Tyrosine Hydroxylase Labeling Research Articles

Immunohistochemical Study of Human Mitochondrial Ferritin in the Substantia Nigra Following Subarachnoid Hemorrhage.

Mitochondrial ferritin (FtMt) is a novel ferritin that sequesters iron and plays a protective role against oxidative stress. FtMt shares a high homology with H-ferritin but is expressed only in the brain, heart, and testis. In the midbrain, FtMt expression is observed in the substantia nigra. FtMt plays a neuroprotective role in the pathology of neurodegenerative diseases such as Parkinson's disease, where excessive iron induces oxidative stress, causing cell death. Herein, we investigated FtMt immunoreactivity in the brains of patients with subarachnoid hemorrhage (SAH). Double immunofluorescence labeling of tyrosine hydroxylase (TH) and FtMt showed high colocalization in the substantia nigra pars compacta (SNc) in control and SAH cases. However, in SAH cases, FtMt immunoreactivity was observed in some TH-negative neurons. Double immunofluorescence labeling of glial cell markers and FtMt showed no apparent colocalization. The number and ratio of FtMt-positive but TH-negative neurons significantly differed between the control and SAH groups. Prussian blue staining in SAH cases showed positive iron staining over a wide surface range and the substantia nigra. Thus, FtMt may be related to iron dynamics in the substantia nigra following subarachnoid hemorrhage.

133 Anatomical evidence for kisspeptin and dopaminergic neuron interaction in the diestrous mare

Dopamine (DA), via dopaminergic neurons, modulates prolactin secretion from the pituitary by tonic inhibition of lactotropes. Previously, kisspeptin (Kp) attenuated the prolactin response to the DA antagonist, sulpiride, in diestrous mares and enhanced the prolactin response to sulpiride in estradiol-primed mares. Whether or not Kp can modulate prolactin by acting on DA neurons has yet to be described in horses; therefore, the objective of this study was to determine if Kp and kisspeptin receptor (Kiss1r) co-localize with DA neurons, thus providing evidence for a direct action of Kp on DA neurons. Hypothalami from 4 light-horse diestrous mares, aged 6 to 21, were obtained following euthanasia. Mares were determined to be diestrous based on presence of corpora lutea and progesterone concentrations. Immediately following euthanasia, the carotid artery was perfused with sodium nitrite and 4% paraformaldehyde before decapitation and dissection of hypothalami. Immunofluorescent double labeling of tyrosine hydroxylase (TH) and Kp, as well as TH and Kiss1r, was carried out on paraffin-embedded hypothalamic sections using validated antibodies. Whole slide scans were analyzed for double-labeled cells; percentage of DA neurons colocalized with Kp-ir cell bodies and fibers, as well as percentage of DA neurons colocalized with Kiss1r-ir cells, were calculated and averaged for multiple sectionsper horse from rostral to caudal throughout the hypothalamus. Similarly, percentage and number of Kp or Kiss1r-ir cell bodies co-colocalized with TH-ir cells were calculated for different hypothalamic regions: the arcuate nucleus (Arc), ventromedial nucleus (VMH), and dorsomedial nucleus (DMH), and compared using Kruskall Wallis with Dunn's multiple comparison test. DA neurons were observed lining the third ventricle and spread from the Arc to the DMH, with little to none present in the posterior nucleus. TH and Kp expressing cell bodies co-localized between 8 and 30%, with less in the caudal DMH (P ≤ 0.05) compared with the Arc. Kp fibers synapsed with TH-expressing cell bodies at a low percentage, between 0 and 16%, with less connections in the DMH (P ≤ 0.05) than in the ARC. TH and Kiss1r co-localized between 20 and 65%, with similar distribution throughout the Arc, VMH, and DMH in rostral sections. In caudal hypothalamic sections, co-localization was greater (P ≤ 0.05) in the Arc compared with other areas that express lower or no DA neurons. Co-localization of TH and Kiss1r as well as co-localization and synaptic attachment of Kp on TH positive cells suggests a direct effect of kisspeptin on DA neurons to modulate prolactin release.

Distribution of the transcription factor islet-1 in the central nervous system of nonteleost actinopterygian fish: Relationship with cholinergic and catecholaminergic systems.

