Methyl parathion increases neuronal activities in the rat locus coeruleus

Exposure to organophosphate insecticides induces undesirable behavioral changes in humans, including anxiety and irritability, depression, cognitive disturbances and sleep disorders. Little information currently exists concerning the neural mechanisms underlying such behavioral changes. The brain stem locus coeruleus (LC) could be a mediator of organophosphate insecticide-induced behavioral toxicities since it contains high levels of acetylcholinesterase and is involved in the regulation of the sleep-wake cycle, attention, arousal, memory, and pathological processes, including anxiety and depression. In the present study, using a multi-wire recording technique, we examined the effects of methyl parathion, a commonly used organophosphate insecticide, on the firing patterns of LC neurons in rats. Systemic administration of a single dose of methyl parathion (1 mg/kg, i.v.) increased the spontaneous firing rates of LC neurons by 240% but did not change the temporal relationships among the activities of multiple LC neurons. This dose of methyl parathion induced a 50% decrease in blood acetylcholinesterase activity and a 48% decrease in LC acetylcholinesterase activity. The methyl parathion-induced excitation of LC neurons was reversed by administration of atropine sulfate, a muscarinic receptor antagonist, indicating an involvement of muscarinic receptors. The methyl parathion-induced increase in LC neuronal activity returned to normal within 30 min while the blood acetylcholinesterase activity remained inhibited for over 1 h. These data indicate that methyl parathion treatment can elicit excitation of LC neurons. Such excitation could contribute to the neuronal basis of organophosphate insecticide-induced behavioral changes in human.     

Subtypes of guanine-nucleotide-binding regulatory proteins at the locus coeruleus involved in fentanyl-induced muscular rigidity in the rat

Previous results from our laboratory have established that the G_o subtype of guanine nucleotide (GTP)-binding regulatory proteins at the locus coeruleus (LC) may participate in the elicitation of muscular rigidity by fentanyl. The present study further examined the involvement of other subtypes of GTP-binding regulatory proteins at the LC in this process, using Sprague-Dawley rats anesthetized with ketamine (120 mg/kg, i.p., with 30 mg/kg/h i.v. infusion supplements) and under mechanical ventilation. Intravenous administration of fentanyl (100 µg/kg) induced a significant increase in electromyographic signals recorded from the sacrococcygeus dorsi lateralis muscle. Power spectral analysis revealed that this was accomplished by a decrease in the mean power frequency and an increase in the root mean square values of the signals. The above responses were appreciably antagonized by pretreating animals with bilateral microinjection into the LC of pertussis toxin (80 or 160 fmol), N-ethylmaleimide (16 pmol) or 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (100 or 200 fmol); but not by cholera toxin (120 or 240 fmol), forskolin (240 or 480 pmol) or N-ethylmaleimide at a higher dose (32 pmol). These results suggest that, in addition to G_o protein, fentanyl-induced muscular rigidity may also involve other pertussis toxin-sensitive GTP-binding regulatory proteins, possibly G_i and G_p subtypes, in the signal transduction processes following activation of -opioid receptors at the LC.     

Histochemical studies on the morphology of the golgi apparatus and its relationship to catecholamine biosynthesis in the locus coeruleus of the rat

Summary Detailed histochemical studies have been performed on the morphology of the Golgi apparatus (GA) by application of the thiamine pyrophosphatase (TPPase) method (Novikoff and Goldfischer, 1961) to the neurons of the locus coeruleus (LC) of normal and catecholamine biosynthesis inhibitors (fusaric acid and D, L--methyl-p-tyrosine methylester HCl) given adult healthy male Wistar strain rats. The neurons were classified into five categories on the basis of the morphology of the Golgi apparatus. The number of cells in individual categories was counted to evaluate the percentage of each category in the whole nucleus.The majority of cells belongs to Types II, III, and IV whose GA goes through cyclic activity, but the remaining neurons belong to Types I and V which may have a strong tendency to be different from the former in character. The latter neurons correspond formally with Types I and V of the rabbit LC, but they do not respond to the drugs administered. The rat LC is very similar to the dorsal vagal nucleus of the rabbit in regard to the dominant category. The present results indicate that the majority of the rat LC neurons may work vigorously and they may be motor neurons.Administration of the drugs caused reduction of TPPase activity, augmentation of disintegration and the budding-off process of the GA of Type IV, a decrease in the percentage of Type IV and an increase in that of Type II. Administration of 100 mg/kg fusaric acid caused maximal morphological change of the GA at the 90th minute; however, administration of 200 mg/kg fusaric acid showed more marked change of the GA, having two peaks and two valleys. The GA revealed much more intense reaction to D,L--methyl-p-tyrosine methylester HCl than to fusaric acid. The present results indicate that tyrosine hydroxylase may be the rate-limiting enzyme in the catecholamine biosynthesis.These noticeable changes of GA caused by administration of the drugs were completely restricted to the neurons of LC and the neurons of the mesencephalic nucleus of the trigeminal nerve did not show any morphological changes of the GA. These results strongly suggest that the GA of the rat LC neurons may have ability to synthesize catecholamine whereas the GA of the rat mesencephalic nucleus of the trigeminal nerve may be completely devoid of this ability and that the role of the GA may be different depending on the anatomical regions.     

