptide Y, and serotonin. Furthermore, we have combined the retrograde transport of Fast Blue and immunofluorescence histochemistry to determine whether enkephalin-containing neurons are catecholaminergic. All pontospinal neurons, irrespective of the neurochemical content, were observed in the ventral and lateral parts of the dorsolateral pontine tegmentum at coronal levels P1.8-P4.0. These neurons were located in the nuclei locus coeruleus alpha and subcoeruleus and the Kolliker-Fuse nucleus. A high concentration of these neurons was evident in the Kolliker-Fuse nucleus when compared to the nuclei locus coeruleus alpha and subcoeruleus. Quantitative data have revealed that enkephalin is contained in a large proportion of the pontospinal catecholaminergic neurons (75%). The observations suggest that catecholaminergic neurons may contain one or more putative peptide neurotransmitters.

Publisher Summary This chapter discusses the current study of the chief afferents and efferents that participate in the major striatal sensory motor function examined in animals not pre-injured. Connectivities included the cortical, nigral, and raphe to the striatal graft, and the striatal graft to the nigra, respectively. The Phaseolus oulgaris agglutinin leuca (PHAL) anterograde-tracing method is used to detect cortical input to the transplants, because the injection of PHAL into host cortex produces more consistent results than the injection of horseradish peroxidase (HRP) into transplants of unknown shape, size, and location. The immunocytochemical staining of serotonin (5-HT) and the catecholaminergic enzyme, tyrosine hydroxylase (TH), are used to detect the 5-HT-raphe and dopamine (DA)-nigral inputs to graft. HRP retrograde-tracing methods are used to detect the innervation of transplant into host nigra, because the HRP-injection into host nigra and labeling in the striatum and striatal graft are quite reproducible.

Catecholamines were measured in microdissected brain nuclei of female rats by a radioenzymatic assay. Catecholamines fluctuated over the estrous cycle in several brain nuclei. Treatment of ovariectomized females with estradiol alone or in combination with progesterone altered catecholamine turnover and also affected cholinergic and serotonergic systems in some discrete areas. Sex differences in catecholamines were detected in several areas, and some of these appeared to be under control of neonatal androgen. Numerous studies have implicated central catecholaminergic systems in the control of reproductive processes such as the release of the pituitary gonadotropins Follicle-Stimulating-Hormone (FSH) and Luteinizing Hormone (LH). Norepinephrine (NE) and dopamine (DA)-containing neurons provide dense innervation to hypothalamic and limbic regions involved in reproduction, yet the discrete sites at which they influence the hypothalamo-hypophyseal axis remain largely conjectural. The present experiments combined the microdissection technique for removal of individual brain nuclei with a sensitive microassay for catecholamines and radioimmunoassays for gonadotropins in preliminary attempts to establish discrete loci for catecholaminergic control over reproductive neuroendocrine processes.