Islet-1 (Isl1) is one of the most conserved transcription factors in the evolution of vertebrates, due to its continuing involvement in such important functions as the differentiation of motoneurons, among other essential roles in cell fate in the forebrain. Although its functions are thought to be similar in all vertebrates, the knowledge about the conservation of its expression pattern in the central nervous system goes as far as teleosts, leaving the basal groups of actinopterygian fishes overlooked, despite their important phylogenetic position. In order to assess the extent of its conservation among vertebrates, we studied its expression pattern in the central nervous system of selected nonteleost actinopterygian fishes. By means of immunohistochemical techniques, we analyzed the Isl1 expression in the brain, spinal cord, and sensory ganglia of the cranial nerves of young adult specimens of the cladistian species Polypterus senegalus and Erpetoichthys calabaricus, the chondrostean Acipenser ruthenus, and the holostean Lepisosteus oculatus. We also detected the presence of the transcription factor Orthopedia and the enzymes tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT) to better locate all the immunoreactive structures in the different brain areas and to reveal the possible coexpression with Isl1. Numerous conserved features in the expression pattern of Isl1 were observed in these groups of fishes, such as populations of cells in the subpallial nuclei, preoptic area, subparaventricular and tuberal hypothalamic regions, prethalamus, epiphysis, cranial motor nuclei and sensory ganglia of the cranial nerves, and the ventral horn of the spinal cord. Double labeling of TH and Isl1 was observed in cells of the preoptic area, the subparaventricular and tuberal hypothalamic regions, and the prethalamus, while virtually all motoneurons in the hindbrain and the spinal cord coexpressed ChAT and Isl1. Altogether, these results show the high degree of conservation of the expression pattern of the transcription factor Isl1, not only among fish, but in the subsequent evolution of vertebrates.

Catecholaminergic axon innervation and morphology in flat-mounts of atria and ventricles of mice.

Sympathetic efferent axons regulate cardiac functions. However, the topographical distribution and morphology of cardiac sympathetic efferent axons remain insufficiently characterized due to the technical challenges involved in immunohistochemical labeling of the thick walls of the whole heart. In this study, flat-mounts of the left and right atria and ventricles of FVB mice were immunolabeled for tyrosine hydroxylase (TH), a marker of sympathetic nerves. Atrial and ventricular flat-mounts were scanned using a confocal microscope to construct montages. We found (1) In the atria: A few large TH-immunoreactive (IR) axon bundles entered both atria, branched into small bundles and then single axons that eventually formed very dense terminal networks in the epicardium, myocardium and inlet regions of great vessels to the atria. Varicose TH-IR axons formed close contact with cardiomyocytes, vessels, and adipocytes. Multiple intrinsic cardiac ganglia (ICG) were identified in the epicardium of both atria, and a subpopulation of the neurons in the ICG were TH-IR. Most TH-IR axons in bundles traveled through ICG before forming dense varicose terminal networks in cardiomyocytes. We did not observe varicose TH-IR terminalsencircling ICG neurons. (2) In the left and right ventricles and interventricular septum: TH-IR axons formed dense terminal networks in the epicardium, myocardium, andvasculature. Collectively, TH labeling is achievable in flat-mounts of thick cardiac walls, enabling detailed mapping of catecholaminergic axons and terminal structures in the whole heart at single-cell/axon/varicosity scale. This approach provides a foundation for future quantification of the topographical organization of the cardiac sympathetic innervation in different pathological conditions.

Prior aerobic physical training modulates neuropeptide expression and central thermoregulation after ovariectomy in the rat

This study compared the effects of aerobic physical training and estradiol (E2) replacement on central pathways involved with thermoregulation in ovariectomized rats. Rats were assigned to untrained ovariectomized treated with placebo (UN-OVX), untrained ovariectomized treated with E2 (E2-OVX), and trained ovariectomized (TR-OVX) groups. Tail skin temperature (TST), internal temperature (Tint), and basal oxygen consumption (VO2) were recorded. Neuronal activity, brain expression of Kiss1, NKB and Prodyn, and central norepinephrine (NE) levels were measured. UN-OVX had the highest TST. Compared to UN-OVX rats, TR-OVX and E2-OVX had lower Fos expression in the paraventricular and arcuate (ARC) nuclei, and lower double labeling for Tyrosine Hydroxylase and Fos in the brainstem. Compared to UN-OVX, only TR-OVX group exhibited lower kisspeptin (Kiss1), neurokinin B (NKB), and prodynorphin expression in the ARC and higher central NE levels. Aerobic physical training before menopause may prevent the heat dissipation imbalance induced by reduction of E2, through central NE release, modulation of Kiss1, NKB and prodynorphin expression in neurons from ARC nucleus.

Preventive Vitamin A Supplementation Improves Striatal Function in 6-Hydroxydopamine Hemiparkinsonian Rats.