Participation of galaninergic neurotransmission at the paraventricular hypothalamic nucleus in the suppression of baroreceptor reflex response by locus ceruleus in the rat

We evaluated the potential participation of galanin (GAL) at the paraventricular nucleus of hypothalamus (PVN) in the suppression of baroreceptor reflex (BRR) response by locus ceruleus (LC), using adult male Sprague-Dawley rats anesthetized with pentobarbital sodium. Microinjection of GAL (100 pmol) bilaterally into the PVN significantly depressed the BRR response. This suppressive effect was appreciably antagonized when GAL (100 pmol) and GAL antiserum (1:20) were coadministered into the bilateral PVN. Whereas bilateral microinjection of GAL antiserum into the PVN by itself elicited minimal effect, it nevertheless significantly attenuated the suppressive effect of either electrical or chemical activation of LC on the BRR response. Pretreatment with the same amount of normal rabbit serum (1:20), on the other hand, was ineffective. These results suggest that a galaninergic projection from the LC to PVN may participate in the suppression of BRR response by this dorsal pontine nucleus.     

Spatiotemporal interaction of alpha(2) autoreceptors and noradrenaline transporters in the rat locus coeruleus: implications for volume transmission

We investigated the roles of alpha(2) autoreceptors and noradrenaline (NA) transporters on NA efflux and uptake in the rat locus coeruleus after electrical stimulation. NA efflux was evoked by various trains (50 pulses, 10-500 Hz) and measured by fast cyclic voltammetry. NA efflux and uptake half-time (t(1/2)) were stimulus-dependent, ranging from 43 +/- 3 nM and 2.45 +/- 0.21 s, respectively, with 500-Hz stimuli to 127 +/- 11 nM and 4.41 +/- 0.34 s, respectively, with 100-Hz trains. Based on these data, we calculate that each transporter removes 0.19 NA molecules from the extracellular space every second, a velocity compatible more with transporter-than channel-mode conduction. Dexmedetomidine (10 nM) decreased NA efflux by approximately 30% on stimulations of < or =1 s in duration. BRL 44408 (1 microM) increased NA efflux on stimuli of > or =2 s (by up to 92 +/- 16%). Desipramine (50 nM) increased NA efflux on stimuli of > or =1 s (by 113 +/- 24%) but slowed NA uptake on all stimuli. When given together, the effects of desipramine and BRL 44408 were additive at stimuli of >or =1 s but showed potentiation on shorter trains. There was a significant time delay for the elevation of NA efflux by blockade of uptake (0.79 s) or autoreceptors (1.14 s), suggesting that both are located extrasynaptically and that NA must diffuse through the extracellular space to these structures. We suggest that released NA may interact with alpha(2) autoreceptors and NA transporters as far as 10 microm from the release sites, an action compatible with a volume transmission role of NA in the locus coeruleus.     

Restoration of evoked responses in the rat dorsomedial thalamic nucleus after amygdaloid lesion

The changing pattern of focal potentials in the thalamic dorsomedial nucleus, produced by stimulating the periamygdaloid cortex between 2 and 90 days after unilateral destruction of the basolateral amygdaloid nuclei, was investigated during semichronic experiments on anesthetized rats. A comparison was made between the parameters and spatio-temporal characteristics of potentials, as revealed at different stages of functional reorganization of thalamo-limbic interaction. The biggest increase in latency to peak of the principal positive-negative component is seen during the first two months after amygdaloid lesion. The original pattern and numerical features of focal potentials are restored in 2.5 months. The potentials formed during the course of the compensatory process differed from those of animals with an intact CNS, however, the amplitude of their test response to paired stimuli being incompletely restored, especially at interstimulus intervals of 40–150 msec. Findings indicate the high functional plasticity of the neural fibers mediating afferents at the level of the above thalamic association nucleus.Institute of Higher Nervous Activity and Neurophysiology, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 18, No. 2, pp. 153–161, March–April, 1986.     

