Dopamine-β-hydroxylase-like immunoreactivity in the fetal cerebral cortex of the rat: Noradrenergic ascending pathways and terminal fields

A topographical analysis of the noradrenergic innervation in the fetal rat cerebral cortex was carried out from embryonic day 15 (E15) until birth using antibodies raised against dopamine-β-hydroxylase (DBH). During late gestation DBH-like immunoreactive axons were coursing through the basal forebrain along three pathways:

1. (1) a medial component reached the medial cortex and then ran caudally along the anlage of the cingulum bundle;

2. (2) a lateral component reached the frontal pole and curved ventro-dorsally in the primordium of the external capsule;

3. (3) a few fibers were observed along the ventral amygdaloid bundle toward the amygdaloid complex and the surrounding cortex. No DBH positive fibers were observed in the main body of the internal capsule.

The first noradrenergic axons were seen at E17 in the frontal pole, the lateral frontal cortex, and in the medial frontal cortex which also receives a dopaminergic input. The innervation then extended caudally, but the dorsal part of the cortex was reached after a 2-day delay when compared to the medial and lateral parts. The arrival of noradrenergic axons did not parallel the gradient of cortical neurogenesis; however, all cortical areas were innervated at birth. DBH positive fibers reached a given cortical region simultaneously through the marginal and intermediate zones and then invaded the cortical plate.     

Dopamine-β-hydroxylase in cerebrospinal fluid and plasma: Effects of α-adrenergic agents

Dopamine-β-hydroxylase (DBH) and norepinephrine are both localized in noradrenergic storage vesicles. When noradrenergic nerves fire, both norepinephrine and DBH are released by exocytosis. DBH released from the peripheral nervous system and the adrenal medullae is found in blood, while DBH in cerebrospinal fluid (CSF) is presumably of central origin. This study was designed to: (1) investigate the effect of drugs which alter central noradrenergic activity on DBH activity in CSF; and (2) compare the effects of these drugs on DBH in CSF and plasma in cats. Phenoxybenzamine was given subcutaneously at 6 mg/kg and DBH was measured 8 h later. This treatment significantly increased DBH activity in CSF (n= 10, P < 0.005). There were no consistent changes in plasma DBH, although there was a tendency for DBH to increase from low basal levels and to decrease from high basal levels. Clonidine was administered in 4 subcutaneous injections (100, 50, 50, 50 μg/kg) over a 19-h period, and blood and CSG were taken 5 h after the last injection. This treatment caused a significant decrease in CSF DBH activity (P < 0.05, n= 8). The effect of clonidine on plasma DBH was strongly dependent on the basal enzyme level. The 3 lowest DBH values increased and the 5 highest DBH values decreased on drug treatment. These results are discussed with respect to the theory that changes in CSF DBH may reflect central noradrenergic activity.     

Descending projections of the locus coeruleus and subcoeruleus/medial parabrachial nuclei in monkey: Axonal transport studies and dopamine-β-hydroxylase immunocytochemistry

Spinal projections originating in the dorsolateral pons in the ventral locus coeruleus and in the subcoeruleus and medial parabrachial nuclei were identified in monkeys (Macaco fascicularis) using the retrograde horseradish peroxidase tracing technique. Anterograde autoradiographic tracing studies were then carried out to determine the brain stem and spinal cord terminations of the neurons. Finally, results obtained with the axonal transport tracing methods were compared with the pattern of staining for noradrenergic cells and terminals revealed immunocytochemically with an antibody to dopamine-β-hydroxylase (DβH), the synthesizing enzyme for norepinephrine. The major findings of these studies are that two presumed noradrenergic cell groups of the dorsolateral pons, one corresponding to the nucleus locus coeruleus, the second to the subcoeruleus/medial parabrachial nuclei, give rise to descending project-ions. They differ significantly in their patterns of termination in the lower brain stem and spinal cord. Among the major terminations of the locus coeruleus pathway are projections to parasympathetic neurons of the dorsal motor nucleus of the vagus, the region of the nucleus ambiguus, and the sacral spinal cord. The terminations of the descending subcoeruleus/medial parabrachial pathway, in contrast, include project-ions to sympathetic preganglionic neurons of the intermediolateral cell column of the thoracic cord and heavier projections to somatic cranial nerve nuclei. Both pathways have additional widespread and bilateral terminations in various nuclei of the reticular formation, in the spinal dorsal horn (including the marginal zone), in the region around the central canal and in the ventral spinal gray matter. Since the origins and terminations of both these pathways correspond closely to the locations and patterns of terminations of noradrenaline-containing neurons, demonstrated here with DβH immunocytochemistry, norepinephrine (or epinephrine) is suggested to be the transmitter in both these descending systems.     

