Locus Coeruleus Cell Bodies Research Articles

Chemical ablation of locus coeruleus terminals in mice dysregulates behavioral features reminiscent of prodromal Alzheimer’s disease

AbstractBackgroundThe prodromal stages of Alzheimer’s disease (AD), which precede cognitive impairment by decades, are characterized by neuropsychiatric symptoms such as anxiety, agitation, impulsivity, and sleep disturbances that significantly impair quality of life. The brainstem noradrenergic locus coeruleus (LC) modulates behaviors that are reminiscent of these prodromal symptoms. Furthermore, the loss of norepinephrine (NE) terminals in LC‐projecting regions is a prominent feature of early AD, implicating LC dysfunction as a possible contributor to prodromal symptoms. However, the behavioral and pathological consequences of LC terminal loss have not been fully investigated.MethodAdult male and female C57BL/6 mice aged 4‐6 months were administered either saline or the LC‐specific neurotoxin, N‐(2‐chloroethyl)‐N‐ethyl‐bromo‐benzylamine (DSP‐4) (50 mg/kg, i.p.) on day 1 and day 7 (12 animals/group). Performance in behavioral tests that assess arousal (sleep latency), locomotor activity (novelty‐induced and circadian), anxiety (novelty‐suppressed feeding), and associative memory (contextual fear conditioning) commenced one week following the last treatment. In addition, brain sections consisting of LC, hippocampus, and cortical areas were immunostained for tyrosine hydroxylase (TH) to evaluate LC integrity, NE transporter (NET) to determine fiber/terminal density to LC‐projecting regions, and glial fibrillary acidic protein (GFAP) to assess astrogliosis. Fluorescence immunoreactivity (IR) was analyzed with ImageJ.ResultAnimals treated with DSP‐4 displayed significantly increased latency to feed in a novel environment compared to saline‐treated animals, with no treatment differences in the home cage, suggesting elevated neophobic behavior. Furthermore, DSP‐4‐treated animals trended towards decreased locomotor activity in the center of the environment over 1 hour and 23 hours, indicating increased thigmotaxis. No behavioral treatment differences were observed for sleep latency and contextual fear conditioning paradigms. Immunofluorescence revealed no treatment differences in TH IR in the LC but reduced NET IR in LC‐projecting regions of DSP‐4‐treated animals, demonstrating preserved LC cell bodies and LC fiber loss. No significant treatment differences were found for GFAP.ConclusionThese data suggest that chemical ablation of LC terminals with DSP‐4 can contribute to neophobia and anxiogenic responses, implicating LC terminal loss in mediating prodromal symptoms relevant to AD. Further studies are warranted to identify the physiological and molecular mechanisms underlying LC dysfunction for alleviating prodromal AD symptoms.

Modulation of Prefrontal Cortex Slow Oscillations by Phasic Activation of the Locus Coeruleus

Cortical slow rhythmic activity, a hallmark of deep sleep, is observed under urethane anesthesia. Synchronized fluctuations of the membrane excitability of a large neuronal population are reflected in the extracellular Local Field Potential (LFP), as high-amplitude slow (∼1 Hz) oscillations (SO). The SO-phase indicates the presence (Up) or absence (Down) of neuronal spiking. The cortical state is controlled by the input from thalamic and neuromodulatory centers, including the brainstem noradrenergic nucleus Locus Coeruleus (LC). The bidirectional modulation of neuronal excitability by noradrenaline (NA) is well known. We have previously shown that LC phasic activation caused transient excitability increase in the medial prefrontal cortex (mPFC). In the present study, we characterized the effect of LC phasic activation on the prefrontal population dynamics at a temporal scale of a single SO cycle. We applied short (0.2 s) trains of electric pulses (0.02–0.05 mA at 20–50 Hz) to the LC cell bodies and monitored a broadband (0.1 Hz–8 kHz) mPFC LFP in urethane-anesthetized rats. The direct electrical stimulation of LC (LC-DES), applied during the Up-phase, enhanced the firing probability in the mPFC by ∼20% and substantially prolonged Up-states in 56% of trials. The LC-DES applied during Down-phase caused a rapid Down-to-Up transition in 81.5% of trials. The LC-DES was more effective at a higher frequency, but not at a higher current. Our results suggest that transient NA release, coupled to SO, may promote synaptic plasticity and memory consolidation by sustaining a depolarized state in the mPFC neurons.

