Catecholamine-synthesizing Neurons Research Articles

EGR-1 Expression in Catecholamine-synthesizing Neurons Reflects Auditory Learning and Correlates with Responses in Auditory Processing Areas

Distinguishing between familiar and unfamiliar individuals is an important task that shapes the expression of social behavior. As such, identifying the neural populations involved in processing and learning the sensory attributes of individuals is important for understanding mechanisms of behavior. Catecholamine-synthesizing neurons have been implicated in sensory processing, but relatively little is known about their contribution to auditory learning and processing across various vertebrate taxa. Here we investigated the extent to which immediate early gene expression in catecholaminergic circuitry reflects information about the familiarity of social signals and predicts immediate early gene expression in sensory processing areas in songbirds. We found that male zebra finches readily learned to differentiate between familiar and unfamiliar acoustic signals (‘songs’) and that playback of familiar songs led to fewer catecholaminergic neurons in the locus coeruleus (but not in the ventral tegmental area, substantia nigra, or periaqueductal gray) expressing the immediate early gene, EGR-1, than playback of unfamiliar songs. The pattern of EGR-1 expression in the locus coeruleus was similar to that observed in two auditory processing areas implicated in auditory learning and memory, namely the caudomedial nidopallium (NCM) and the caudal medial mesopallium (CMM), suggesting a contribution of catecholamines to sensory processing. Consistent with this, the pattern of catecholaminergic innervation onto auditory neurons co-varied with the degree to which song playback affected the relative intensity of EGR-1 expression. Together, our data support the contention that catecholamines like norepinephrine contribute to social recognition and the processing of social information.

Eff ect of a single asenapine treatment on Fos expression in the brain catecholamine-synthesizing neurons: impact of a chronic mild stress preconditioning.

Fos protein expression in catecholamine-synthesizing neurons of the substantia nigra (SN) pars compacta (SNC, A8), pars reticulata (SNR, A9), and pars lateralis (SNL), the ventral tegmental area (VTA, A10), the locus coeruleus (LC, A6) and subcoeruleus (sLC), the ventrolateral pons (PON-A5), the nucleus of the solitary tract (NTS-A2), the area postrema (AP), and the ventrolateral medulla (VLM-A1) was quantitatively evaluated aft er a single administration of asenapine (ASE) (designated for schizophrenia treatment) in male Wistar rats preconditioned with a chronic unpredictable variable mild stress (CMS) for 21 days. Th e aim of the present study was to reveal whether a single ASE treatment may 1) activate Fos expression in the brain areas selected; 2) activate tyrosine hydroxylase (TH)-synthesizing cells displaying Fos presence; and 3) be modulated by CMS preconditioning. Control (CON), ASE, CMS, and CMS+ASE groups were used. CMS included restraint, social isolation, crowding, swimming, and cold. Th e ASE and CMS+ASE groups received a single dose of ASE (0.3 mg/kg, s.c.) and CON and CMS saline (300 μl/rat, s.c.). The animals were sacrificed 90 min aft er the treatments. Fos protein and TH-labeled immunoreactive perikarya were analyzed on double labeled histological sections and enumerated on captured pictures using combined light and fluorescence microscope illumination. Saline or CMS alone did not promote Fos expression in any of the structures investigated. ASE alone or in combination with CMS elicited Fos expression in two parts of the SN (SNC, SNR) and the VTA. Aside from some cells in the central gray tegmental nuclei adjacent to LC, where a small number of Fos profiles occurred, none or negligible Fos occurrence was detected in the other structures investigated including the LC and sLC, PON-A5, NTS-A2, AP, and VLM-A1. CMS preconditioning did not infl uence the level of Fos induction in the SN and VTA elicited by ASE administration. Similarly, the ratio between the amount of free Fos and Fos colocalized with TH was not aff ected by stress preconditioning in the SNC, SNR, and the VTA. Th e present study provides an anatomical/functional knowledge about the nature of the acute ASE treatment on the catecholamine-synthesizing neurons activity in certain brain structures and their missing interplay with the CMS preconditioning.