Orthograde and retrograde axonal transport of dopamine-β-hydroxylase in ileal mesenteric nerves of rats with chronic streptozotocin diabetes

Rats with chronic streptozotocin-induced diabetes develop a neuropathy involving the ileal mesenteric nerves. Distal portions of these postganglionic sympathetic axons develop markedly dilated, dopamine-β-hydroxylase (DBH)-containing dystrophic swellings. These findings led us to develop a quantitative method to examine orthograde and retrograde axonal transport of DBH in ileal mesenteric nerves. Surprisingly, no significant alteration in orthograde or retrograde axonal transport of DBH was identified.     

In vivo role for CREB signaling in the noradrenergic differentiation of sympathetic neurons

Signaling pathways involving cAMP and CREB have been implicated in several aspects of sympathetic neuron differentiation. Here, we used in vivo loss-of-function approaches in both mouse and chick embryos to characterize the physiological role of cAMP/CREB. Whereas sympathetic neuron development proceeds normally in CREB-deficient mouse embryos, a decrease in noradrenergic differentiation (TH, DBH) was observed in chick sympathetic ganglia in response to ACREB, a dominant-negative CREB variant which interferes with the function of all CREB family members. In contrast, expression of the generic neuronal marker SCG10 was not affected by ACREB. As the decrease in noradrenergic gene expression is compensated at later stages of development and TH expression in differentiated neurons is not CREB-dependent, a transient role for CREB is proposed, accelerating noradrenergic but not generic neuronal differentiation of sympathetic neurons.     

Anatomical evidence for genetic differences in the innervation of the rat spinal cord by noradrenergic locus coeruleus neurons

Pontospinal noradrenergic neurons located in the A5, A6 (locus coeruleus, LC), and A7 cell groups are the major source of the noradrenergic innervation of the spinal cord. We have recently examined the specific terminations of these three cell groups in the spinal cord and found that the LC provides the major noradrenergic innervation of the ventral horn, while the A7 and A5 cell groups innervate the dorsal horn and intermediate zone, respectively. However, the results of similar experiments from another laboratory have shown that noradrenergic neurons in the locus coeruleus primarily innervate the dorsal horn, while the A5 and A7 innervate the intermediate zone and the ventral horn. These conflicting results may be due to fundamental genetic differences between the rats used in our experiments (Sasco Sprague-Dawley) and those used by the other laboratory (Harlan Sprague-Dawley). This possibility was examined by determining the projections of coeruleospinal neurons in these two rat substrains using the anterograde tracer Phaseolus vulgaris leucoagglutinin. The results indicate that in Sasco rats the LC neurons project through the ipsilateral ventromedial funiculus and terminate almost exclusively in the medial part of laminae VII and VIII, the motoneuron pool of lamina IX, and lamina X. In contrast, LC neurons in Harlan rats project bilaterally through the superficial dorsal horn and the dorsolateral funiculus and terminate most heavily in dorsal horn laminae I-IV. In addition, the LC neurons of Sasco rats innervate cervical spinal cord segments more densely than lumbar spinal cord segments, while in Harlan rats the lumbar spinal cord is more densely innervated than the cervical spinal cord. These results indicate that the projections of coeruleospinal neurons in Sasco rats are fundamentally different from those in Harlan rats and suggest that noradrenergic LC neurons may have different physiological functions in these two rat substrains.     

Identification of catecholamine cell bodies in the pons and pons-mesencephalon junction of the cat brain, using tyrosine hydroxylase and dopamine-β-hydroxylase immunohistochemistry

Using a double labeling immunohistochemical method with antibodies to tyrosine hydroxylase and dopamine-beta-hydroxylase, we reexamined the topographical distribution of dopaminergic (DA) and noradrenergic (NA) cells in the pons-mesencephalon junction of the cat. Besides DA cells of SN and its extensions, DA cells were also observed more caudally in nucleus raphe linearis intermedius and dorsalis, decussation of brachium conjunctivum, fasciculus longitudinalis medialis and periaqueductal gray. In nucleus locus coeruleus, cells exhibited variable levels of DBH, but dopaminergic cells were not evidenced in this structure.     

