Tyrosine Hydroxylase Labeling Research Articles

Morphofunctional and the Hodological Characteristics of the Ascending Dopaminergic System of the Amur Bitterling (Rhodeus sericeus, Cyprinidae)

Immunofluorescent labeling of tyrosine hydroxylase (TH) combined with labeling of fibers with the carbocyanine stain DiI (1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate) was used to study the distribution of catecholaminergic (CE) neurons and their ascending projections to the brain in Amur bitterlings (Rhodeus sericeus). A highly specialized complex of CE cells was observed in the diencephalon. Six populations of TH-immunopositive neurons were seen in the posterior tuberculum, two of which showed double labeling, evidencing the existence of dopaminergic projections to the ventral part of the telencephalon (striatum). The posterior tuberal area contained two populations of CE cells (small round and large piriform), possibly corresponding to the sensorimotor and limbic cells in mammals.

Morphological pattern of mouse intrinsic cardiac nerve plexus

The study was aimed to determine the 3D organization and immunohistochemistry of intrinsic neural plexus in mouse heart. In a whole heart preparation, intrinsic neural structures were revealed by a histochemical staining for acetylcholinesterase, while the double labeling of tyrosine hydroxylase (TH), choline acetyltransferase (ChAT), substance P (SP) and calcitonin gene related peptide (CGRP) was applied to identify immunohistochemically the distribution of adrenergic, cholinergic and peptidergic neural components, respectively. Our findings demonstrate that extrinsic cardiac nerves access the ganglionated nerve plexus of the mouse heart hilum at the right and left cranial veins. Nerves and bundles of nerve fibers extend epicardially from this plexus to atria and ventricles by 5 neural pathways or subplexuses. Generally, the examined hearts from 56 mice contained 19±3 ganglia, in which 1.082±160 neurons reside. Majority of intrinsic neurons (83%) are ChAT(+), 3% ‐ TH(+); 14% – bifenotypic, i.e. they were immunoreactive both for ChAT and TH. The TH(+) epicardial nerve fibers are predominant on the dorsal and ventral sides of left atrium, whereas the most ChAT(+) axons proceed on the heart base toward the large intrinsic ganglia and within epicardium of the root of the right cranial vein. Peptidergic nerve fibers were abundant in the epicardium and within intrinsic ganglia adjacent to the heart hilum. In conclusion, although majority of nerves and neural bundles in the mouse heart are mixed, some of them express either adrenergic or cholinergic phenotype. This study was supported by the Research Council of Lithuania (No. MIP‐11184).

Neurochemical Characterization of Pterygopalatine Ganglion Branches in Humans

Pterygopalatine ganglion (PPG) branches, seem to be involved in the pathophysiology of facial pain. The functions of these branches, including a recently discovered orbital branch, are not completely known but could be of clinical significance. This study was designed to characterize PPG branches through immunohistochemical stain and study their anatomy, specifically the orbital branches. In a cadaver study of four specimens, the pterygopalatine fossa (PPF) was dissected out of its bony surroundings as a tissue block. Subsequently, cryostat sectioning of these blocks was performed. In one specimen the PPF was microscopically dissected. Recently discovered neural structures were identified, dissected out of the tissue block, and cryosectioned. All cryostat sectionings were immunohistochemically stained for protein gene product (PGP) 9.5, nitric oxide synthase (NOS), and tyrosine hydroxylase (TH). A recently discovered neural connection between the PPG and the ophthalmic nerve could be confirmed and classified as an orbital PPG branch. The connection stained throughout for PGP 9.5 and partially stained for NOS. In other orbital branches, both NOS and TH(+) nerve fibers were found. The PPG contained NOS(+) cells. TH labeling was also found in nerve fibers running through the PPG and the vidian nerve. The recently discovered orbital PPG branch is of a mixed parasympathetic and sensory nature. In the other orbital branches, sympathetic fibers were shown as well. This knowledge may add to understanding the symptomatology and therapies of headache syndromes such as nerve block.