Selective Lifelong Destruction of Brain Monoaminergic Nerves Through Perinatal DSP-4 Treatment.

N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) is a highly selective neurotoxin for noradrenergic projections originating from the locus coeruleus (LC). The outcome of the systemic DSP-4 treatment of newborn rats is an alteration in postnatal development of the noradrenergic system, involving the permanent denervation of distal noradrenergic projection areas (neocortex, hippocampus, spinal cord), accompanied by noradrenergic hyperinnervation in regions proximal to the LC cell bodies (cerebellum, pons-medulla). DSP-4 is well tolerated by developing rats and does not increase the mortality rate. Permanent noradrenergic denervation in the cerebral cortex and spinal cord is present at all developmental stages, although this effect is more pronounced in rats treated with DSP-4 at an early age, i.e., up to postnatal day 5 (PND 5). Notably, regional hyperinnervation is a hallmark of neonatal DSP-4 treatment, which is not observed after either prenatal or adult DSP-4 application. In contrast to robust biochemical changes in the brain, DSP-4 treatment of newborn rats has a marginal effect on arousal and cognition functions assessed in adulthood, and these processes are critically influenced by the action of the noradrenergic neurotransmitter, norepinephrine (NE). Conversely, neonatal DSP-4 does not significantly affect 5-hydroxytryptamine (serotonin; 5-HT), dopamine (DA), gamma-aminobutyric acid (GABA), and histamine levels in brain. However, as a consequence of altering the functional efficacy of 5-HT1A, 5-HT1B, DA, and GABA receptors, these neurotransmitter systems are profoundly affected in adulthood. Thus, the noradrenergic lesion obtained with neonatal DSP-4 treatment represents a unique neurobiological technique for exploring the interplay between various neuronal phenotypes and examining the pathomechanism of neurodevelopmental disorders.

Heightened transcription for enzymes involved in norepinephrine biosynthesis in the rat locus coeruleus by immobilization stress

Background: The locus coeruleus (LC), a target for CRH neurons, is critically involved in responses to stress. Various physiological stresses increase norepinephrine turnover, tyrosine hydroxylase (TH) enzymatic activity, protein and mRNA levels in LC cell bodies and terminals; however, the effect of stress on other enzymes involved in norepinephrine biosynthesis in the LC is unknown. Methods: Rats were exposed to single (2 hour) or repeated (2 hour daily) immobilization stress (IMO). Recombinant rat dopamine b–hydroxylase (DBH) cDNA was expressed in E. coli and used to generate antisera for immunohistochemistry and immunoblots in LC. Northern blots were used to assess changes in mRNA levels for TH, DBH, and GTP cyclohydrolase I (GTPCH) in the LC in response to the stress. Conditions were found to isolate nuclei from LC and to use them for run–on assays of transcription. Results: Repeated stress elevated the DBH immunoreactive protein levels in LC. Parallel increases in TH, DBH and GTPCH mRNA levels of about 300% to 400% over control levels were observed with single IMO, and remained at similar levels after repeated IMO. This effect was transcriptionally mediated, and even 30 min of a single IMO significantly increased the relative rate of transcription. Conclusions: This study is the first to reveal transcriptional activation of the genes encoding catecholamine biosynthetic enzymes in the LC by stress. In addition to TH, changes in DBH and GTPCH gene expression may also contribute to the development of stress–triggered affective disorders.

Brain monoamines, exercise, and behavioral stress: animal models.

This paper summarizes our studies examining whether changes in levels of brain monoamines after chronic exercise are associated with altered behavioral and endocrine responses to stressors other than exercise. The focus is on using animal models relevant for understanding reports by humans that regular physical activity reduces depression and anxiety. We studied the effects of chronic activity wheel running or treadmill exercise training on levels of norepinephrine (NE) measured in brain cell bodies and terminal regions at rest and after behavioral stress. We also measured brain levels of serotonin, i.e., 5-hydroxytryptamine (5-HT), dopamine (DA), and gamma aminobutyric acid (GABA), which function as both antagonists and synergists with NE. In general, we found that chronic activity wheel running increased NE levels in the pons medulla at rest and protected against NE depletion in locus coeruleus cell bodies after footshock; the concomitant reduction in escape-latency was consistent with an antidepressant effect. Wheel running also decreased the density of GABAA receptors in the corpus striatum while increasing open-field locomotion, consistent with an anxiolytic effect, but had no effect on hypothalamic-pituitary-adrenal cortical response to footshock measured by plasma levels of adrenocorticotropic hormone (ACTH), corticosterone, and prolactin. In contrast, treadmill exercise training increased the metabolism of NE in brain ascending terminal areas for NE, increased the secretion of ACTH after footshock and immobilization stress and had no effect on GABAA receptor density or open field locomotion. The validity of animal models for studying depression and anxiety after forced versus voluntary exercise is discussed. Recommendations are offered for improving the methods used in this area of research.