Retrograde transport of dopamine β-hydroxylase antibodies in sympathetic neurons: Effects of drugs modifying noradrenergic transmission

Antibodies to dopamine beta-hydroxylase (anti-D beta H) were taken up by noradrenergic nerve terminals in the iris following attachment to D beta H, and were transported back to, and accumulated in, the superior cervical ganglion (SCG). Concurrent, or prior destruction of noradrenergic terminals with 6-hydroxydopamine, injected intraocularly, blocked the retrograde transport of anti-D beta H. However, recovery was rapid, reaching 50% of control values within 1 day. Such transport was characterized by a shorter time period before accumulation could be detected in the SCG and by a slower rate of accumulation. These results suggest that noradrenergic neurons recover their ability to turn over synaptic vesicles by exocytosis and transport these back to the ganglion early during the period of axonal regeneration when the axonal length is shorter than normal. The uptake and transport of anti-D beta H was regulated by alpha-adrenergic agents administered locally in the vicinity of noradrenergic nerve terminals. Thus intraocular injection of phentolamine resulted in an increased accumulation of anti-D beta H in the SCG, while amphetamine and the postsynaptic alpha-receptor antagonist, phenylephrine, decreased accumulation. Clonidine and desipramine, which have a predominant presynaptic action, failed to influence the transport of anti-D beta H. These results suggest that in vivo the uptake of anti-D beta H can be increased more by local postsynaptic reflex actions than by a mechanism depending on the inhibition of presynaptic alpha-receptors.     

Ultrastructural analysis of noradrenergic nerve terminals in the cat lumbosacral spinal dorsal horn: a dopamine-β-hydroxylase immunocytochemical study

Noradrenaline-containing nerve terminals within the cat spinal dorsal horn were studied by immunocytochemical localization of dopamine-β-hydroxylase. Immunoreactive terminals formed symmetrical (Gray type II) synaptic specializations with dendrites and somata throughout laminae I–IV, but no junctions were formed with other axons. These findings suggest that noradrenaline regulates sensory transmission through the dorsal horn via a postsynaptic action.     

Comparison of Dopamine β-hydroxylase activity in normal rat adrenal medulla and rat pheochromocytoma cells

Dopamine β-hydroxylase activity is reportedly negligible in malignant rat adrenal cell lines. However, in two pheochromocytoma cell lines, PC12 and PCG2, considerable amounts of this catecholamine enzyme exist but its characteristics differ from the enzyme found in normal rat tissue in two ways. First, in normal adrenal medullary tissue, dopamine β-hydroxylase activity increases linearly with increasing protein concentration. Second, there is a stringent requirement for copper. Concentrations of copper above or below the optimum inhibit enzymatic activity. In contrast, in pheochromocytoma cells, dopamine β-hydroxylase exhibits a sigmoidal response with increasing tissue content. At dilute protein concentrations where considerable dopamine β-hydroxylase activity is observed in normal adrenal medullary extracts, enzymatic activity is negligible in the pheochromocytoma cell lines. As the protein concentration is increased, activation of the enzyme occurs, and enzymatic activity increases linearly with further increases in protein concentration. At very high concentrations of protein, enzymatic activity plateaus. For both PC12 and PCG2 cells, dopamine β-hydroxylase activity shows minimal copper dependency, suggesting that endogenous inhibitors present in normal tissue are absent in these malignant cell lines. Under conditions of maximum activation, the activity of the enzyme in PC12 cells becomes equivalent to that in normal rat adrenal medulla but remains 45-fold greater than that in PCG2 cells.     

Catecholamine innervation of the piriform cortex: a tracing and immunohistochemical study in the rat

In order to determine the origin of the Catecholamine innervation of the rat piriform cortex (PC), we combined retrograde transport of the B subunit of the cholera toxin (CTb) with tyrosine hydroxylase (TH) immunohistochemistry. A substantial number of CTb retrogradely labeled cells was found in the parabrachial pigmented, paranigral and interfascicular nuclei of the ventral tegmental area and the dorsal part of the locus coeruleus, whereas nearly no labeling was noted in the substantia nigra. Following TH immunohistochemistry on the same sections, most if not all of the CTb labeled cells were also TH immunoreactive. Occasional double-labeled cells were also observed in the anterior part of the raphe dorsal nucleus. As visualized with dopamine β-hydroxylase, dopamine or TH immunohistochemistry, che noradrenaline fibers were homogeneously distributed whereas the dopamine fibers showed rostro-caudal and latero-medial differences. The distribution of TH fibers overlapped both patterns. Our report suggests that the heterogeneous distribution of the DA fibers could support a differential centrifugal modulation of the olfactory information processing throughout the PC.     