Social affiliation relates to tyrosine hydroxylase immunolabeling in male and female zebra finches (Taeniopygia guttata)

The catecholamines dopamine and norepinephrine are implicated in affiliative behaviors, yet few studies have addressed the extent to which affiliative behaviors within distinct social settings rely upon similar or distinct catecholaminergic mechanisms. To explore the role of catecholamines in affiliative behavior within distinct long-term social contexts, we examined the density of the catecholamine synthetic enzyme tyrosine hydroxylase (TH) in brain regions within both the mesolimbic dopaminergic system and “social behavior network” in male and female zebra finches (Taeniopygia guttata) paired for 21 days with either a same- or opposite-sex conspecific. On days 16–21 after pairing, members of both same- and mixed-sex pairs produced similar rates of affiliative behaviors. Measures of affiliation related to TH labeling in the ventral tegmental area (VTA), nucleus accumbens (Ac), medial preoptic nucleus (POM), and ventromedial nucleus of the hypothalamus (VMH). Relationships between TH labeling density and specific measures of affiliative behavior differed in rostral compared to caudal subregions of Ac and VTA, suggesting distinct roles for these subregions in the regulation of affiliative behavior. Finally, TH labeling density in the VMH and rostral VTA were positively related to the amount of courtship received from the partner and TH labeling in Ac was denser in opposite-sex pairs compared to same-sex pairs, indicative of socially induced brain plasticity. Overall, results highlight a complex region- and behavior-specific role for catecholamines in vertebrate affiliation.

Sexual dimorphism in locus coeruleus dendritic morphology: A structural basis for sex differences in emotional arousal

Stress-related psychiatric disorders, such as depression and anxiety, affect a disproportionate number of women. We previously demonstrated that the major brain norepinephrine (NE)-containing nucleus, locus coeruleus (LC) is more sensitive to stressors and to the stress-related neuropeptide, corticotropin-releasing factor (CRF) in female compared to male rats. Because the LC–NE system is a stress-responsive system that is thought to be dysregulated in affective disorders, sex differences in LC structure or function could play a role in female vulnerability to these diseases. The present study used different approaches to compare LC dendritic characteristics between male and female rats. Immunofluorescence labeling of tyrosine hydroxylase, the norepinephrine synthetic enzyme, revealed that LC dendrites of female rats extend further into the peri-LC region, covering a significantly greater area than those of males. Optical density measurements of dendrites in the peri-LC revealed increased dendritic density in females compared to their male counterparts. Additionally, immunoreactivity for synaptophysin, a synaptic vesicle protein, was significantly greater in the LC in female rats, suggesting an increased number of synaptic contacts onto LC processes. Individual LC neurons were juxtacellularly labeled with neurobiotin in vivo for morphological analysis. LC dendritic trees of females were longer and had more branch points and ends. Consistent with this, Sholl analysis determined that, compared to males, LC dendrites of females had a more complex pattern of branching. The greater dendritic extension and complexity seen in females predicts a higher probability of communication with diverse afferents that terminate in the peri-LC. This may be a structural basis for heightened arousal in females, an effect which may, in part, account for the sex bias in incidence of stress-related psychiatric disorders.

Reliability in the Identification of Midbrain Dopamine Neurons

Brain regions typically contain intermixed subpopulations of neurons with different connectivity and neurotransmitters. This complicates identification of neuronal phenotypes in electrophysiological experiments without using direct detection of unique molecular markers. A prime example of this difficulty is the identification of dopamine (DA) neurons in the midbrain ventral tegmental area (VTA). Although immunocytochemistry (ICC) against tyrosine hydroxylase (TH) is widely used to identify DA neurons, a high false negative rate for TH ICC following ex vivo electrophysiology experiments was recently reported, calling into question the validity of comparing DA and non-DA VTA neurons based on post-hoc ICC. However, in whole cell recordings from randomly selected rat VTA neurons we have found that TH labeling is consistently detected in ∼55% of neurons even after long recording durations (range: 2.5–150 min). This is consistent with our prior anatomical finding that 55% of VTA neurons are TH(+). To directly estimate a false negative rate for our ICC method we recorded VTA neurons from mice in which EGFP production is driven by the TH promoter. All 12 EGFP(+) neurons recorded with a K-gluconate internal solution (as used in our rat recordings) were strongly labeled by TH ICC (recording duration 16.6±1.8 min). However, using recording electrodes with an internal solution with high Cl− concentration reduced the intensity of TH co-labeling, in some cases to background (recording duration 16.7±0.9 min; n = 10). Thus TH is a highly reliable molecular marker for DA neurons in VTA patch clamp recordings provided compatible microelectrode solutions are used.