Influence of corticotropin-releasing hormone on electrophysiological activity of locus coeruleus neurons

These experiments examined the effects of corticotropin-releasing hormone (CRH) on single-unit electrophysiological activity of locus coeruleus (LC) neurons. As has been reported previously, infusion of CRH into the ventricular system of the brain (i.c.v.) of halothane-anesthetized adult male rats increased spontaneous discharge rate of LC neurons while producing no increase, and possibly a decrease, in sensory-evoked activity. However, when i.c.v. CRH was given to female rats or immature male rats, which had not been studied previously, LC activity was not altered. To attempt to understand this sex and age difference, potential mechanisms by which i.c.v. CRH elevates LC spontaneous activity in adult male rats were examined; in that i.c.v. CRH activates the pituitary-adrenal axis and autonomic nervous system, these response systems were manipulated. Adrenalectomy (with or without corticosterone replacement by pellet) did not affect the ability of i.c.v. CRH to increase LC spontaneous activity in adult male animals, but blockade of sympathetically-mediated autonomic responses, either by chlorisondamine or the beta adrenergic receptor blocker timolol, blocked this increase, indicating that afferent feedback from peripheral autonomic responses was critical for activating LC neurons following i.c.v. CRH. To determine whether CRH neurotransmission might play a role in this feedback pathway, the CRH antagonist alpha-helical CRH (α-hCRH) was microinjected into several brain regions including LC prior to i.c.v. CRH. α-hCRH microinjected into LC reduced the increase in LC activity caused by i.c.v. CRH; however, blockade of this increase was total when α-hCRH was microinjected into the lateral parabrachial nucleus ipsilateral to the LC recording site, suggesting that increased LC activity following i.c.v. CRH is mediated by CRH acting in the parabrachial region. During these studies, it was also observed that microinjection of α-hCRH into LC increased LC spontaneous discharge rate; consequently, CRH was microinjected into LC, and produced a dose-dependent decrease in LC spontaneous activity in both male and female rats, which could be blocked by microinjection of α-hCRH — these data indicated that the direct influence of CRH on LC neurons is to decrease their spontaneous activity. To reconcile this with the original report that CRH applied to LC neurons increases their activity, one possibility suggested is that the CRH microinjection procedure used in the present study stimulated inhibitory receptors on LC dendrites whereas the original study stimulated excitatory receptors on LC cell bodies. It is concluded that an inhibitory influence of CRH on LC activity is consistent with recent data indicating that decreased LC activity increases anxiety and stress-related responses, but that direct influences of CRH appear rather minor in determining LC neuronal activity in comparison to other inputs to LC such as are seen after i.c.v. CRH infusion.

Restoration of ascending noradrenergic projections by residual locus coeruleus neurons: compensatory response to neurotoxin-induced cell death in the adult rat brain.