BackgroundThe mechanisms leading to a loss of dopaminergic (DA) neurons from the substantia nigra pars compacta (SNc) in Parkinson's disease (PD) have multifactorial origins. In this context, nutrition is currently investigated as a modifiable environmental factor for the prevention of PD. In particular, initial studies revealed the deleterious consequences of vitamin A signaling failure on dopamine-related motor behaviors. However, the potential of vitamin A supplementation itself to prevent neurodegeneration has not been established yet.ObjectiveThe hypothesis tested in this study is that preventive vitamin A supplementation can protect DA neurons in a rat model of PD.MethodsThe impact of a 5-week preventive supplementation with vitamin A (20 IU/g of diet) was measured on motor and neurobiological alterations induced by 6-hydroxydopamine (6-OHDA) unilateral injections in the striatum of rats. Rotarod, step test and cylinder tests were performed up to 3 weeks after the lesion. Post-mortem analyses (retinol and monoamines dosages, western blots, immunofluorescence) were performed to investigate neurobiological processes.ResultsVitamin A supplementation improved voluntary movements in the cylinder test. In 6-OHDA lesioned rats, a marked decrease of dopamine levels in striatum homogenates was measured. Tyrosine hydroxylase labeling in the SNc and in the striatum was significantly decreased by 6-OHDA injection, without effect of vitamin A. By contrast, vitamin A supplementation increased striatal expression of D2 and RXR receptors in the striatum of 6-OHDA lesioned rats.ConclusionsVitamin A supplementation partially alleviates motor alterations and improved striatal function, revealing a possible beneficial preventive approach for PD.

Immunolabeling Provides Evidence for Subregions in the Songbird Nucleus Accumbens and Suggests a Context-Dependent Role in Song in Male European Starlings (Sturnus vulgaris)

Birdsong is well known for its role in mate attraction during the breeding season. However, many birds, including European starlings (Sturnus vulgaris), also sing outside the breeding season as part of large flocks. Song in a breeding context can be extrinsically rewarded by mate attraction; however, song in nonbreeding flocks, referred to here as gregarious song, results in no obvious extrinsic reward and is proposed to be intrinsically rewarded. The nucleus accumbens (NAC) is a brain region well known to mediate reward and motivation, which suggests it is an ideal candidate to regulate reward associated with gregarious song. The goal of this study was to provide new histochemical information on the songbird NAC and its subregions (rostral pole, core, and shell) and to begin to determine subregion-specific contributions to gregarious song in male starlings. We examined immunolabeling for tyrosine hydroxylase (TH), neurotensin, and enkephalin (ENK) in the NAC. We then examined the extent to which gregarious and sexually motivated song differentially correlated with immunolabeling for the immediate early genes FOS and ZENK in each subdivision of the NAC. We found that TH and ENK labeling within subregions of the starling NAC was generally similar to patterns seen in the core and shell of NACs in mammals and birds. Additionally, we found that gregarious song, but not sexually motivated song, positively correlated with FOS in all NAC subregions. Our observations provide further evidence for distinct subregions within the songbird NAC and suggest the NAC may play an important role in regulating gregarious song in songbirds.

Dopamine-Producing Neurons in Rat Ontogeny: Phenotypic Features Underlying Molecular Mechanisms of Secretion and Regulation

Dopamine (DA), which is synthesized in DA-ergic neurons and so-called monoenzymatic neurons, is involved in the regulation of neuronal differentiation during the critical period of morphogenesis. The aim of the present study was to examine phenotypic features of DA-producing neurons in several brain regions, including the striatum, suprachiasmatic nucleus, and arcuate nucleus, during rat ontogeny starting from embryonic day 18 (E18) to postnatal day 60 (P60). The enzymes of DA synthesis tyrosine hydroxylase (TH) and aromatic amino acid decarboxylase (AAAD), vesicular monoamine transporters (VMAT), and D1 and D2 DA receptors were used as markers for functional activity of DA neurons. Expression of these markers was studied using double immunohistochemical labeling of TH and AAAD or PCR assay of TH, AAAD, VMAT1, VMAT2, and D1 and D2 DA receptors. In the striatum, mRNAs of all mentioned markers were revealed within the whole period of ontogeny studied, bienzymatic TH and AAAD-immunopositive neurons were observed at E18, whereas monoenzymatic TH- or AAAD-containing neurons were observed at P60 only. In the suprachiasmatic nucleus, monoenzymatic AAAD neurons coexist with monoenzymatic TH-immunoreactive fibers, which innervate the ventral part of the nucleus, in the early postnatal period (P10). At P10 in the suprachiasmatic nucleus, the TH, VMAT1, and VMAT2 genes are not expressed; however, transcripts of the D1 and D2 genes were detected. The arcuate nucleus of rat embryos contained only monoenzymatic TH neurons in the ventrolateral area and AAAD neurons in the dorsomedial area in the late prenatal period (E21). At the same time TH, AAAD, VMAT2, D1, and D2 mRNAs were expressed in both parts of the arcuate nucleus. Thus, during rat ontogeny, nondopaminergic neurons containing one of the enzymes, TH or AAAD, are present in the striatum, suprachiasmatic nucleus, and arcuate nucleus, which allows for suggesting the cooperative synthesis of dopamine in these brain structures.