The effect of air puff stress on c‐Fos expression in rat hypothalamus and brainstem: central circuitry mediating sympathoexcitation and baroreflex resetting

Psychological stress evokes increases in sympathetic activity and blood pressure, which are due at least in part to an upward resetting of the baroreceptor-sympathetic reflex. In this study we determined whether sympathetic premotor neurons in the rostral ventrolateral medulla (RVLM), which have a critical role in the reflex control of sympathetic activity, are activated during air puff stress, a moderate psychological stressor. Secondly, we identified neurons that are activated by air puff stress and that also project to the nucleus tractus solitarius (NTS), a key site for modulation of the baroreceptor reflex. Air puff stress resulted in increased c-Fos expression in several hypothalamic and brainstem nuclei, including the paraventricular nucleus (PVN), dorsomedial hypothalamus, perifornical area (PeF), periaqueductal gray (PAG), NTS and rostral ventromedial medulla, but not in the RVLM region that contains sympathetic premotor neurons. In contrast, neurons in this RVLM region, including catecholamine-synthesizing neurons, did express c-Fos following induced hypotension, which reflexly activates RVLM sympathetic premotor neurons. The highest proportion of NTS-projecting neurons that were double-labelled with c-Fos after air puff stress was in the ventrolateral PAG (29.3 ± 5.5%), with smaller but still significant proportions of double-labelled NTS-projecting neurons in the PVN and PeF (6.5 ± 1.8 and 6.4 ± 1.7%, respectively). The results suggest that the increased sympathetic activity during psychological stress is not driven primarily by RVLM sympathetic premotor neurons, and that neurons in the PVN, PeF and ventrolateral PAG may contribute to the resetting of the baroreceptor-sympathetic reflex that is associated with psychological stress.

Prolonged Activation of cAMP-response Element-binding Protein and ATF-2 Needed for Nicotine-triggered Elevation of Tyrosine Hydroxylase Gene Transcription in PC12 Cells

Phosphorylation (P-) of cAMP-response element-binding protein (CREB) by protein kinase A or mitogen-activated protein kinases was implicated in mediating the increased tyrosine hydroxylase (TH) gene expression after prolonged exposure to nicotine in vivo and in cell culture. We examined the time course and signaling pathways for phosphorylation of CREB and possible involvement of ATF-2. Treatment of PC12 cells with 200 microm nicotine triggered rapid but transient elevation of P-CREB followed by a second sustained rise after 2-5 h of continuous nicotine. In contrast, ERK1/2 was only phosphorylated with short term nicotine exposure. MEK inhibitor U0126 abolished nicotine-induced rise in P-ERK1/2, but not P-CREB, nor did it inhibit nicotine-evoked elevation in TH promoter activity, indicating that ERK1/2 was not needed for induction of TH gene expression by nicotine. In contrast, protein kinase A inhibitor H-89 or Ca(2+)/calmodulin-activated protein kinase inhibitor KN-93 reduced the nicotine-triggered rise in P-CREB and TH promoter activity. There was a delayed elevation of P-ATF-2 after 1 h of nicotine treatment, accompanied by increased ATF-2 protein. Upstream kinase JNK, but not p38, was phosphorylated especially after 5 min to 2 h of nicotine exposure. To examine the requirement for CREB and ATF-2, cells were transfected with dominant negative forms of ATF-2 or CREB. Both reduced the basal TH promoter activity and the response to nicotine. Knockdown of ATF-2 or CREB with siRNA did not alter basal TH promoter activity or mRNA but greatly attenuated the response to nicotine. The results suggest that both ATF-2 and CREB mediate activation of TH gene transcription by nicotine.

Distribution of tyrosine hydroxylase, dopamine, and serotonin in the central nervous system of amphioxus (Branchiostoma lanceolatum): implications for the evolution of catecholamine systems in vertebrates.