There is clinical and experimental evidence that monoamine neurons respond to lesions with a wide range of compensatory adaptations aimed at preserving their functional integrity. Neurotoxin-induced lesions are followed by increased synthesis and release of transmitter from residual monoamine fibers and by axonal sprouting. However, the fate of lesioned neurons after long survival periods remains largely unknown. Whether regenerative sprouting may contribute significantly to recovery of function following lesions which induce cell loss has been questioned. We have previously analyzed the response of locus coeruleus (LC) neurons to systemic administration of the noradrenergic (NE) neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) to adult rats. This drug causes ablation of nearly all LC axon terminals within 2 weeks after administration, followed by a profound loss of LC cell bodies 6 months later. The present study was conducted to determine the fate of surviving LC neurons and to characterize their potential for regenerative sprouting during a 16 month period after DSP-4 treatment. The time-course and extent of LC neuron degeneration were analyzed quantitatively in Nissl-stained sections, and the regenerative response of residual neurons was characterized by dopamine-beta-hydroxylase immunohistochemistry. The results document that LC neurons degenerate gradually after DSP-4 treatment, cell loss reaching on average 57% after 1 year. LC neurons which survive the lesion exhibit a vigorous regenerative response, even in those animals in which cell loss exceeds 60-70%. This regenerative process leads progressively to restoration of the NE innervation pattern in the forebrain, with some regions becoming markedly hyperinnervated. In stark contrast to the forebrain, very little reinnervation takes place in the brainstem, cerebellum and spinal cord. These findings suggest that regenerative sprouting of residual neurons is an important compensatory mechanism by which the LC may regain much of its functional integrity in the presence of extensive cell loss. Furthermore, regeneration of LC axons after DSP-4 treatment is region-specific, suggesting that the pattern of reinnervation is controlled by target areas. Elucidation of the factors underlying recovery of LC neurons after DSP-4 treatment may provide insights into the compensatory mechanisms of central neurons after injury and in disease states.

Experimentally-induced neuron loss in the locus coeruleus of adult rats

Systemic administration of the noradrenergic neurotoxin N-(2-chloroethyl)- N-ethyl-2-bromobenzylamine (DSP-4) to adult rats causes widespread degeneration of locus coeruleus (LC) axon terminals. The present study was conducted to determine the effects of DSP-4-induced LC axon lesions on LC cell bodies. Six months after DSP-4 treatment, quantitative analysis of Nisslstained sections revealed a profound loss of LC perikarya, ranging from 20 to 73% of control. The remaining LC neurons appeared shrunken, but stained strongly with dopamine β-hydroxylase immunohistochemistry. These findings support the conclusion that DSP-4-induced LC axon lesions cause retrograde degeneration of LC neurons. DSP-4 may serve as a useful tool in studies of the mechanisms of LC neuron degeneration.

Light-microscopic localization of somatostatin binding sites in the locus coeruleus of the rat

Somatostatin (SS14) binding sites within locus coeruleus (LC) were localized at the light microscope level by [ 125I][Tyr 0, d-Trp 8]SS14 radioautography combined with an immunohistochemical/neurotoxic lesioning approach. In intact rats, the dense accumulation of SS14 binding sites of LC conspicuously overlapped with the cluster of tyrosine hydroxylase (TH) immunoreactive neurons; SS14 specific binding was directly proportional to the number of TH immunostained (TH+) cell bodies per mg of tissue throughout LC. Complete lesion of catecholaminergic nerve cell bodies of LC by intracerebroventricular injection of 6-hydroxydopamine (6-OHDA) resulted in the total abolition of SS14 specific binding in the structure. In addition, specifically bound [ 125I][Tyr 0, d-Trp 8]SS14 and TH+ cell density were quantified serially in a set of rats bearing various partial neurotoxic lesions; a highly significant correlation was found between the two parameters at each of the 16 coronal levels of LC examined. The coefficient of proportionality was identical at all levels. These results strongly suggest that somatostatin binding sites are uniformly localized on all noradrenergic neurons of LC.

Retrograde axonal transport specificity in the locus coeruleus neurons after [ 3H]noradrenaline injection into the rat olfactory bulb