The role of glutamate in the locus coeruleus during opioid withdrawal and effects of H-7, a protein kinase inhibitor, on the action of glutamate in rats

To investigate the role of glutamate in the locus coeruleus (LC) during opioid withdrawal, rats were continuously infused with morphine (a -opioid receptor agonist, 26 nmol/µl/h) or butorphanol (a //-mixed opioid receptor agonist, 26 nmol/µl/h) intracerebroventricularly (i.c.v.) via osmotic minipumps for 3 days. A direct LC injection of glutamate (1 or 10 nmol/5 µl) or naloxone (an opioid receptor antagonist, 24 nmol/5 µl) induced withdrawal signs in morphine- or butorphanol-dependent animals. However, these agents failed to precipitate any withdrawal signs in saline-treated control animals. On the other hand, the expression of withdrawal signs precipitated by the administration of glutamate or naloxone in opioid-dependent animals was completely blocked by concomitant infusion with 1 or 10 nmol/µl/h of an inhibitor of adenosine 3,5-cyclic monophosphate (cAMP)-dependent protein kinase and protein kinase C, H-7 [1-(5-isoquinolinesulfonyl)-2-methylpiperazine]. In animals that had been infused with opioids in the same manner, i.c.v. injection of naloxone (48 nmol/5 µl) precipitated withdrawal signs and increased extracellular fluid levels of glutamate in the LC of morphine- or butorphanol-dependent rats measured by in vivo microdialysis method. However, concomitant infusion with H-7 inhibited the increases of glutamate levels in the LC. These results strongly suggest that an expeditious release of glutamate in the LC region plays an important role in the expression of physical dependence on opioids. Furthermore, the action on glutamate release might be increased by the enhancement of cAMP-dependent protein kinase and/or protein kinase C activity.     

Potentiation of the glutamatergic synaptic input to rat locus coeruleus neurons by P2X7 receptors

Locus coeruleus (LC) neurons in a rat brain slice preparation were superfused with a Mg²⁺-free and bicuculline-containing external medium. Under these conditions, glutamatergic spontaneous excitatory postsynaptic currents (sEPSCs) were recorded by means of the whole-cell patch-clamp method. ATP, as well as its structural analogue 2-methylthio ATP (2-MeSATP), both caused transient inward currents, which were outlasted by an increase in the frequency but not the amplitude of the sEPSCs. PPADS, but not suramin or reactive blue 2 counteracted both effects of 2-MeSATP. By contrast, α,β-methylene ATP (α,β-meATP), UTP and BzATP did not cause an inward current response. Of these latter agonists, only BzATP slightly facilitated the sEPSC amplitude and strongly potentiated its frequency. PPADS and Brilliant Blue G, as well as fluorocitric acid and aminoadipic acid prevented the activity of BzATP. Furthermore, BzATP caused a similar facilitation of the miniature (m)EPSC (recorded in the presence of tetrodotoxin) and sEPSC frequencies (recorded in its absence). Eventually, capsaicin augmented the frequency of the sEPSCs in a capsazepine-, but not PPADS-antagonizable, manner. In conclusion, the stimulation of astrocytic P2X7 receptors appears to lead to the outflow of a signalling molecule, which presynaptically increases the spontaneous release of glutamate onto LC neurons from their afferent fibre tracts. It is suggested, that the two algogenic compounds ATP and capsaicin utilise separate receptor systems to potentiate the release of glutamate and in consequence to increase the excitability of LC neurons.     

Maternal attenuation of hypothalamic paraventricular nucleus norepinephrine switches avoidance learning to preference learning in preweanling rat pups