To investigate the evolutionary transition that has shaped the catecholaminergic systems of vertebrates, the organization of catecholamine-synthesizing neurons and the nature of the catecholamines were examined in the central nervous system of adult amphioxus (Branchiostoma lanceolatum), a cephalochordate. We isolated a gene transcript encoding tyrosine hydroxylase (TH), the limiting enzyme of catecholamine biosynthesis, and studied its distribution together with that of dopamine and serotonin. Dopamine and TH are found in the same neurons of which they are three separate populations. Two are located in the anterior brain, the first being dorsal and lying in a row and the second being more posterior and lateral. A third population comprising a few dorsal commissural neurons was found in the posterior brain. The anterior dopaminergic cells innervate the ventral commissure of the cephalic vesicle, the hindbrain, and the spinal cord. A serotonin-containing cell group is located in the same plane as the second dopaminergic cell population but is more caudal, marking the probable transition between anterior brain and hindbrain, as deduced from gene expression patterns. The overall distribution of dopaminergic and serotoninergic systems is similar in amphioxus and vertebrate central nervous system and could be an ancestral character of chordates. As assayed by high-performance liquid chromatography and electrochemical detection, significant amounts of dopamine and octopamine, but not of noradrenaline, are present in amphioxus head. This finding is consistent with data obtained from most prostomian species. We conclude that the noradrenergic system is probably an innovation of vertebrates that appeared along with the neural crest and specific hindbrain nuclei.

Stress-induced Fos-like Immunoreactivity in the Pons and the Medulla Oblongata of Rats

Immunoreactivity of the immediate early gene c-fos was used to investigate changes in the activity of brainstem neurons in response to acute stressors like immobilization, formalin-induced pain, cold exposure, hemorrhage and insulin-induced hypoglycemia. Different stressors induced Fos-like immunoreactivity in different pontine and medullary neurons. A single, 3 hour immobilization was found to be a very strong stimulus that activated brainstem cate-cholaminergic (tyrosine hydroxylase-immunopositive) neurons and cells in the raphe and certain pontine tegmental nuclei, as well as in the reticular formation. Pain, induced by a subcutaneous injection of formalin was also effective on catecholamine-synthesizing neurons and on others cells in the nucleus of the solitary tract. Cold exposure activated cells mainly in the sensory spinal trigeminal and parabrachial nuclei and in the so-called “pontine thermoregulatory area”. Moderate Fos-like immunoreactivity was induced by a hypotonic (25%) hemorrhage in medullary catecholaminergic neurons, the nucleus of the solitary tract and the Barrington nucleus. Among stressful stimuli used, insulin-induced hypoglycemia elicited die smallest Fos activation in the lower brainstem. The present observations indicate that different stressors may use different neuronal pathways in the central organization of the stress response.

Organization of presumptive catecholamine-synthesizing neurons in the canine medulla oblongata