Pulmonary Vein Ganglia Are Remodeled in the Diabetic Heart.

BackgroundCardiac autonomic neuropathy is thought to cause adverse cardiovascular effects in diabetes mellitus. Pulmonary vein ganglia (PVG), which have been implicated in normal and abnormal heart rhythm regulation, have not been fully investigated in type 1 diabetes mellitus (T1D). We examined the functional and anatomical effects of T1D on PVG and studied the details of T1D‐induced remodeling on the PVG structure and function.Methods and ResultsWe used a mouse model of T1D (Akita mouse), immunofluorescence, isolated Langendorff‐perfused hearts, and mathematical simulations to explore the effects of T1D on PVG. Whole‐mount atrial immunofluorescence of choline acetyltransferase and tyrosine hydroxylase labeling showed that sympathetic and parasympathetic somas of the PVG neurons were significantly hypotrophied in T1D hearts versus wild type. Stimulation of PVG in isolated Langendorff‐perfused hearts caused more pronounced P‐P interval prolongation in wild type compared with Akita hearts. Propranolol resulted in a comparable P‐P prolongation in both phenotypes, and atropine led to more pronounced P‐P interval shortening in wild type compared with Akita hearts. Numerical modeling using network simulations revealed that a decrease in the sympathetic and parasympathetic activities of PVG in T1D could explain the experimental results.ConclusionsT1D leads to PVG remodeling with hypotrophy of sympathetic and parasympathetic cell bodies and a concomitant decrease in the PVG sympathetic and parasympathetic activities.

Intravascular imaging, histopathological analysis, and catecholamine quantification following catheter-based renal denervation in a swine model: the impact of prebifurcation energy delivery.

The purpose of this study was to evaluate the impact of prebifurcation renal denervation in a swine model and assess its safety through optical coherence tomography (OCT). Prebifurcation renal denervation with a multi-electrode catheter was performed in one renal artery of 12 healthy pigs, with the contralateral artery and kidney being used as controls. Angiograms and OCT pullbacks were obtained peri-procedurally and 1 month post procedure. Renal tissue catecholamines were quantified, and the arterial wall and peri-adventitial tissue were analyzed histologically. Intraluminal changes (endothelial swelling, spasm, and thrombus formation) were observed acutely by OCT in most of the treated arteries and were no longer visible at follow-up. Histology revealed a statistically significant accumulation of collagen (fibrosis) and a near absence of tyrosine hydroxylase labeling in the denervated artery, suggesting a clear reduction in nervous terminals. Renal tissue catecholamine levels were similar between both sides, probably due to the low number of ablation points and the renorenal reflex. The present study demonstrates that renal denervation is associated with acute intimal disruptions, areas of fibrosis, and a reduction in nervous terminals. The lack of difference in renal tissue catecholamine levels is indicative of the need to perform the highest and safest number of ablation points in both renal arteries. These findings are important because they demonstrate the histological consequences of radiofrequency energy application and its medium-term safety.