Retrograde axonal transport process was investigated in the afferent systems to the rat olfactory bulb, after [ 3H]noradrenaline ([ 3H]NA) injection into the olfactory bulb, in order to provide evidence regarding its specificity and the biochemical basis supporting this specificity. Radioautographs showed that [ 3H]NA unilaterally injected into the olfactory bulb at a concentration of 10 −3 M, resulted in labeling of the structures afferent to the olfactory bulb, mainly on the injected side: locus coeruleus (LC), dorsal and central raphes, nucleus of the lateral olfactory tract and piriform cortex. Heavy labeling was observed on the noradrenergic LC cell bodies, whereas the radioautographic reaction was less intense on the other structures. After 10 −4 M injection, the labeling intensity of the LC cell bodies was lower while very rare weakly labeled cell bodies persisted in the dorsal raphe, nucleus of the lateral olfactory tract and piriform cortex. The LC cell bodies were exclusively labeled when the concentration of [ 3H]NA injection was 10 −5 M. All the other structures were devoid of labeling. It was still possible to detect labeled cell bodies in the LC for a 10 −6 M concentration. Following bilateral injections of [ 3H]NA (10 −3 M) the total radioactivity retrogradely transported to the LC represented about 4 times the total radioactivity measured in the periaqueductal gray substance (as control tissue of the tracer diffusion). Fractional study by ethanol of LC tissue homogenate and liquid scintillation counting of each fraction showed that 60% of the total radioactivity (about 2.5 times the control value) was in the supernatant and 40% (about 20 times the control value) was associated with the precipitate. In the other regions such as the dorsal and central raphes and periaqueductal gray substance, radioactivity was mainly found in the supernatant, except for the dorsal raphe whose precipitate contained a low amount of radioactivity (about 4 times the control value). Colchicine (an axonal transport inhibitor) bilaterally injected into the medial forebrain bundle and systemic administration of desipramine (a noradrenaline uptake inhibitor) decreased the radioactivity associated with the LC precipitate by 90 and 85% and the LC supernatant radioactivity by 55 and 35%, respectively. These pretreatments did not significantly affect the radioactivity amounts measured in the different fractions of dorsal and central raphes and periaqueductal gray substance. Radioautographic study after colchicine treatment showed a large decrease in the labeling intensity of the LC cell bodies as compared to the non-treated side. Therefore, we suggest that low concentrations (10 −5 M) of [ 3H]NA injected in the olfactory bulb determine specific conditions of noradrenergic pathway labeling. This specific labeling after migration could be supported by the radioactive ethanol precipitate which would appear to contain [ 3H]NA- and/or 3H-derivatives-binding protein. Such a 3H-macromolecular complex, which could represent the specific carrier, may well undergo retrograde transport from the nerve terminals towards the cell bodies.

Effects of locus coeruleus lesions on auditory, long-latency, event- related potentials in monkey

It has previously been demonstrated that monkeys exhibit certain event-related potential (ERP) components showing latency, polarity, and contingency similarities to those observed in humans. In the present study, monkey P300-like components were studied in order to evaluate the hypothesis that the noradrenergic locus coeruleus (LC) system participates in their generation or modulation. ERPs were recorded from untrained squirrel monkeys (Saimiri sciureus) twice a week for 4 weeks before and after bilateral LC lesions and interruption of dorsal bundle (DB) fibers. Stimuli consisted of 2 and 6 kHz tone pips (40 msec duration, 60 dB above nHL) presented once a second in random order. In most sessions, one tone constituted 90% of the stimuli and the other tone 10%, while in some sessions tones were made equiprobable to test the effects of manipulating stimulus probability. LC and DB lesions were made by first localizing the nucleus and creating an electrolytic lesion. Then, the electrode was placed at the anterior pole of the nucleus and a knife cut effected. The extent of damage to LC perikarya and ascending axons was assessed by reconstructing lesions from Nissl-stained sagittal sections through the brain stems. The effect of lesions on cortical noradrenergic axons was immunohistochemically verified utilizing antisera directed against dopamine-B-hydroxylase and tyrosine hydroxylase to label noradrenergic and dopaminergic axons, respectively. The prelesion ERP results replicated previous findings of P300-like components recorded in response to low-probability tones. The postlesion ERP data indicated that following damage to LC cell bodies, combined with interruption of histochemically detectable ascending noradrenergic axons, monkey P300-like potentials exhibited decreased areas, altered brain-surface distribution, and reduced sensitivity to stimulus probability. The correlation between the extent of cell body lesions and percentage reduction in the magnitude of P300-like responses was significant. However, interruption of DB fibers alone did not have similar effects. Neither type of lesion had any effect on amplitudes, latencies, or brain-surface distributions of P52, P172, or N250-900. There was, however, a significant effect on N106. Stimulus probability effects on the frontally distributed P52 and N106 were not altered by the lesions. These data support the hypothesis that the integrity of the LC nucleus and its ascending fibers is important in the generation and modulation of surface-recorded P300-like activity.