Immunocytochemical methods were used to identify cells and processes containing two major catecholamine (CA)-biosynthetic enzymes in areas of the canine medulla implicated in autonomic control. Antisera were employed against tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT). These enzymes respectively catalyze the conversions of tyrosine to L-DOPA and noradrenaline to adrenaline. Immunocytochemical studies laid the groundwork for subsequent investigations in conscious dog in which we characterized an area of cardiovascular control in the rostral ventrolateral medulla (RVLM). In the anatomical studies, previously unidentified neuronal somata and processes were demonstrated in the canine medulla. Presumptive adrenergic (CI) neurons in the canine RVLM were subjacent to the nucleus ambiguous (NA) and most numerous at a level where the compact and semicompact divisions of NA merged. In contrast to their distribution in rodents, C1 neurons were skewed caudally and did not extend rostrally to the caudal pole of the facial nucleus. C1 neurons were also relatively less concentrated in the RVLM. A large number of C1 neurons extended dorsally into the lateral tegmental field (LTF). Most C1 neurons in the LTF (like those in the A1 area) were aligned with catecholaminergic (TH- and PNMT-ir) processes traversing the intermediate reticular zone. Since the numbers and locations of TH- and PNMT-ir neurons in the C1 area of the RVLM and rostral LTF were virtually identical on adjacent sections, it can be implicitly inferred that the enzymes are co-localized to the same somata and that these neurons are capable of biosynthesizing adrenaline. The C1 and A5 areas were clearly separated by a transitional zone, sparsely populated by TH-ir somata (1-2 cells per section), where the facial nucleus and rostral pole of the NA pars compacta (NAc) occupied the same level. A5 neurons were more abundant and complexly organized than suggested by previous CA-histofluorescence data. In addition, a new parvicellular subgroup was identified and composed of neurons containing TH but not PNMT. In contrast to other species, the A1 cell group was not confined to the VLM. A large number of A1 neurons extended into the caudal LTF and were situated between the nucleus tractus solitarii-motor vagal complex (NTS-X) and caudal VLM (CVLM). In contrast to previous reports, presumptive adrenergic (TH- and PNMT-ir) cell groups were more densely represented in the C2-3 areas of the canine NTS and dorsomedial reticular formation.(ABSTRACT TRUNCATED AT 400 WORDS)

Neuropeptide FF is colocalized with catecholamine-synthesizing enzymes in neurons of the nucleus of the solitary tract.

Neuropeptide FF (NFF) is an amidated octapeptide of bovine origin. It has some antiopioid-like effects and it elevates blood pressure when injected intravenously in rats. NFF-immunoreactive nerve cells and terminals are localized in large numbers in the dorsomedial caudal brainstem which is a region involved in central regulation of blood pressure. We compared the localization of NFF-immunoreactive neurons with medullary catecholamine-synthesizing neurons by using immunohistochemical double-labeling and light microscopic mirror methods. NFF and tyrosine hydroxylase coexisted in a minor portion of the NFF neurons in the nucleus of the solitary tract and occasional cell bodies were stained with both NFF and PNMT (phenylethanolamine N-methyltransferase) antisera. The results have anatomical correlation with previous pharmacological reports, suggesting that NFF is present in neuronal systems involved in cardiovascular reflex arcs.

SUBSTANCE P-CONTAINING NEURONS IN THE MESOPONTINE TEGMENTUM ARE SEVERELY AFFECTED IN PARKINSON'S DISEASE

Substance P immunoreactive (SP+) neurons were analysed quantitatively in serial sections of the mesopontine tegmentum in 6 patients with idiopathic Parkinson's disease and 5 age-matched normal controls. In the tegmentum of the Parkinson's disease brains many SP+ neurons contained swollen, twisted neuronal processes as well as Lewy bodies. There were significant reductions in the total number of SP+ neurons in the pedunculopontine tegmental nucleus (loss 43%), in the laterodorsal tegmental nucleus (loss 28%), in the oral pontine reticular nucleus (loss 41%) and in the median raphe nucleus (loss 76%). It was the large SP+ (greater than 20 microns) neurons that were particularly affected. In our control group we did not document a significant relationship between age at death and number of SP+ neurons in these tegmental nuclei or between age at death and number of pigmented neurons in the locus coeruleus. In contrast, in patients with Parkinson's disease, there was a strong inverse relationship between age at death and numbers of SP+ and pigmented neurons. Our findings suggest an interaction between the pathophysiological mechanisms initiated by Parkinson's disease and other processes related to ageing. Since tegmental SP+ neurons are affected by the primary pathological processes underlying Parkinson's disease as severely as catecholamine-synthesizing neurons are affected, theories of pathogenesis and therapeutic strategies in Parkinson's disease will need to take into account the involvement of these SP+ neurons.