RENAL SYMPATHETIC AND SENSORY NERVES DO NOT DRIVE HYPERTENSION IN A POLYCYSTIC KIDNEY DISEASE RAT MODEL

Objective: Catheter-based renal denervation (RDN) is used to treat hypertension however recent clinical trials have cast doubt on previously reported effects. This study aims to determine if RDN improves systolic blood pressure, renal function and autonomic dysfunction in a hypertensive rodent model due to polycystic kidney disease (PKD).Design and method: Lewis polycystic kidney rats (LPK) underwent total, selective sensory or sham RDN by periaxonal application of phenol, capsaicin or normal saline, respectively, at 6-weeks-old. Animals then underwent 1) euthanasia at 7-weeks-old, 10-weeks-old or 14-weeks-old or 2) repeat procedure at 10-weeks-old and euthanasia at 14-weeks-old. Blood pressure was measured by radiotelemetry and treatment efficacy determined using renal immunohistochemistry for sensory [calcitonin gene-related peptide [(CGRP)] and sympathetic nerve [tyrosine hydroxylase (TH)] markers. Renal function was determined by plasma creatinine and urea. Autonomic function was assessed using baroreflex sensitivity (BRS at low frequency, αLF and high frequency, αHF). Results: Cortical TH labelling was significantly depleted at 1-week, but returned to ∼23% and ∼32% of control levels by 4-week and 8-week post-total RDN respectively. Pelvic CGRP labelling was largely abolished by 1-week, but returned to ∼43% and ∼80% of control levels by 4-week and 8-week post-total RDN, and to 67% and 97% by 4-week and 8-week post-sensory RDN. Neither total RDN nor sensory RDN reduced SBP in the LPK rats (total vs. sensory vs. sham, 202 ± 22 vs. 213 ± 7 vs. 205 ± 12 mmHg, P = 0.5) between 7–10 weeks. Repeat total RDN did not have any additional impact on SBP compared to a single procedure or shams between 11–14 weeks (P = 0.48). Neither total nor sensory RDN affected αLF (total vs. sensory vs. sham, 1.1 ± 0.1 vs. 1.1 ± 0.0 vs. 1.2 ± 0.0 ms/mmHg, P = 0.6) or αHF (total vs. sensory vs. sham, 2.2 ± 0.2 vs. 1.8 ± 0.1 vs. 2.1 ± 0.1 ms/mmHg, P = 0.1) between 7–10 weeks. Between 11–14 weeks, there was no significant difference in BRS between all groups. Neither procedure worsened plasma creatinine or urea. Conclusions: We conclude that renal sympathetic and sensory nerves do not drive hypertension or autonomic dysfunction in the LPK model of PKD. Furthermore, PKD patients as a cohort might not benefit from the RDN procedure.

Vulnerability to a Metabolic Challenge Following Perinatal Asphyxia Evaluated by Organotypic Cultures: Neonatal Nicotinamide Treatment.

The hypothesis of enhanced vulnerability following perinatal asphyxia was investigated with a protocol combining in vivo and in vitro experiments. Asphyxia-exposed (AS) (by 21min water immersion of foetuses containing uterine horns) and caesarean-delivered control (CS) rat neonates were used at P2-3 for preparing triple organotypic cultures (substantia nigra, neostriatum and neocortex). At DIV 18, cultures were exposed to different concentrations of H2O2 (0.25-45mM), added to the culture medium for 18h. After a 48-h recovery period, the cultures were either assessed for cell viability or for neurochemical phenotype by confocal microscopy. Energy metabolism (ADP/ATP ratio), oxidative stress (GSH/GSSG) and a modified ferric reducing/antioxidant power assay were applied to homogenates of parallel culture series. In CS cultures, the number of dying cells was similar in substantia nigra, neostriatum and neocortex, but it was several times increased in AS cultures evaluated under the same conditions. A H2O2 challenge led to a concentration-dependent increase in cell death (>fourfold after 0.25mM of H2O2) in CS cultures. In AS cultures, a significant increase in cell death was only observed after 0.5mM of H2O2. At higher than 1mM of H2O2 (up to 45mM), cell death increased several times in all cultures, but the effect was still more prominent in CS than in AS cultures. The cell phenotype of dying/alive cells was investigated in formalin-fixed cultures exposed to 0 or 1mM of H2O2, co-labelling for TUNEL (apoptosis), MAP-2 (neuronal phenotype), GFAP (astroglial phenotype) and TH (tyrosine hydroxylase; for dopamine phenotype), counterstaining for DAPI (nuclear staining), also evaluating the effect of a single dose of nicotinamide (0.8nmol/kg, i.p. injected in 100μL, 60min after delivery). Perinatal asphyxia produced a significant increase in the number of DAPI/TUNEL cells/mm3, in substantia nigra and neostriatum. One millimolar of H202 increased the number of DAPI/TUNEL cells/mm3 by ≈twofold in all regions of CS and AS cultures, an effect that was prevented by neonatal nicotinamide treatment. In substantia nigra, the number of MAP-2/TH-positive cells/mm3 was decreased in AS compared to CS cultures, also by 1mM of H202, both in CS and AS cultures, prevented by nicotinamide. In agreement, the number of MAP-2/TUNEL-positive cells/mm3 was increased by 1mM H2O2, both in CS (twofold) and AS (threefold) cultures, prevented by nicotinamide. The number of MAP-2/TH/TUNEL-positive cells/mm3 was only increased in CS (>threefold), but not in AS (1.3-fold) cultures. No TH labelling was observed in neostriatum, but 1mM of H2O2 produced a strong increase in the number of MAP-2/TUNEL-positive cells/mm3, both in CS (>2.9-fold) and AS (>fourfold), decreased by nicotinamide. In neocortex, H2O2 increased the number of MAP-2/TUNEL-positive cells/mm3, both in CS and AS cultures (≈threefold), decreased by nicotinamide. The ADP/ATP ratio was increased in AS culture homogenates (>sixfold), compared to CS homogenates, increased by 1mM of H202, both in CS and AS homogenates. The GSH/GSSG ratio was significantly decreased in AS, compared to CS cultures. One millimolar of H2O2 decreased that ratio in CS and AS homogenates. The present results demonstrate that perinatal asphyxia induces long-term changes in metabolic pathways related to energy and oxidative stress, priming cell vulnerability with both neuronal and glial phenotype. The observed effects were region dependent, being the substantia nigra particularly prone to cell death. Nicotinamide administration in vivo prevented the deleterious effects observed after perinatal asphyxia in vitro, a suitable pharmacological strategy against the deleterious consequences of perinatal asphyxia.