Morphologic pattern of the intrinsic ganglionated nerve plexus in mouse heart

Both normal and genetically modified mice are excellent models for investigating molecular mechanisms of arrhythmogenic cardiac diseases that may be associated with an imbalance between sympathetic and parasympathetic nervous input to the heart. The purpose of this study was to (1) determine the structural organization of the mouse cardiac neural plexus, (2) identify extrinsic neural sources and their relationship with the cardiac plexus, and (3) reveal any anatomic differences in the cardiac plexus between mouse and other species. Cardiac nerve structures were visualized using histochemical staining for acetylcholinesterase (AChE) on whole heart and thorax-dissected preparations derived from 25 mice. To confirm the reliability of staining parasympathetic and sympathetic neural components in the mouse heart, we applied a histochemical method for AChE and immunohistochemistry for tyrosine hydroxylase (TH) and/or choline acetyltransferase (ChAT) on whole mounts preparations from six mice. Double immunohistochemical labeling of TH and ChAT on AChE-positive neural elements in mouse whole mounts demonstrated equal staining of nerves and ganglia for AChE that were positive for both TH and ChAT. The extrinsic cardiac nerves access the mouse heart at the right and left cranial veins and interblend within the ganglionated nerve plexus of the heart hilum that is persistently localized on the heart base. Nerves and bundles of nerve fibers extend epicardially from this plexus to atria and ventricles by left dorsal, dorsal right atrial, right ventral, and ventral left atrial routes or subplexuses. The right cranial vein receives extrinsic nerves that mainly originate from the right cervicothoracic ganglion and a branch of the right vagus nerve, whereas the left cranial vein is supplied by extrinsic nerves from the left cervicothoracic ganglion and the left vagus nerve. The majority of intrinsic cardiac ganglia are localized on the heart base at the roots of the pulmonary veins. These ganglia are interlinked by interganglionic nerves into the above mentioned nerve plexus of the heart hilum. In general, the examined hearts contained 19 ± 3 ganglia, giving a cumulative ganglion area of 0.4 ± 0.1 mm(2). Despite substantial anatomic differences in ganglion number and distribution, the structural organization of the intrinsic ganglionated plexus in the mouse heart corresponds in general to that of other mammalian species, including human.

In vitro identification and electrophysiological characterization of dopamine neurons in the ventral tegmental area

Dopamine (DA) neurons in the ventral tegmental area (VTA) have been implicated in brain mechanisms related to motivation, reward, and drug addiction. Successful identification of these neurons in vitro has historically depended upon the expression of a hyperpolarization-activated current (I h) and immunohistochemical demonstration of the presence of tyrosine hydroxylase (TH), the rate-limiting enzyme for DA synthesis. Recent findings suggest that electrophysiological criteria may be insufficient for distinguishing DA neurons from non-DA neurons in the VTA. In this study, we sought to determine factors that could potentially account for the apparent discrepancies in the literature regarding DA neuron identification in the rodent brain slice preparation. We found that confirmed DA neurons from the lateral VTA generally displayed a larger amplitude I h relative to DA neurons located in the medial VTA. Measurement of a large amplitude I h (>100 pA) consistently indicated a dopaminergic phenotype, but non-dopamine neurons also can have I h current. The data also showed that immunohistochemical TH labeling of DA neurons can render false negative results after relatively long duration (>15 min) whole-cell patch clamp recordings. We conclude that whole-cell patch clamp recording in combination with immunohistochemical detection of TH expression can guarantee positive but not negative DA identification in the VTA.