Oxygen dependence of dopamine-β-hydroxylase activity and lactate metabolism in synaptosomes from rat brain

Dopamine-β-hydroxylase activity can be assayed in vitro in suspensions of rat hypothalamic synaptosomes by following tritium release from [2-³H]dopamine in the presence of monoamine oxidase inhibitors. The activity is inhibited by diethyldithiocarbamate, fusaric acid, amphetamine, reserpine, and desipramine. It is not sensitive to addition of tyrosine at the concentration present in cerebrospinal fluid, to added fumarate or to a rat heart extract known to contain potent inhibitors of the isolated enzyme. The reaction in air follows simple saturation kinetics with respect to variation in dopamine concentration with aK_m of 0.075 μM. There is reversible inhibition of activity when it is assayed at low oxygen tension. The apparent oxygen affinity is such that the enzyme does not appear saturated with respect to oxygen even at arterial oxygen tensions. Other reactions assayed exhibit less sensitivity to hypoxia: production of lactic acid from glucose becomes stimulated at 10–12 mm Hg; and oxidation of lactic acid is not inhibited at oxygen tensions above 3–4 mm Hg. Comparison of these data with the distribution of oxygen tensions present in brain suggests a basis for classifying oxygen dependent neurochemical reactions.     

Inhibition of the retrograde axonal transport of dopamine-β-hydroxylase antibodies by the calcium ionophore A23187

High levels of calcium, as well as calcium ionophores, have been reported to inhibit the anterograde transport of proteins. The effect of the calcium ionophore, A23187, on the retrograde axonal transport of proteins was therefore investigated. The uptake of antibodies to dopamine-beta-hydroxylase (anti-D beta H) by sympathetic nerve terminals in the iris and their subsequent accumulation in the superior cervical ganglion was inhibited by up to 65% by A23187 (6 nmol, i.o.). At this dose, catecholamine fluorescence in the iris was reduced, indicating a high rate of exocytosis, but tyrosine hydroxylase levels and the capacity of the treated irides to take up noradrenaline were unaffected. Higher amounts of A23187 (28 nmol, i.o.) did not cause a greater degree of inhibition of retrograde transport. However, this dose was toxic to the neurons, as shown by a 68% decrease in the ability of the nerve terminals in the iris to take up [3H]noradrenaline. This loss of function occurred gradually over a 12-h period. On the other hand, tyrosine hydroxylase levels were unaffected by 28 nmol A23187. The toxicity of A23187 may be a consequence of a build up in intracellular calcium, but such toxicity did not lead to any apparent loss of nerve terminals within a 3-day period.     

Genotype-controlled analysis of serum dopamine β-hydroxylase activity in civilian post-traumatic stress disorder

Background

Norepinephrine (NE) plays a central role in post-traumatic stress disorder (PTSD). Dopamine β-hydroxylase (DβH) converts dopamine (DA) to NE and its activity varies widely across individuals. Mustapic et al. (2007) reported a PTSD-associated deficit in serum DβH activity in a genotype-controlled analysis of combat veterans. We tested whether such a deficit would occur in a sample of civilians.

Methods

The severity of current adult PTSD symptoms and current DSM-IV diagnosis of PTSD were determined by the PTSD Symptom Scale (PSS). Adulthood trauma exposure was assessed using the Traumatic Experience Inventory (TEI). Serum DβH activity (sDβH) was assayed by HPLC with electrochemical detection and genotypes were determined using the Taqman® platform.

Results

Two hundred and twenty seven African American (AA) subjects were enrolled in this study, with a mean age (± SD) of 42.9 (± 12.9) years. We found a strong association between rs1611115 genotype and sDβH (p < 0.0001). After controlling for adulthood trauma exposure, there were no significant differences of sDβH between subjects who met a PTSD diagnosis and those who did not (p > 0.05) in any genotype group. No significant correlations were found between sDβH and PTSD severity, but sDβH significantly associated with the status of comorbid depression based on the cutoff of HAMD (p = 0.014) in subjects with PTSD.