Defining Subpopulations of Arcuate Nucleus GABA Neurons in Male, Female, and Prenatally Androgenized Female Mice

Background/Aims: Arcuate nucleus (ARN) γ-aminobutyric acid (GABA) neurons are implicated in many critical homeostatic mechanisms, from food intake to fertility. To determine the functional relevance of ARN GABA neurons, it is essential to define the neurotransmitters co-expressed with and potentially co-released from ARN GABA neurons. Methods: The present study investigated the expression of markers of specific signaling molecules by ARN GABA neurons in brain sections from male, female, and, in some cases, prenatally androgen-treated (PNA) female, vesicular GABA transporter (VGaT)-ires-Cre/tdTomato reporter mice. Immunofluorescence for kisspeptin, β-endorphin, neuropeptide Y (NPY), tyrosine hydroxylase (TH) and neuronal nitric oxide synthase (nNOS) was detected by confocal microscopy, and co-localization with tdTomato VGaT reporter expression throughout the ARN was quantified. Results: GABA neurons rarely co-localized with kisspeptin (<2%) or β-endorphin (<1%), and only a small proportion of kisspeptin (∼10%) or β-endorphin (∼3%) neurons co-localized with VGaT in male and female mice. In contrast, one-third of ARN GABA neurons co-localized with NPY, and nearly all NPY neurons (>95%) co-localized with VGaT across groups. Both TH and nNOS labeling was co-localized with ∼10% of ARN GABA neurons. The proportion of TH neurons co-localized with VGaT was significantly greater in males than either control or PNA females, and the proportion of nNOS neurons co-localizing VGaT was higher in control and PNA females compared with males. Conclusion: These data highlight NPY as a significant subpopulation of ARN GABA neurons, demonstrate no significant impact of PNA on signal co-expression, and, for the first time, show sexually dimorphic co-expression patterns of TH and nNOS with ARN GABA neurons.

Complex reinnervation pattern after unilateral renal denervation in rats.

Renal denervation (DNX) is a treatment for resistant arterial hypertension. Efferent sympathetic nerves regrow, but reinnervation by renal afferent nerves has only recently been shown in the renal pelvis of rats after unilateral DNX. We examined intrarenal perivascular afferent and sympathetic efferent nerves after unilateral surgical DNX. Tyrosine hydroxylase (TH), CGRP, and smooth muscle actin were identified in kidney sections from 12 Sprague-Dawley rats, to distinguish afferents, efferents, and vasculature. DNX kidneys and nondenervated kidneys were examined 1, 4, and 12 wk after DNX. Tissue levels of CGRP and norepinephrine (NE) were measured with ELISA and mass spectrometry, respectively. DNX decreased TH and CGRP labeling by 90% and 95%, respectively (P < 0.05) within 1 wk. After 12 wk TH and CGRP labeling returned to baseline with a shift toward afferent innervation (P < 0.05). Nondenervated kidneys showed a doubling of both labels within 12 wk (P < 0.05). CGRP content decreased by 72% [3.2 ± 0.3 vs. 0.9 ± 0.2 ng/gkidney; P < 0.05] and NA by 78% [1.1 ± 0.1 vs. 0.2 ± 0.1 pmol/mgkidney; P < 0.05] 1 wk after DNX. After 12 wk, CGRP, but not NE, content in DNX kidneys was fully recovered, with no changes in the nondenervated kidneys. The use of phenol in the DNX procedure did not influence this result. We found morphological reinnervation and transmitter recovery of afferents within 12 wk after DNX. Despite morphological evidence of sympathetic regrowth, NE content did not fully recover. These results suggest a long-term net surplus of afferent influence on the DNX kidney may be contributing to the blood pressure lowering effect of DNX.