Periaqueductal gray afferents synapse onto dopamine and GABA neurons in the rat ventral tegmental area

The midbrain central gray (periaqueductal gray; PAG) mediates defensive behaviors and is implicated in the rewarding effects of opiate drugs. Projections from the PAG to the ventral tegmental area (VTA) suggest that this region might also regulate behaviors involving motivation and cognition. However, studies have not yet examined the morphological features of PAG axons in the VTA or whether they synapse onto dopamine (DA) or GABA neurons. In this study, we injected anterograde tracers into the rat PAG and used immunoperoxidase to visualize the projections to the VTA. Immunogold-silver labeling for tyrosine hydroxylase (TH) or GABA was then used to identify the phenotype of innervated cells. Electron microscopic examination of the VTA revealed axons labeled anterogradely from the PAG, including myelinated and unmyelinated fibers and axon varicosities, some of which formed identifiable synapses. Approximately 55% of these synaptic contacts were of the symmetric (presumably inhibitory) type; the rest were asymmetric (presumably excitatory). These findings are consistent with the presence of both GABA and glutamate projection neurons in the PAG. Some PAG axons contained dense-cored vesicles indicating the presence of neuropeptides in addition to classical neurotransmitters. PAG projections synapsed onto both DA and GABA cells with no obvious selectivity, providing the first anatomical evidence for these direct connections. The results suggest a diverse nature of PAG physiological actions on midbrain neurons. Moreover, as both the VTA and PAG are implicated in the reinforcing actions of opiates, our findings provide a potential substrate for some of the rewarding effects of these drugs.

Lateral habenula projections to dopamine and GABA neurons in the rat ventral tegmental area

Ventral tegmental area (VTA) dopamine (DA) neurons and their forebrain projections are critically involved in reward processing and cognitive functions. Descending projections from the lateral habenula (LHb) play a central role in inhibiting DA cell activity in response to the absence of expected rewards. As LHb efferents are reportedly glutamatergic, their ability to inhibit DA cells would theoretically require a disynaptic connection involving VTA GABA neurons and their local collateral inputs to DA cells. We therefore used anterograde tract-tracing from the LHb to investigate the relative selectivity of LHb synapses onto GABA versus DA VTA neurons. LHb axons were visualized using immunoperoxidase, and DA and GABA cells were marked by immunogold-silver labeling for tyrosine hydroxylase (TH) or GABA, respectively. By ultrastructural analysis, 16% of LHb axons were observed to form synaptic contacts in the VTA, and most of these were of an intermediate morphological type that did not exhibit definitive asymmetric or symmetric character. LHb axons synaptically targeted TH- and GABA-labeled dendrites to a comparable extent (45 and 52% observed incidence, respectively). Pre-embedding immunogold labeling for the vesicular glutamate transporter type 2 and post-embedding immunogold staining for GABA confirmed that approximately 85% of LHb terminals were glutamatergic and not GABAergic. These results suggest that the robust inhibition of DA cells evoked by the LHb is unlikely to arise from a selective innervation of VTA GABA neurons. Moreover, the LHb may mediate a direct excitation of DA cells that is over-ridden by indirect inhibition originating from an extrinsic source.

Effects of calpain inhibition on dopaminergic markers and motor function following intrastriatal 6-hydroxydopamine administration in rats