Conclusions

We have replicated in this sample the prior finding that DBH rs1611115 genotype strongly associates with sDβH. No associations between sDβH and PTSD diagnosis or symptom severity were found in this civilian sample.     

Visual P3-like potentials in squirrel monkey: Effects of a noradrenergic agonist

Event-related potentials (ERPs) were recorded from squirrel monkeys (Saimiri sciureus) in a 90-10 visual "oddball" paradigm. A small, blue rectangle was presented every 2 s on 90% of the trials (background), whereas a yellow rectangle occurred on 10% on the trials (oddball). Electrical activity time-locked to these stimulus events was recorded from epidural electrodes before and following systemic administration of the alpha-2 noradrenergic agonist clonidine (0.1 mg/kg intramuscularly, IM). Baseline data in response to oddball stimuli showed a large, P3-like potential exhibiting a fronto-central maximum along midline electrodes and a parietal maximum along lateral electrodes. A frontally dominant, long-latency, negative slow wave (SN) consistently followed this P3-like potential. Amplitudes for P3 were larger following 10% than 50% probable oddball events. These results suggest that monkeys exhibit large, probability-sensitive P3-like potentials similar to the visual potentials reported in humans. Administration of clonidine had no effect on the amplitude, area, or latency of the monkey P3 component. This contrasts with our previous findings that the same dose of clonidine significantly decreases auditory P3s in these monkeys. Such differences may reflect distinct functional roles for norepinephrine in the processing of low-probability acoustic versus visual signals and argues against the hypothesis that norepinephrine is a common neurotransmitter substrate for auditory and visual P3-like potentials.     

Origin of noradrenergic projections to GnRH perikarya-containing areas in the medial septum-diagonal band and preoptic area

The purpose of the present study was to identify the sites of origin of the noradrenergic fibers that project to areas containing gonadotropin-releasing hormone (GnRH) perikarya since norepinephrine (NE) is known to influence the activity of GnRH neurons. Fluorescent retrograde tracers were used in combination with immunohistochemistry for dopamine-β-hydroxylase (DBH) and GnRH. Small volumes of either Fluoro-gold (FG) or Fluoro-Ruby (FR) were pressure injected into areas that contain the largest number of GnRH cell bodies, i.e., the medial septum-diagonal band complex or preoptic area. Retrogradely labeled neurons were observed ipsilaterally in the following noradrenergic cell groups: A2 (in the nucleus tractus solitarii), A1 (in the ventrolateral medulla) and locus coeruleus. Approximately 8% of all DBH-positive neurons within the A2-cell group were retrogradely labeled, while 12% of DBH-ir neurons in the A1-group were double-labeled. Only a few retrogradely labeled DBH-ir neurons were observed in the locus coeruleus (<1%). Double-labeled neurons were not organized into discrete cell groups, but were dispersed among other NE-neurons within the A2- and A1-cell groups. The highest concentrations of double-labeled neurons were located in the central one-third of both the A2 and A1 cell groups. The results suggest that most noradrenergic terminals in the region of the GnRH perikarya in the medial septum-diagonal band/rostral preoptic area originate from ipsilateral neurons in areas A1 and A2. These data support the view that the medullary cell groups A1 and A2, and not the locus coeruleus, are the primary sites of origin of the noradrenergic component that is crucial for the direct regulation of GnRH perikarya.     

Bradykinin elevates tyrosine hydroxylase and dopamine β-hydroxylase mRNA levels in PC12 cells

Bradykinin is known to rapidly elevate intracellular calcium leading to secretion of neurotransmitters and short term activation of tyrosine hydroxylase (TH). In this study we examined the effect of bradykinin on mRNA levels of two catecholamine biosynthetic enzymes. Treatment of PC12 cells with 1 μM bradykinin for 3 h markedly elevated both TH and dopamine β-hydroxylase (DBH) mRNA levels.     