Variations in 3,4-dihydroxyphenylacetic acid concentration are correlated to single cell firing changes in the rat locus coeruleus

The purpose of this study is to see whether variations in the catechol oxidation peak in the locus coeruleus would be closely related with variations in single cell firing, and would be independent of the mechanism triggering the variation of neuronal activity. Single unit and electrochemical (differential pulse voltammetry) recordings were performed, respectively on the left and right locus coeruleus of anaesthetized rats. The variations in single unit activity were induced using well known stimulatory mechanisms (yohimbine, hypovolemia, indirect activation using a 5-hydroxytryptamine central agonist RU 24969, RU 24722 known to activate noradrenergic metabolism), or inhibitory (clonidine, morphine) models. This was done in addition to successive activation or inhibition (reversal of oxotremorine with scopolamine, antagonism of clonidine by piperoxane). In all the experimental conditions, variations in the catechol oxidation current followed variations in single cell activity. Furthermore, the catechol oxidation current variations correlated significantly to changes in the firing rate. Such a close correlation between a catechol oxidation peak, and the electrical activity of the locus coeruleus cell bodies supports the use of catechol oxidation current variations as a good indirect index of functional activity in noradrenergic locus coeruleus perikarya. This suggests a close relationship between dopamine metabolism and electrical activity in locus coeruleus cell bodies.

Efferent projections of nucleus locus coeruleus: Morphologic subpopulations have different efferent targets

This study quantitatively addresses the hypothesis that there is a systematic relationship between the morphologic characteristics of locus coeruleus neurons and the particular target regions they innervate. Following horseradish peroxidase injections into selected terminal fields, locus coeruleus cell bodies are heavily labeled by retrograde transport so that somata size and shape, and in many cases primary dendritic pattern can be observed. This allows the classification of neurons as one of six cell types: large multipolar cells within ventral locus coeruleus, large multipolar cells in the anterior pole of locus coeruleus, fusiform cells in dorsal LC, posterior pole cells, medium-sized multipolar cells (termed core cells in this report), and small round cells. It was found that while core cells contribute to the innervation of all terminal fields examined, other cell types project to more restricted sets of targets. The contributions of each type to selected efferents are presented in detail. In particular, fusiform cells project to hippocampus and cortex, large multipolar cells in ventral locus coeruleus project to spinal cord and cerebellum, and small round cells in central and anterior locus coeruleus, as well as large multipolar cells in anterior locus coeruleus, project to hypothalamus. These results, in conjunction with those described in the preceding report, indicate that locus coeruleus is intrinsically organized with respect to efferent projections with much more specificity than has previously been evident. This high degree of organization is consistent with other recent demonstrations of functional specificity exhibited by locus coeruleus neurons.

Decreased activity of locus coeruleus neurons in the rat after DSP-4 treatment

The activity of noradrenergic neurons of the rat locus coeruleus was investigated at 10 and 50 days after the administration of DSP-4 (N-(2-chloroethyl)-N-ethy-l-2-bromobenzyl-amine), a selective noradrenergic neurotoxin. The mean neuronal firing rate in control animals was 2.4 Hz. In contrast, DSP-4 animals had lower rates of 1.2 Hz at 10 days and 1.7 Hz at 50 days. Histological examinations revealed no morphological changes of locus coeruleus cell bodies at either the 10- or 50-day time points. These results suggest that DSP-4 can impair neuronal activity of the locus coeruleus without altering the structural appearance of locus coeruleus perikarya.

Catecholamine metabolism in the rat locus coeruleus as studied by in vivo differential pulse voltammetry. I. Nature and origin of contributors to the oxidation current at +0.1v

