Tyrosine Hydroxylase Innervation Research Articles

Evaluation of the Cardiometabolic Disorders after Spinal Cord Injury in Mice.

Simple SummaryThe present study demonstrates severity-dependent effects of a thoracic T8 injury on cardiometabolic dysfunctions in adult mice. While these chronic cardiometabolic issues can be multifactorial, the data indicate that systemic inflammatory response is likely to be involved. These findings are supportive of the role of systemic inflammation following SCI being critical in identifying therapeutic targets and predicting long-term outcomes.Changes in cardiometabolic functions contribute to increased morbidity and mortality after chronic spinal cord injury. Despite many advancements in discovering SCI-induced pathologies, the cardiometabolic risks and divergences in severity-related responses have yet to be elucidated. Here, we examined the effects of SCI severity on functional recovery and cardiometabolic functions following moderate (50 kdyn) and severe (75 kdyn) contusions in the thoracic-8 (T8) vertebrae in mice using imaging, morphometric, and molecular analyses. Both severities reduced hindlimbs motor functions, body weight (g), and total body fat (%) at all-time points up to 20 weeks post-injury (PI), while only severe SCI reduced the total body lean (%). Severe SCI increased liver echogenicity starting from 12 weeks PI, with an increase in liver fibrosis in both moderate and severe SCI. Severe SCI mice showed a significant reduction in left ventricular internal diameters and LV volume at 20 weeks PI, associated with increased LV ejection fraction as well as cardiac fibrosis. These cardiometabolic dysfunctions were accompanied by changes in the inflammation profile, varying with the severity of the injury, but not in the lipid profile nor cardiac or hepatic tyrosine hydroxylase innervation changes, suggesting that systemic inflammation may be involved in these SCI-induced health complications.

Spatial relationship between the c-Fos distribution and enkephalinergic, substance P, and tyrosine hydroxylase innervation fields after acute treatment with neuroleptics olanzapine, amisulpride, quetiapine, and aripiprazole in the rat septum.

The aim of the present study was to demonstrate the spatial relationship between the c-Fos immunoreactive cells elicited by an acute treatment with neuroleptics including amisulpride (AMI), olanzapine (OLA), quetiapine (QUE), and aripiprazole (ARI) and enkephalinergic (ENK), substance P (SP), and tyrosine hydroxylase (TH) innervation fields in the rat septum. Male Sprague Dawley rats received a single injection of OLA (5 mg), ARI (10 mg), AMI (20 mg), QUE (15 mg/kg/b.w.). Ninety min after antipsychotics administration, the animals were transcardially perfused with a fixative and the brains cryocut into serial coronal sections of 35 µm thickness. The sections were processed for c-Fos staining using an avidin-biotin-peroxidase complex and visualized by nickel intensified diaminobenzidine to reach black endproduct. Afterwards, the sections were exposed to ENK, SP, and TH antibodies and the reaction product visualized by biotin-labeled fluorescent AlexaFluor 564 dye. The data were evaluated from the sections either simultaneously illuminated with fluorescent and transmission microscope beams or after merging the separately illuminated sections in the Adobe Photoshop 7.0 software. ENK, SP, and TH displayed characteristic spatial images formed by a dense accumulation of immunoreactive fibers and terminals on the both sides of the septum. A dense plexus of axons formed by ENK and SP immunopositive terminals were situated predominantly in the lateral, while TH ones more medial portion of the septum. QUE and AMI activated distinct amount of c-Fos expression in cells located within the SP-immunoreactive principal innervation field. The OLA effect on the c-Fos expression was very pronounced in the ventral TH-labeled principal innervation field including the space between the ENK field ventral portion and the dorsal margin of the accumbens nucleus shell. Generally, the occurrence of c-Fos cells in the ENK-immunoreactive principal innervation field, in comparison with the surrounding septal area, was less abundant after all of the four antipsychotics treatments. The data of the present study indicate that ENK, SP, and TH innervation fields may influence separate populations of septal cells activated by AMI, OLA, QUE, and ARI and that each of these region-differently innervated cells may be associated with the functional heterogeneity of the individual lateral septal nuclei.