Infant rats learn to prefer stimuli paired with pain, presumably due to the importance of learning to prefer the caregiver to receive protection and food. With maturity, a more 'adult-like' learning system emerges that includes the amygdala and avoidance/fear learning. The attachment and 'adult-like' systems appear to co-exist in older pups with maternal presence engaging the attachment system by lowering corticosterone (CORT). Specifically, odor-shock conditioning (11 odor-0.5 mA shock trials) in 12-day-old pups results in an odor aversion, although an odor preference is learned if the mother is present during conditioning. Here, we propose a mechanism to explain pups ability to 'switch' between the dual learning systems by exploring the effect of maternal presence on hypothalamic paraventricular nucleus (PVN) neural activity, norepinephrine (NE) levels and learning. Maternal presence attenuates both PVN neural activity and PVN NE levels during odor-shock conditioning. Intra-PVN NE receptor antagonist infusion blocked the odor aversion learning with maternal absence, while intra-PVN NE receptor agonist infusion permitted odor aversion learning with maternal presence. These data suggest maternal control over pup learning acts through attenuation of PVN NE to reduce the CORT required for pup odor aversion learning. Moreover, these data also represent pups' continued maternal dependence for nursing, while enabling aversion learning outside the nest to prepare for pups future independent living.     

Increased locus coeruleus glutamate levels are associated with naloxone-precipitate withdrawal from butorphanol in the rat

Extracellular fluid levels of glutamate were measured in the locus coeruleus during butorphanol (a mixed agonist at -, -, and -opioid receptors) withdrawal by using microdialysis in conscious butorphanol-dependent Sprague-Dawley rats. Guide cannulae were implanted chronically and rats were given intracerebroventricular (i.c.v.) infusions of butorphanol (26 nmol/l l/hr) or saline (1 l/hr) for 3 days. Microdialysis probes (2 mm tip) were inserted into the locus coeruleus 24 hr before precipitation of withdrawal by i.c.v. injection of naloxone (48 nmol/5 l). A separate series of rats was rendered dependent by peripheral injection of butorphanol (20 mg/kg, s.c., b.i.d.) for 5 days and naloxone (5 mg/kg, i.p.) was given to precipitate withdrawal. Single injections of butorphanol (26 nmol/5 l, i.c.v.) had no effect on the extracellular fluid levels of glutamate, compared to rats injected with vehicle. Behavioral evidence of withdrawal was detected following naloxone challenge in butorphanol-dependent rats (both i.c.v. and s.c. models), but not in nondependent, vehicle-treated rats. Significant increases (P<0.05) in levels of glutamate were noted after naloxone-precipitated withdrawal only in the butorphanol group. The glutamate levels in the locus coeruleus increased from 8.37±2.01 before, to 21.93±4.58 M in the first 15 min sample following i.c.v. injections of 48 nmol/5 l naloxone and from 10.84±1.74 before, to 26.01±6.19 M in the 15–30 min sample following i.p. injections of 5 mg/kg naloxone in the butorphanol-dependent rats, respectively. These results provide direct evidence to support the role of excitatory amino acids within the locus coeruleus in butorphanol withdrawal.     

蓝斑核、中缝大核和迷走神经背核在胃运动调节中的关系

目的：探讨蓝斑（LC）、中缝大核（NRM）和迷走神经背核（DMV）,及其相关递质和受体对胃运动的调节途径及机制,阐明它们在调节胃运动中的相互关系。方法：实验采用了核团定位电刺激、损毁和核团微量注射等实验方法,以记录胃内压,统计胃收缩幅度作为胃运动变化的指标。结果：①刺激LC显著降低胃收缩幅度（P〈0.01）,损毁DMV可以减弱此效应,而阻断DMV上的肾上腺素能α受体,可以反转此抑胃效应。②刺激NRM显著降低胃收缩幅度（P〈0.01）,损毁DMV后此效应被消除;阻断DMV上的5-HT2A受体使胃收缩幅度大幅度降低（P〈0.01）,此时再刺激NRM不能进一步的抑制胃运动;而损毁LC后刺激NRM,可消除NRM的抑胃效应,在LC注射5-HT2A受体阻断剂也可以消除该效应。结论：①LC可能通过DMV的5-HT2A受体和α受体对生理条件下正常胃的运动起着重要的双向调节作用;②NRM通过LC上的5-HT2A受体而发挥其对胃运动的抑制效应。     

Organization and interrelationship of neuropeptides in the central amygdaloid nucleus of the rat

The organization and interactions of neuropeptides in the central nucleus of the amygdala (Ce) were studied using single and double label immunocytochemical techniques. Immunocytochemical localization of substance P (SP), neurotensin (NT), met-enkephalin (m-ENK), somatostatin (SS) and vasoactive intestinal polypeptide (VIP) revealed all of these peptides within discrete regions of the Ce. The regions differed from the classical medial and lateral anatomical divisions reported for the Ce. Instead, three easily recognizable neuropeptidergic subdivisions were evident: a medial zone, a central zone and a lateral capsular zone. Two types of interrelationships between peptides were noted. The first involved a peptidergic fiber in apposition to a peptidergic perikarya. The most prevalent peptidergic interaction of this type occurred between SP and NT. The second interrelationship involved two different peptidergic fibers in apposition to an immunonegative cell. Two interactions of this type were commonly observed. The first involved NT and m-ENK fibers simultaneously apposed to an unstained cell. The second involved SP and m-ENK fibers adjacent to the same immunonegative cell. The interactions between peptidergic systems may suggest a role of these substances in the regulation of autonomic functions in the Ce.     