Undifferentiated Catecholaminergic and NO-Producing Cells of Forebrain Matrix Zones and Intercellular Relationships in Periventricular Diencephalon of Juvenile Oncorhynchus masou

Localization of TH- and NO-producing systems in the diencephalon of juvenile masu salmon Oncorhynchus masou was investigated by using histofluorescence labeling of glyoxylic acid (GA), and ICH labeling of tyrosine hydroxylase (TH) and neuronal nitric oxide synthase (nNOS). High density distribution of catecholaminergic and NO-ergic cells has been found in preoptic, posterior tuberal and hypothalamic areas. Cells revealed in above mentioned diencephalic areas were assigned to three main types: pear-shaped, bipolar and rounded. Most of the TH- and NO-producing cells had the phenotype of undifferentiated elements localized on territory of diencephalic matrix zones. Paracrine and autocrine relationships between TH- and NO-producing cells in periventricular diencephalon of juvenile O. masou before formation of blood-brain barrier have been hypothesized. The lack of differentiated cells and the presence of a large variety of size groups of cells indicate a possible heterochrony in growth and differentiation of O. masou diencephalic areas. Location of catecholaminergic and NO-producing cells in the territory of diencephalic matrix zones suggests that catecholamines and nitric oxide are involved in the regulation of post-embryonic neurogenesis in diencephalon of O. masou.

Mapping dopaminergic deficiencies in the substantia nigra/ventral tegmental area in schizophrenia.

Previous work from our laboratory showed deficits in tyrosine hydroxylase protein expression within the substantia nigra/ventral tegmental area (SN/VTA) in schizophrenia. However, little is known about the nature and specific location of these deficits within the SN/VTA. The present study had two aims: (1) test if tyrosine hydroxylase deficits could be explained as the result of neuronal loss; (2) assess if deficits in tyrosine hydroxylase are sub-region specific within the SN/VTA, and thus, could affect specific dopaminergic pathways. To achieve these objectives: (1) we obtained estimates of the number of dopaminergic neurons, total number of neurons, and their ratio in matched SN/VTA schizophrenia and control samples; (2) we performed a qualitative assessment in SN/VTA schizophrenia and control matched samples that were processed simultaneously for tyrosine hydroxylase immunohistochemistry. We did not find any significant differences in the total number of neurons, dopaminergic neurons, or their ratio. Our qualitative study of TH expression showed a conspicuous decrease in labeling of neuronal processes and cell bodies within the SN/VTA, which was sub-region specific. Dorsal diencephalic dopaminergic populations of the SN/VTA presented the most conspicuous decrease in TH labeling. These data support the existence of pathway-specific dopaminergic deficits that would affect the dopamine input to the cortex without significant neuronal loss. Interestingly, these findings support earlier reports of decreases in tyrosine hydroxylase labeling in the target areas for this dopaminergic input in the prefrontal and entorhinal cortex. Finally, our findings support that tyrosine hydroxylase deficits could contribute to the hypodopaminergic state observed in cortical areas in schizophrenia.

Neuroanatomy of the murine cardiac conduction system

The mouse heart is a popular model to study the function and autonomic control of the specialized cardiac conduction system (CCS). However, the precise identity and anatomical distribution of the intrinsic cardiac nerves that modulate the function of the mouse CCS have not been adequately studied. We aimed at determining the organization and distribution of the intrinsic cardiac nerves that supply the CCS of the mouse. In whole mouse heart preparations, intrinsic neural structures were revealed by histochemical staining for acetylcholinesterase (AChE). Adrenergic, cholinergic and peptidergic neural components were identified, respectively, by immunohistochemical labeling for tyrosine hydroxylase (TH), choline acetyltransferase (ChAT), calcitonin gene related peptide (CGRP), substance P (SP), and protein gene product 9.5 (PGP 9.5). Myocytes of the CCS were identified by immunolabeling of hyperpolarization activated cyclic nucleotide-gated potassium channel 4 (HCN4). In addition, the presence of CCS myocytes in atypical locations was verified using fluorescent immunohistochemistry performed on routine paraffin sections. The results demonstrate that four microscopic epicardial nerves orientated toward the sinuatrial nodal (SAN) region derive from both the dorsal right atrial and right ventral nerve subplexuses. The atrioventricular nodal (AVN) region is typically supplied by a single intrinsic nerve derived from the left dorsal nerve subplexus at the posterior interatrial groove. SAN myocytes positive for HCN4 were widely distributed both on the medial, anterior, lateral and even posterior sides of the root of the right cranial (superior caval) vein. The distribution of HCN4-positive myocytes in the AVN region was also wider than previously considered. HCN4-positive cells and thin slivers of the AVN extended to the roots of the ascending aorta, posteriorly to the orifice of the coronary sinus, and even along both atrioventricular rings. Notwithstanding the fact that cholinergic nerve fibers and axons clearly predominate in the mouse CCS, adrenergic nerve fibers and axons are abundant therein as well. Altogether, these results provide new insight into the anatomical basis of the neural control of the mouse CCS.