Innervation of the medial prefrontal cortex by tyrosine hydroxylase immunoreactive fibers during adolescence in male and female rats.

Adolescence is associated with continued maturation of the cerebral cortex, particularly the medial prefrontal cortex (mPFC). We have previously documented pruning in the number of neurons, dendrites, and synapses in the rat mPFC from preadolescence to adulthood, with the period of pubertal onset being particularly important. We hypothesized that dopaminergic innervation of this region, critical for executive functions, would also be influenced by pubertal onset. Here, we measured changes in the volume of tyrosine hydroxylase (TH) immunoreactive axons in all layers of the male and female mPFC from preadolescence to adulthood (postnatal Day (P) 25, 35, 45, 60, and 90) as a marker of dopaminergic innervation. Assessing both total fiber volume and length, TH fibers were quantified by multiplying the mPFC volume by fiber density. While there were subtle layer-specific changes, TH fiber volume and length increased between P25 and P90 in both males and females. Contrary to our hypothesis, a role for pubertal onset in TH innervation of this region was not discernable. In summary, axons immunoreactive for TH increase with similar trajectories in the mPFC of male and female rats from pre-puberty to young adulthood.

Abstract 003: Selective Splenic Denervation Inhibits the Egression of T Cell From Spleen, Induced by Angiotensinii, and Protects From Hypertension

It has been elucidated that immunity plays a role in both etiology and target organ damage of hypertension (HTN) induced by different stimuli (AngII, salt, etc.). In the very last year, we found that AngII activates a neuroimmune pathway, passing through the celiac ganglion (CG). Here, we aimed at dissecting this neuroimmune drive, in order to examine the specific role of splenic sympathetic nerve activity (SSNA) in the onset of HTN. We tested the hypothesis that chronic AngII infusion activates SSNA, in order to recruit T cells. In splenic nerve bundles, we recorded SSNA with a bipolar electrode in mice infused with AngII for 3 days (before BP increase) or with vehicle. SSNA was higher in AngII mice, as compared to vehicle (355±18 vs 141±24 spikes/10min) (p<0.001). We next selectively denervated splenic nerve (SDN) by thermoablation, in order to evaluate whether the AngII-induced increase in SSNA could be detrimental for activation of immunity and onset of HTN. We confirmed the efficacy of SDN by injecting a retrograde neurotracer in the spleen, which was effective in labeling the CG neurons only in sham mice and not in SDN. Furthermore, the tyrosine hydroxylase innervation in the splenic artery of SDN mice was reduced as compared to sham, as well as noradrenaline content in the spleen (sham: 1000±274 vs SDN: 172±44 pg/mg tissue, p<0.01). After 28 days from AngII infusion, SDN mice were protected from HTN as compared to sham (SBP: 107±2 vs 129±3 mmHg) and vehicle treated mice (veh-sham 110±1 vs veh-SDN 106±1 mmHg) (p<0.001). Moreover, SDN hampered the T cell egression from splenic reservoir, evaluated as CD3+ cell content, induced by AngII. In the end, we assessed the amount of T cells infiltration, finding that AngII-SDN mice were significantly protected in aorta (CD8+/mm2: veh-sham 197±17; veh-SDN 143±14; AngII-sham 293±36*; AngII-SDN 156±8) and kidney (CD8+/mm2: veh-sham 40±4; veh-SDN 34±4; AngII-sham 231±27*; AngII-SDN 29±5), *p<0.01 vs all other groups. Similar results were found for CD4+ infiltrates. Our results demonstrate that the neuroimmune drive activated by chronic AngII infusion is mediated by the activation of the SSNA, converging into T cells activation in the spleen and their egression toward target organs, where they contribute to the onset of HTN.