Systematic glutamate induces degeneration of a subpopulation of tyrosine hydroxylase-immunoreactive neurons in the rat area postrema

Neuronal damage in the area postrema (AP) of 12–14-week-old male rats was induced by subcutaneous administration of monosodium glutamate (MSG). An immunocytochemical method was used to visualize catecholaminergic neurons in the AP after MSG-treatment. Some tyrosine hydroxylase-immunoreactive neurons exhibited marked signs of degeneration, while others appeared undamaged. We conclude that catecholamine-synthesizing neurons in the AP are differentially sensitive to the neuroexcitotoxic effect of systemic glutamate.

PNMT-containing neurons in the rostral medulla oblongata (C1, C3 groups) are transneuronally labelled after injection of herpes simplex virus type I into the adrenal gland

Herpes simplex virus type 1 (HSV1) was injected into the rat adrenal gland. After 3 days the rat CNS was processed immunohistochemically to demonstrate viral antigen. In the lower thoracic spinal cord viral antigen was found in neurons in the intermediolateral column. In the medulla oblongata HSV1-positive neurons were found in the raphe pallidus and in the C1 and C3 regions of the rostral medulla. Approximately 50% of HSV1-positive neurons in the C1 and all the HSV1-positive neurons in the C3 area also contained phenylethanolamine N-methyltransferase (PNMT) and were thus identified as C1 and C3 cells. The HSV1-positive neurons in the C1 region which did not contain PNMT were also negative for tyrosine hydroxylase and were therefore not catecholamine-synthesizing neurons. The HSV1-positive neurons in the medulla oblongata were presumably transsynaptically labelled from the adrenal gland and our study therefore provides neuroanatomical evidence supporting the view that some C1 neurons are involved in controlling the function of the adrenal gland.

Distribution of monoamine-synthesizing neurons in the human medulla oblongata.

We have employed immunohistochemical and morphometric procedures to study the distribution of monoamine-synthesizing neurons in the medulla oblongata of the adult human, utilizing antibodies to tyrosine hydroxylase (TH), phenylethanolamine N-methyltransferase (PNMT), and phenylalanine hydroxylase (PH8). In the human brain, the antigen with which PH8 reacts occurs within neurons that presumably synthesize serotonin (Haan et al., '87). Neurons containing these antigens were mapped and counted in successive coronal sections with the aid of a computer-assisted procedure. The results indicate that monoamine-synthesizing neurons are distributed in the human brain in patterns broadly similar to those described for other species. TH-immunoreactive cells extended caudorostrally for approximately 32 mm commencing at the spinomedullary junction and ending 8 mm caudal to the pontomedullary junction. In coronal sections these TH-immunoreactive neurons were seen in the lateral medulla dorsal to the inferior olive extending in a continuous band to the dorsomedial medulla. Above the obex the majority of these cells apparently synthesize adrenaline since many PNMT-immunoreactive cells were also found in this region. There were few or no PNMT-immunoreactive cells caudal to the obex, indicating that the TH-immunoreactive cells in this region synthesize either noradrenaline or dopamine. Approximately 65% of these TH-immunoreactive neurons contained melanin pigment, whereas few or no PNMT-immunoreactive cells contained melanin pigment. PH8-immunoreactive cells extended throughout the rostrocaudal extent of the medulla oblongata (approximately 40 mm). In coronal sections the majority were found in the medullary raphe nuclei. However, many cells throughout the rostrocaudal extent of the medulla were found laterally intermingled with catecholamine-synthesizing neurons. Occasional neurons in the lateral medulla appeared to contain both PH8- and TH-immunoreactivity.