Behavioral indices of breeding readiness in female European starlings correlate with immunolabeling for catecholamine markers in brain areas involved in sexual motivation

In seasonally-breeding songbirds, lengthening photoperiod, increases in estradiol and exposure to male courtship facilitate breeding behavior in females in spring. However, there is extreme variability in the extent to which spring-condition females are attracted by male courtship or engage in nesting behavior. Here we explore possible links between catecholamines and individual differences in behaviors indicative of breeding readiness. Female European starlings were placed in conditions typical of the breeding season (spring-like) or the non-breeding season (fall-like). Although many females examined nesting locations, only a subset of spring-like females occupied nest sites. Labeling for dopamine-beta-hydroxylase (DBH; the enzyme involved in norepinephrine synthesis) in the ventromedial nucleus of the hypothalamus (VMH) was densest in females that acquired nest sites compared to spring-like females without nest sites or fall-like females. Within the group of spring-like females, nesting behaviors correlated positively with DBH labeling in VMH. Females with nest sites had the lowest density of DBH labeling in the ventral tegmental area, and labeling correlated negatively with spring-like female nesting behaviors. Labeling for tyrosine hydroxylase (TH; the rate limiting enzyme for catecholamine synthesis) in putative nucleus accumbens was lowest in spring-like females without nest sites, and labeling correlated positively with nesting behavior in spring-like females. TH labeling density in the medial preoptic nucleus was highest in fall-like females, but a trend was observed for a positive correlation between TH labeling and spring-like female nesting behaviors. These results link distinct patterns of catecholamine activity in brain regions implicated in sexual motivation to female breeding readiness.

Catecholaminergic System of the Medulla Oblongata of the Amur Bitterling (Bony Fishes, Family Cyprinidae)

Catecholaminergic (CA-ergic) systems in the medulla of the Amur bitterling (Rhodeus sericeus) were studied using immunohistochemical labeling of tyrosine hydroxylase (ТН), the main enzyme of catecholamine synthesis. The peculiarities of localization of medullary neurons, morphology of the dendrites, and trajectories of the axon projections in the medulla of the Amur bitterling allow us to differentiate three groups of ТН-positive neurons, namely interfascicular cells, units related to the lobus vagus, and cells localized within the area postrema. Interfascicular medullary neurons form a longitudinal column of multipolar cells with broadly branching dendrites localized on both sides with respect to the midline. Neurons of the second group are smaller than interfascicular cells; their dendrites form branchings within the reticular formation and lobus vagus. On apical parts of the cells of the second group, there are branched processes directed toward the clearance of the cerebral ventricle. The third group consists of bipolar ТН-positive cells; their density of distribution is maximal near the area postrema. Terminal axon fields of medullary CA-ergic neurons in the Amur bitterling are connected with sensory systems of the medulla. In the periventricular region of this cerebral zone, we found phenotypically immature forms of the cells, highly immunopositive processes, and elements of radial glia expressing ТН. We hypothesize that, in fishes, dopamine functions as an inductor of development (morphogenetic factor) and is involved in postembryonal neurogenesis in matrix zones of the brain.

Phosphodiesterase 2 Inhibitors Promote Axonal Outgrowth in Organotypic Slice Co-Cultures

The development of appropriate models assessing the potential of substances for regeneration of neuronal circuits is of great importance. Here, we present procedures to analyze effects of substances on fiber outgrowth based on organotypic slice co-cultures of the nigrostriatal dopaminergic system in combination with biocytin tracing and tyrosine hydroxylase labeling and subsequent automated image quantification. Selected phosphodiesterase inhibitors (PDE-Is) were studied to identify their potential growth-promoting capacities. Immunohistochemical methods were used to visualize developing fibers in the border region between ventral tegmental area/substantia nigra co-cultivated with the striatum as well as the cellular expression of PDE2A and PDE10. The quantification shows a significant increase of fiber density in the border region induced by PDE2-Is (BAY60-7550; ND7001), comparable with the potential of the nerve growth factor and in contrast to PDE10-I (MP-10). Analysis of tyrosine hydroxylase-positive fibers indicated a significant increase after treatment with BAY60-7550 and nerve growth factor in relation to dimethyl sulfoxide. Additionally, a dose-dependent increase of intracellular cGMP levels in response to the applied PDE2-Is in PDE2-transfected HEK293 cells was found. In summary, our findings show that PDE2-Is are able to significantly promote axonal outgrowth in organotypic slice co-cultures, which are a suitable model to assess growth-related effects in neuro(re)generation.