Differential Postnatal Development of Catecholamine and Serotonin Inputs to Identified Neurons in Prefrontal Cortex of Rhesus Monkey

The monoaminergic innervation of cerebral cortex has long been implicated in its development. Methods now exist to examine catecholamine and serotonin inputs to identified neurons in the cerebral cortex. We have quantified such inputs on pyramidal and nonpyramidal cells in prefrontal cortex of rhesus monkeys ranging in age from 2 weeks to 10 years. Individual layer III neurons were filled with Lucifer yellow and double-immunostained with axons containing either tyrosine hydroxylase (TH) or 5-hydroxytryptamine (5-HT). The filled cells were reconstructed, and putative appositions between the axons and dendritic spines and shafts were quantified at high magnification using light microscopy. The density of catecholamine appositions on pyramidal neurons matures slowly, reaching only half the adult level by 6 months of age and thereafter rising gradually to adult levels by 2 years of age. By contrast, the density of serotonin appositions on pyramidal cells reaches the adult level before the second week after birth. The average adult pyramidal neuron in layer III of area 9m receives three times stronger input from catecholaminergic than from serotoninergic axons. The overall density of both inputs to interneurons does not appear to change during postnatal development. Selective changes in the TH innervation of pyramidal cells against a backdrop of constant TH innervation of interneurons suggest that the balance between excitation and inhibition may change developmentally in the prefrontal cortex. By contrast, 5-HT innervation of both types of neurons remains relatively constant over the age range studied.

Regionally selective effects of gonadectomy on cortical catecholamine innervation in adult male rats are most disruptive to afferents in prefrontal cortex.

Changes in gonadal hormones induced early in life produce substantial, seemingly permanent decreases in tyrosine hydroxylase (TH)-immunoreactive axon density in sensory, motor and prefrontal regions in the rat cerebral cortex. Less is known, however, about the responsiveness of cortical catecholamines to hormone stimulation during adulthood. In this study we expanded upon an earlier analysis of the effects of acute (4 day) and chronic (28 day) gonadectomy in adult male rats on TH innervation in right hemifield of the cingulate cortex to include assessment of sensorimotor areas previously examined following perinatal gonadectomy, the left cingulate hemifield, and one additional prefrontal area - the dorsal anterior insular cortex. Qualitative and quantitative analyses of immunoreactivity revealed modest, transient declines in innervation in sensorimotor areas 4 days after gonadectomy, and a return to normal innervation densities by 28 days after surgery. In cingulate and insular cortices, however, strikingly depleted axon densities observed following acute gonadectomy rebounded to significantly higher than normal levels of innervation 3 weeks later. All effects were attenuated in gonadectomized animals supplemented with testosterone. Thus, for cortical catecholamine innervation, as for other endpoints of hormone stimulation, gonadal steroid sensitivity appears to change dramatically with lifestage. In adult male rats, this sensitivity is also marked by a seemingly selective vulnerability of catecholamine innervation in prefrontal areas to changes in the hormone environment induced by gonadectomy.

Afferent and efferent connections of the caudolateral neostriatum in the pigeon (Columba livia): a retro- and anterograde pathway tracing study.