Topographic,morphologic and developmental characterization of the nucleus loci coerulei in the chicken

Summary Golgi- and fluorescence-histochemical studies in the chicken show the presence of a sharply delimited group of aminergic neurons beneath the floor of the fourth ventricle at the mesen-metencephalic boundary. According to the observations reported in other avian species a homology can be established between the mammalian locus coeruleus (LC) and this fluorescent cell mass of the chicken brainstem. Golgi studies revealed an isodendritic pattern of ramification of the neurons in this nucleus.In addition, a developmental study on the morphological maturation of the LC in the chick embryo was carried out by means of the histochemical-fluorescence method for biogenic amines and the rapid Golgi method. The time of the first onset of catecholamine synthesis and storage has been shown to correspond to the 9th day of incubation (stage HH 35), just when these cells display a well-established and peculiar dendritic pattern. All maturational events in the LC of the chick embryo thus occur earlier than in the fetal rat brain, the prenatal development of which is accomplished in a period of comparable length.This investigation was partly supported by grants from the Italian National Research Council (CNR) No. 79.01890.04 and No. 80.00442.04     

Estradiol acts in the medial preoptic area, arcuate nucleus, and dorsal raphe nucleus to reduce food intake in ovariectomized rats

Estradiol (E2) exerts an inhibitory effect on food intake in a variety of species. While compelling evidence indicates that central, rather than peripheral, estrogen receptors (ERs) mediate this effect, the exact brain regions involved have yet to be conclusively identified. In order to identify brain regions that are sufficient for E2's anorectic effect, food intake was monitored for 48 h following acute, unilateral, microinfusions of vehicle and two doses (0.25 and 2.5 μg) of a water-soluble form of E2 in multiple brain regions within the hypothalamus and midbrain of ovariectomized rats. Dose-related decreases in 24-h food intake were observed following E2 administration in the medial preoptic area (MPOA), arcuate nucleus (ARC), and dorsal raphe nucleus (DRN). Within the former two brain areas, the larger dose of E2 also decreased 4-h food intake. Food intake was not influenced, however, by similar E2 administration in the paraventricular nucleus, lateral hypothalamus, or ventromedial nucleus. These data suggest that E2-responsive neurons within the MPOA, ARC, and DRN participate in the estrogenic control of food intake and provide specific brain areas for future investigations of the cellular mechanism underlying estradiol's anorexigenic effect.     

Ultrastructural characteristics of vasopressin-containing neurons in the paraventricular nucleus of the hypothalamus

Summary Vasopressin-containing neurons, identified by immunocytochemistry, are located predominantly in the posterior magnocellular division of the paraventricular nucleus of the rat hypothalamus. By electron microscopy, the immunoreaction product is seen within the cell bodies and neuronal processes. In the perikarya and dendritic processes, the immunoreactive material is associated primarily with neurosecretory granules. Axonal processes, identified by their content of microtubules and accumulation of neurosecretory granules, show the immunoreaction product in association with both of these organelles. Afferent axo-dendritic, axo-somatic and putative axo-axonic synapses with immunostained vasopressinergic neurons can be identified. The presynaptic profiles do not contain immunoreactive material. This study contributes to the ultrastructural characterization of vasopressinergic neurons in the paraventricular nucleus and of their afferent synaptic input.Supported by NIH Grants HD-12956 and 2SO7RR05403     

Co-localization of midbrain projections, progestin receptors, and mating-induced fos in the hypothalamic ventromedial nucleus of the female rat