Neurons in the area postrema are the only catecholamine-synthesizing cells in the medulla or pons with projections to the rostral ventrolateral medulla (C 1-area) in the rabbit

We have identified, in the rabbit medulla and pons, neurons which project to the C 1-region of the rostral ventrolateral medulla. By combining tyrosine hydroxylase immunohistochemistry with retrograde transport of Fluoro-Gold we determined whether any of the retrogradely labelled neurons synthesize catecholamines. The only doubly labelled cells were located in the area postrema. No other group of catecholamine-synthesizing neurons in either the medulla or the pons was found to project to the C 1-area of the rostral ventrolateral medulla. Pharmacological agents which lower arterial pressure by stimulating adrenoceptors in the rostral ventrolateral medulla may act on receptors which are not innervated by catecholamine-synthesizing perikarya located outside the C 1-region.

Distribution of tyrosine hydroxylase and neuropeptide Y–like immunoreactive neurons in rabbit medulla oblongata, with attention to colocalization studies, presumptive adrenaline‐synthesizing perikarya, and vagal preganglionic cells

We studied the distribution, within the rabbit medulla oblongata, of neuronal cell bodies containing either tyrosine hydroxylase or neuropeptide Y-like immunoreactivity. Both avidin-biotin and immunofluorescence procedures were used. Because the two primary antibodies were raised in different species it was possible to perform simultaneous colocalization studies with the immunofluorescence procedure. Tyrosine hydroxylase-containing neurons in the rostral medulla were demonstrated to contain a catecholamine by the colchicine-enhanced FAGLU (formaldehyde-glutaraldehyde) fluorescence histochemical procedure. These neurons are presumably adrenergic, corresponding to the C1 and C2 groups described in the rat. No C3 group was found in the rabbit. The distribution of tyrosine hydroxylase-containing neurons in the caudal medulla was in accordance with previous descriptions of the A1 and A2 groups based on the unenhanced FAGLU procedure. Neuropeptide Y-like immunoreactivity was observed in cell groups corresponding to those already described in the rat, but additional groups were discovered in the rabbit. Some neurons containing neuropeptide Y-like immunoreactivity were observed in nucleus raphe pallidus and these also contained serotonin (5-HT). In the nearby nucleus reticularis gigantocellularis there were occasional neurons that contained neuropeptide Y-like immunoreactivity without any colocalized 5-HT. Neuropeptide Y-like immunoreactivity was also observed in the dorsal motor nucleus of the vagus, rostral to the obex, and these neurons were demonstrated to be true vagal preganglionic cells by colocalization of neuropeptide Y-like immunoreactivity and Fast Blue retrogradely transported from the cervical vagus. We found that neuropeptide Y-like immunoreactivity was colocalized in approximately 75% of the tyrosine hydroxylase-containing neurons in the rostral medulla (C1 and C2 cells). A smaller proportion of the A1 cells also contained this peptide but it was absent from both the most caudal A1 cells and from the A2 cells. Some tyrosine hydroxylase-containing neurons occur in direct apposition to vagal preganglionic cells in both the dorsal motor nucleus of the vagus and the nucleus ambiguous. However, colocalization studies revealed that none of these neurons contained Fast Blue when this dye was retrogradely transported from the cervical vagus. Medullary catecholamine-synthesizing neurons apparently do not contribute axons to the vagus nerve. This finding is consistent with our own studies in the rat but is in contrast to studies in this species published by other workers.

Evidence that catecholamine-synthesizing perikarya in rat medulla oblongata do not contribute axons to the vagus nerve

We attempted to confirm reports that medullary catecholamine-synthesizing neurons in the rat contribute axons to the vagus nerve. Vagal preganglionic neurons in the medulla were identified by the retrograde intra-axonal transport of Fast Blue from the cervical vagus. Catecholamine-synthesizing neurons were identified using a specific antibody against tyrosine hydroxylase. A rhodamine-labelled second antibody was used to ensure that Fast Blue and tyrosine hydroxylase could be viewed entirely independently. We did not find any medullary neurons which contained both tyrosine hydroxylase and Fast Blue. Although further investigations by other laboratories are necessary, we believe that previous studies, using punctate versus diffuse horseradish peroxidase staining to doubly label neurons may have produced false positive results.