Noradrenergic innervation of vasopressin-and oxytocin-containing neurons in the hypothalamic paraventricular nucleus of the macaque monkey: Quantitative analysis using double-label immunohistochemistry and confocal laser microscopy

Previous reports on the rat and monkey hypothalamus have revealed a dense noradrenergic innervation within the hypothalamic paraventricular nucleus as assessed by dopamine-β-hydroxylase immunohistochemistry. These single-label analyses were unable to delineate the cellular structures which receive this catecholaminergic innervation. Double-label preparations in the rat hypothalamic paraventricular nucleus have demonstrated synaptic interactions between noradrenergic varicosities and magnocellular neurons. However, the density and distribution of varicosities contacting chemically identified magnocellular neurons have not been assessed at the light or electron microscopic level. In this report, single-label immunohistochemistry was used to assess the morphology and distribution of vasopressin - and oxytocin-immunoreactive neurons within the macaque hypothalamic paraventricular nucleus. In addition, double-label immunohistochemistry was combined with confocal laser scanning microscopy to quantify the number of dopamine-β-hydroxylase-immunoreactive varicosities in apposition to magnocellular neurons expressing vasopressin or oxytocin immunoreactivity. The morphology of chemically identified neurons was also compared to magnocellular neurons in the monkey hypothalamic paraventricular nucleus which were filled with Lucifer Yellow in order to assess the somatodendritic labeling of the immunohistochemical preparation. Qualitative assessment of immunohistochemically identified magnocellular cells indicated that vasopressin- and oxytocin-containing neurons are observed throughout the rostrocaudal extent of the monkey hypothalamic paraventricular nucleus, demarcating this structure from the surrounding anterior hypothalamus. The distribution of the two nonapeptides is complementary, with vasopressin-immunoreactive neurons having a greater somal volume and located in a more medial aspect of the mid and caudal hypothalamic paraventricular nucleus relative to oxytocin-immunoreactive perikarya. For the double-label preparations, a series of confocal optical sections was assessed through the total somal volume of vasopressin- and oxytocin-immunoreactive neurons along with the corresponding dopamine-β-hydroxylase-immunoreactive varicosities in the same volume of tissue, generating a varicosity-to-neuron ratio which was further characterized morphologically to assess afferent input to the soma and proximal dendrites. Quantitative analysis revealed that vasopressin-immunoreactive neurons received approximately two thirds of their dopamine-β-hydroxylase-immunoreactive varicosities in apposition to the proximal dendrites and one third in apposition to the somata. Furthermore, vasopressin-immunoreactive neurons received a greater innervation density than oxytocin-immunoreactive neurons, which did not have a differential distribution of varicosities on the proximal dendrites and somata. The distribution of dopamine-β-hydroxylase-immunoreactive afferents on magnocellular neurons in the hypothalamic paraventricular nucleus may reflect a physiological role of this circuit in terms of preferential release of vasopressin from magnocellular neurons upon noradrenergic stimulation.     

A new double labeling technique using tyrosine hydroxylase and dopamine-β-hydroxylase immunohistochemistry: Evidence for dopaminergic cells lying in the pons of the beef brain

Despite numerous data on the organization of the central catecholaminergic (CA) system, the nature of many CA terminal fields, fiber pathways and cell bodies, remains unknown. The discrepancies between the results obtained by CA histofluorescence and dopamine-ß-hydroxylase (DBH) immunofluorescence method suggest that certain areas described as noradrenergic (NA) may be in fact dopaminergic (DA). A method to solve this problem is to compare the localization of the two CA synthetizing enzymes: tyrosine hydroxylase (TH) and DBH. The first enzyme is a marker of all CA neurons, whereas the second one is a specific marker of noradrenergic and adrenergic neurons. We decided to develop a technique which allows visualization of both antigens on a same section. We chose to combine immunofluorescence and immunoperoxidase techniques which yield very different staining. The optimal conditions for this double labeling technique were developed in bovine brain, the homologous system for our antibodies. The results obtained by this procedure provide evidence that DA cells together with NA cells extend throughout the rostral part of the pons, and outline a successful technique for a more extensive investigation of the DA system. The same method should be applicable to other investigations involving other antigens.     

Induction of food intake by a noradrenergic system using clonidine and fusaric acid in the neonatal chick

To clarify noradrenergic systems on food intake of the neonatal chicks, we examined the effects of i.c.v injection of clonidine (CLON), an alpha2-receptor agonist, and fusaric (5-butylpicolinic) acid (FA), a dopamine (DA)-beta-hydroxylase (DBH) inhibitor. Although a high dose (250 ng) of CLON induced a narcoleptic response and reduced food intake, food intake at 30 min post-injection was enhanced by lower doses (25 and 50 ng) of CLON. Central administration of FA (25, 50 and 100 microg) increased food intake in a dose-dependent fashion. It is suggested that feeding behavior is stimulated by low levels of CLON and decreased by further production of norepinephrine (NE), and FA may play the disturbance of sleeping and then enhance food intake.