The avian caudolateral neostriatum (NCL) was first identified on the basis of its dense dopaminergic innervation. This fact and data from lesion studies have led to the notion that NCL might be the avian equivalent of prefrontal cortex (PFC). A key feature of the PFC is the ability to integrate information from all modalities needed for the generation of motor plans. By using antero- and retrograde pathway tracing techniques, we investigated the organization of sensory afferents to the NCL and the connections with limbic and somatomotor centers in the basal ganglia and archistriatum. Data from all tracing experiments were compared with the distribution of tyrosine-hydroxylase (TH)-immunoreactive fibers, serving as a marker of dopaminergic innervation. The results show that NCL is reciprocally connected with the secondary sensory areas of all modalities and with at least two parasensory areas. Retrograde tracing also demonstrated further afferents from the deep layers of the Wulst and from the frontolateral neostriatum as well as the sources of thalamic input. Efferents of NCL project onto parts of the avian basal ganglia considered to serve somatomotor or limbic functions. Projections to the archistriatum are mainly directed to the somatomotor part of the intermediate archistriatum. In addition, cells in caudal NCL were found to be connected with the ventral and posterior archistriatum, which are considered avian equivalents of mammalian amygdala. All afferents and projection neurons were confined to the plexus of densest TH innervation. Our results show that the NCL is positioned to amalgamate information from all modalities and to exert control over limbic and somatomotor areas. This organization might comprise the neural basis for such complex behaviours as working memory or spatial orientation.

Differential effects of chemical sympathectomy on expression and activity of tyrosine hydroxylase and levels of catecholamines and DOPA in peripheral tissues of rats.

Tyrosine hydroxylase (TH) mRNA and activity and concentrations of 3,4-dihydroxyphenylalanine (DOPA) and catecholamines were examined as markers of sympathetic innervation and catecholamine synthesis in peripheral tissues of sympathectomized and intact rats. Chemical sympathectomy with 6-hydroxydopamine (6-OHDA) markedly decreased norepinephrine and to a generally lesser extent TH activities and dopamine in most peripheral tissues (stomach, lung, testis, duodenum, pancreas, salivary gland, spleen, heart, kidney, thymus). Superior cervical ganglia, adrenals and descending aorta were unaffected and vas deferens showed a large 92% decrease in norepinephrine, but only a small 38% decrease in TH activity after 6-OHDA. Presence of chromaffin cells or neuronal cell bodies in these latter tissues, indicated by consistent expression of TH mRNA, explained the relative resistance of these tissues to 6-OHDA. Stomach also showed consistent expression of TH mRNA before, but not after 6-OHDA, suggesting that catecholamine synthesizing cells in gastric tissue are sensitive to the toxic effects of 6-OHDA. Tissue concentrations of DOPA were mainly unaffected by 6-OHDA, indicating that much of the DOPA in peripheral tissues is synthesized independently of local TH or sympathetic innervation. The differential effects of chemical sympathectomy on tissue catecholamines, DOPA, TH mRNA and TH activity demonstrate that these variables are not simple markers of sympathetic innervation or catecholamine synthesis. Other factors, including presence of neuronal cell bodies, parenchymal chromaffin cells, non-neuronal sites of catecholamine synthesis and alternative sources of tissue DOPA, must also be considered when tissue catecholamines, DOPA and TH are examined as markers of sympathetic innervation and local catecholamine synthesis.

Intrinsic Organization and Connectivity of Intrastriatal Striatal Transplants in Rats as Revealed by DARPP-32 Immunohistochemistry: Specificity of Connections with the Lesioned Host Brain.