Differential pulse voltammetry was used together with treated carbon fiber microelectrodes to study the in vivo catecholamine (CA) metabolism in the locus coeruleus (LC), a brain region densely packed with noradrenergic neurons. In chronically implanted rats, an in vivo oxidation current that peaks at +0.1 V has been detected inside the LC comples. This current whose potential is characteristic of the oxidation of the catechols, had the same anatomical localization as the noradrenergic cells. Pharmacological experiments have been made to ascertain which catechols contribute to this in vivo current. Monoamine oxidase inhibition by pargyline was followed by a total and rapid suppression of the in vivo signal. Blockade of dopamine-β-hydroxylase by FLA-63 induced a significant increase in the electrochemical signal. Post-mortem analysis of LC catechol levels after administration of this drug revealed a considerable decrease in NA and its major catechol metabolite, 3, 4-dihydroxyphenylglycol (DOPEG) although DA and its metabolite 3, 4-dihydroxyphenylacetic acid (DOPAC) were significantly increased. Comparison of these results led us to conclude that DOPAC is probably the most important contributor to the in vivo oxidation current. This assertion is corroborated by results obtained after tyrosine hydroxylase inhibition with α-methyl-p-tyrosine: the in vivo catechol current was rapidly suppressed and post-mortem levels of DOPAC were significantly reduced while DOPEG remained almost normal. An attempt was made to selectively destroy the LC cell bodies by a unilateral injection of ibotenic acid (10 μg). Eight to 15 days after injection, no current was detectable in the injected side although it was still present in the contralateral intact side. Post-mortem levels of DOPAC and DOPEG levels of the lesioned side were 29% and 17%, respectively, of those in the intact side. Thus, we assumed that the in vivo catechol current in the LC comes from the oxidation of DOPAC most probably synthesized by the noradrenergic cell bodies.

The locus coeruleus: actions of psychoactive drugs.

The locus coeruleus is one of the most thoroughly investigated mammalian brain areas. Its fibers innervate large parts of the neuraxis, in particular, areas involved in cognitive functions such as the cortex and the hippocampus. A role of locus coeruleus has been proposed in such processes as memory, the control of vigilance, blood pressure and others. Results obtained in this and other laboratories demonstrate that the firing rate of locus coeruleus neurons is affected by a great number of psychoactive agents such as antidepressants, minor tranquillizers, neuroleptics, psychostimulants and certain psychogeriatric drugs. We have attempted to correlate the data obtained on the cell bodies of locus coeruleus with studies reporting effects on terminal areas and thereby gain an overall view of the action of the above mentioned drugs on this cell system. The activity of noradrenergic neurons in locus coeruleus is thought to correlate with the level of cortical vigilance. Special emphasis is placed on the finding that a number of drugs which exert a positive effect on cognitive functions in man and animals increase the firing rate of the rat locus coeruleus neurons.

Specific uptake and retrograde flow of antibody to dopamine-β-hydroxylase by central nervous system noradrenergic neurons in vivo

This study sought to determine whether the administration in vivo of antibody to dopamine-β-hydroxylase (ADβH) is taken up by central noradrenergic neurons and transported by retrograde flow to the cell bodies of origin. ADβH serum or pre-immune serum (control) in volumes of 1–20 μl were stereotaxically injected into the lateral ventricle. Rats were sacrificed at times ranging from 1 h to 8 days. Cryostat sections were stained with fluorescein conjugated IgG. After 24 h, a bilateral granular fluorescence was seen only in neuronal cell bodies corresponding to noradrenergic cell groups A 1–A 7 with the most intense fluorescence localized within perikarya and processes of the locus coeruleus (A 6) and subcoeruleus. This technique also permitted the visualization of the ascending dorsal and ventral noradrenergic bundles as well as varicose fibers and terminals in a pattern identical to that reported with hitofluorescence, autoradiographic, biochemical and classical immunofluorescence techniques for the identification of noradrenergic fiber distributions. At 3 and 6 h, the first detectable fluorescence was observed in forebrain noradrenergic terminals and in fibers of the dorsal and ventral noradrenergic bundles. At 10 h fluorescent varicosities were first visualized within the caudal dorsal bundle and some cytoplasmic fluorescent particles were seen within locus coeruleus cell bodies. After 18 h locus coeruleus and subcoeruleus cell bodies were heavily stained, whereas medullary noradrenergic cell groups and nerve fibers were not labeled until after 24 h. An intense locus coeruleus fluorescence remained for 3 days and was completely absent after 6 days. Bilateral transection of the dorsal noradrenergic bundle in the rostral mesencephalon, at the time of injection, effectively blocked the retrograde transport of fluorescing material to the locus coeruleus. The overall staining pattern suggests that, in vivo, central noradrenergic fibers are capable of taking up antibody to dopamine-β-hydroxylase. The ability of a dorsal bundle transection to abolish locus coeruleus staining, as well as the time course of ADβH staining in noradrenergic neurons, suggests that AD/gbH is transported via a rapid retrograde flow process. This technique combines retrograde transport of a marker protein with the sensitivity and specificity of immunocytochemical procedures to provide a new tool for the neuroanatomical study of neurotransmitter systems.