In female rats, sexual behavior requires the convergence of ovarian hormone signals, namely estradiol and progesterone, and sensory cues from the male on a motor output pathway. Estrogen and progestin receptors (ER and PR) are found in neurons in the hypothalamic ventromedial nucleus (VMH), a brain region necessary for lordosis, the stereotypic female copulatory posture. A subset of VMH neurons sends axonal projections to the periaqueductal gray (PAG) to initiate a motor output relay, and some of these projection neurons express PR. Previous studies showed that VMH neurons are activated during mating, based on the expression of the immediate early gene Fos. Many of the activated neurons expressed ER; however, it is not known if such activated neurons co-express PR. Fluorogold, a retrograde tracer, was injected into the PAG of ovariectomized rats to label neurons projecting from the VMH. Hormone-treated animals then were mated, and their brains were immunohistochemically stained for PR and Fos. Of the Fos-positive neurons, 33% were double-labeled for PR, 19% were double-labeled with Fluorogold, and 5% were triple-labeled for Fos, PR, and the retrograde tracer. The majority of triple-labeled neurons were found in the rostral, rather than caudal, portion of the VMH. These results show that PR-containing neurons are engaged during sexual behavior, which suggests that these neurons are the loci of hormonal-sensory convergence and hormonal-motor integration.     

Immunohistochemical and electron microscopic study of the rat hypothalamic nuclei and cell clusters under various experimental conditions

Summary In untreated, pregnant and thirsting rats the neurosecretory hypothalamic areas were investigated by means of the immunoperoxidase technique in order to demonstrate vasopressin- and oxytocin containing elements at the light- and electron microscopic level. In addition, chromalum-hematoxylinphloxin (CHP) staining and conventional double staining of ultrathin sections were used. The areas investigated included the anterior and posterior supraoptic nuclei, the paraventricular nuclei, the numerous accessory cell clusters in the region between the tractus opticus and the third ventricle as well as the median eminence. In all nuclei and in the accessory cell clusters, the number of vasopressin-reactive neurons exceeds that of oxytocin-reactive neurons. Compared with the anterior supraoptic nucleus, the posterior supraoptic nucleus and the accessory cell clusters react more heavily to prolonged thirst. In the median eminence the neurosecretory axons display close contacts with the portal vessels not only in its lateral portion but in thirsting animals also around the mid-line. There the internal layer is broadened and vasopressin-positive tanycytic processes reach the external zone. Parasagittally, fine vasopressin-positive material can be traced from the internal layer to small deposits at the portal vessels. In long term thirsting animals the typical feature of swollen axons exhibits a characteristic distribution in the median eminence and renders a distinct positive reaction to anti-vasopressin. The release of peptide hormones from the perikarya and from the axons within the nuclei as well as the mode of release within the median eminence are discussed. The significance of the positive immunostaining of the ependymal tanycytes and of some perikarya of the suprachiasmatic nucleus must be reconsidered by further studies.Supported by the Deutsche Forschungsgemeinschaft (Grant Nr. Kr 569/1) and Stiftung VolkswagenwerkDedicated to Professor Berta Scharrer on the occasion of her 70th birthdayThe author wishes to express her special gratitude to Dr. L.A. Sternberger for supplying the peroxidaseantiper oxidase-complex and to Dr. H. Stein (Pathologisches Institut der Universität Kiel) for supplying Anti-IgG. The skilful technical assistance of Mrs. H. Prien and Mrs. H. Schöning is thankfully acknowledged     

BMPs, FGF8 and Wnts regulate the differentiation of locus coeruleus noradrenergic neuronal precursors

In the present study, we investigated the involvement of rhombomere 1 patterning proteins in the regulation of the major noradrenergic centre of the brain, the locus coeruleus. Primary cultures of rat embryonic day 13.5 locus coeruleus were treated with fibroblast growth factor-8, noggin and members of the bone morphogenetic and Wnt protein families. We show that bone morphogenetic proteins 2, 5 and 7 increase and noggin decreases the number of tyrosine hydroxylase-positive locus coeruleus neurons. Interestingly, from all Wnts expressed in the first rhombomere by embryonic day 12.5 in the mice, we only found expression of wnt5a mRNA in the vicinity of the locus coeruleus. In agreement with this finding, from all Wnts studied in vitro, only Wnt5a increased the number of tyrosine hydroxylase-positive neurons in locus coeruleus cultures. Finally, we also found that fibroblast growth factor-8 increased the number of tyrosine hydroxylase-positive cells in locus coeruleus cultures. Neither of the identified factors affected the survival of tyrosine hydroxylase-positive locus coeruleus noradrenergic neurons or the proliferation of their progenitors or neurogenesis. Instead, our results suggest that these patterning signals of rhombomere 1 may work to promote the differentiation of noradrenergic progenitors at later stages of development.