Intrastriatal grafts of tissue obtained from the striatal or neocortical primordia of rat fetuses have been studied with respect to their intrinsic organization and connectivity using antibodies to DARPP-32 in combination with acetylcholinesterase (AChE) histochemistry, tyrosine hydroxylase (TH) immunocytochemistry, and anterograde and retrograde axonal tracing techniques. The striatal grafts were characterized by distinct patches of DARPP-32-immunoreactive neurons, which were identical to the densely AChE-positive patches stained in adjacent sections from the same specimens. The non-patch areas possessed only few DARPP-32-positive neurons and contained only sparse AChE-positive fibres. The cortical grafts, by contrast, contained no neurons with clear-cut DARPP-32-positivity and they exhibited a sparse, evenly distributed AChE fibre network, similar to that seen in the non-patch areas of the striatal grafts. The host dopaminergic afferents, as revealed by TH immunostaining, had grown selectively into the DARPP-32-positive patches in the striatal grafts, where they formed a dense terminal network around the DARPP-32-positive cell bodies. The non-patch areas, as well as the cortical grafts, received only sparse TH innervation. By contrast, the host cortical afferents, labelled by Phaseolus vulgaris leucoagglutinin from the host frontal cortex, were seen to extend into both the patch and non-patch areas of the striatal grafts. Transplant neurons projecting into the host brain were labelled by Fluoro-Gold injections into the ipsilateral host globus pallidus. These injections labelled large numbers of medium-sized neurons within the striatal grafts and the vast majority of them (over 85%) were confined to the DARPP-32-positive patches. Similar Fluoro-Gold injections labelled only few graft neurons in the cortical grafts. The results indicate that the striatal grafts are composed of a mixture of striatal and non-striatal tissue, and that the striatal graft compartment selectively establishes afferent and efferent connections with the host nigro-pallidal system. These graft connections demonstrate a remarkable specificity in the formation of graft - host connectivity. The results, moreover, suggest that developmental properties of the grafted striatal primordium are retained and expressed in the implanted cell suspension, and that the neuronal systems of the lesioned adult host brain, at least to some extent, remain responsive to growth regulating mechanisms normally operating during ontogenetic development.

Ontogeny of the dopamine and cyclic adenosine-3′: 5′ -monophosphate-regulated phosphoprotein (DARPP-32) in the pre- and postnatal mouse central nervous system

The ontogeny of a dopamine and cyclic adenosine-3′:5′-monophosphate-regulated phosphoprotein with an apparent molecular weight of 32 kilodaltons (DARPP-32) has been studied in the central nervous system of the prenatal, newborn and adult mouse.DARPP-32-immunoreactive somata were first identified at day 12 of gestation, in the primary olfactory cortex and in the ventrolateral medulla oblongata. On day 14 of gestation, neurons containing DARPP-32-like immunoreactivity became apparent in the caudate nucleus, olfactory tubercle, nucleus accumbens, frontoparietal cortex and the ventral medulla oblongata. During the period up to and including birth, the number of cell bodies and fibres in all these areas increased markedly. In addition, DARPP-32-positive neurons became visible in the olfactory nucleus, the arcuate nucleus, and DARPP-32-positive cells appeared in the choroid plexus of the lateral, third and fourth ventricles. DARPP-32-containing fibres could be seen in the median eminence, the ventrolateral thalamus, and in the striatonigral projection, descending in the internal capsule to ramify extensively in the substantia nigra. Only in the cerebellum and suprachiasmatic nucleus did the development of DARPP-32-like immunoreactivity occur postnatally.The development of tyrosine hydroxylase, the rate-limiting enzyme for catecholamine synthesis, was simultaneously examined. The arrival of the tyrosine hydroxylase-containing projection to the caudate nucleus, the olfactory tubercle and the nucleus accumbens apparently occurred 1–2 days after the appearance of DARPP-32-immunoreactive cells within these regions. In the ventral and ventrolateral medulla oblongata, and the primary olfactory cortex, no tyrosine hydroxylase innervation was seen near the DARPP-32-positive neurons at days 12–14. The organization of the DARPP-32-containing somata of the caudate nucleus into aggregates of 5–15 neurons was partly paralleled spatially by an increased density of tyrosine hydroxylase-positive fibres.Many DARPP-32-immunoreactive cells in the immature mouse brain are present by the day of birth, particularly in the areas known to receive a dopaminergic innervation. The development of these presumptive dopaminoceptive DARPP-32-containing neurons does not seem to be dependent on the presence, however, of a dopaminergic input, since in all regions examined DARPP-32-LI preceded the appearance of tyrosine hydroxylase-like immunoreactivity by at least 1–2 days. Indeed, the results suggest that the existence of DARPP-32-like immunoreactivity in cell bodies and dendrites may be a pre-requisite for the formation or subsequent stabilization of dopaminergic synapses.