The neurotoxin 6-hydroxydopamine has been widely used to model aspects of Parkinson's disease in rodents, but the mechanisms underlying toxin-induced dopaminergic degeneration and functional impairment have not been fully elucidated. The main aim of the present study was to assess a possible role for calpains in neurochemical and behavioral deficits following unilateral infusion of intrastriatal 6-hydroxydopamine in adult rats. Toxin administration produced a profound dopaminergic denervation, as indicated by a 90–95% reduction in dopamine transporter radiolabeling measured in the caudate-putamen at 2 weeks post-lesion. Treatment with 6-hydroxydopamine also resulted in calpain activation in both caudate-putamen and substantia nigra, as measured by the appearance of calpain-specific spectrin breakdown products. Calpain activation peaked at 24 h after 6-hydroxydopamine infusion and remained elevated at later time points. In contrast, caspase-3-mediated spectrin cleavage subsided within 48 h in both brain areas. In a subsequent experiment, calpain inhibition was achieved by intrastriatal infusion of an adenovirus expressing the endogenous calpain inhibitor, calpastatin. Calpastatin delivery abolished the lesion-induced calpain-mediated spectrin cleavage and alleviated forelimb asymmetries resulting from unilateral intrastriatal 6-hydroxydopamine. Unexpectedly, dopamine transporter and tyrosine hydroxylase labeling revealed significant neuroprotection, not in the nigrostriatal pathway but rather in the ventral tegmental area. These findings support a role for calpain activation in 6-hydroxydopamine-induced degeneration of dopaminergic neurons. However, after near-total dopaminergic depletion, the primary benefit of calpain inhibition may not occur within the nigrostriatal dopaminergic pathway itself.

Differential involvement of striosome and matrix dopamine systems in a transgenic model of dopa-responsive dystonia

Dopa-responsive dystonia (DRD) is a hereditary dystonia characterized by a childhood onset of fixed dystonic posture with a dramatic and sustained response to relatively low doses of levodopa. DRD is thought to result from striatal dopamine deficiency due to a reduced synthesis and activity of tyrosine hydroxylase (TH), the synthetic enzyme for dopamine. The mechanisms underlying the genesis of dystonia in DRD present a challenge to models of basal ganglia movement control, given that striatal dopamine deficiency is the hallmark of Parkinson's disease. We report here behavioral and anatomical observations on a transgenic mouse model for DRD in which the gene for 6-pyruvoyl-tetrahydropterin synthase is targeted to render selective dysfunction of TH synthesis in the striatum. Mutant mice exhibited motor deficits phenotypically resembling symptoms of human DRD and manifested a major depletion of TH labeling in the striatum, with a marked posterior-to-anterior gradient resulting in near total loss caudally. Strikingly, within the regions of remaining TH staining in the striatum, there was a greater loss of TH labeling in striosomes than in the surrounding matrix. The predominant loss of TH expression in striosomes occurred during the early postnatal period, when motor symptoms first appeared. We suggest that the differential striosome-matrix pattern of dopamine loss could be a key to identifying the mechanisms underlying the genesis of dystonia in DRD.

Enhanced glutamatergic phenotype of mesencephalic dopamine neurons after neonatal 6-hydroxydopamine lesion

There is increasing evidence that a subset of midbrain dopamine (DA) neurons uses glutamate as a co-transmitter and expresses vesicular glutamate transporter (VGLUT) 2, one of the three vesicular glutamate transporters. In the present study, double in situ hybridization was used to examine tyrosine hydroxylase (TH) and VGLUT2 mRNA expression during the embryonic development of these neurons, and postnatally, in normal rats and rats injected with 6-hydroxydopamine (6-OHDA) at P4 to destroy partially DA neurons. At embryonic days 15 and 16, there was a regional overlap in the labeling of TH and VGLUT2 mRNA in the ventral mesencephalon, which was no longer found at late embryonic stages (E18–E21) and postnatally. In normal pups from P5 to P15, only 1–2% of neurons containing TH mRNA in the ventral tegmental area (VTA) and substantia nigra, pars compacta, also displayed VGLUT2 mRNA. In contrast, after the cerebroventricular administration of 6-OHDA at P4, 26% of surviving DA neurons in the VTA of P15 rats expressed VGLUT2. To search for a colocalization of TH and VGLUT2 protein in axon terminals of these neurons, the nucleus accumbens of normal and 6-OHDA-lesioned P15 rats was examined by electron microscopy after dual immunocytochemical labeling. In normal rats, VGLUT2 protein was found in 28% of TH positive axon terminals in the core of nucleus accumbens. In 6-OHDA-lesioned rats, the total number of TH positive terminals was considerably reduced, and yet the proportion also displaying VGLUT2 immunoreactivity was modestly but significantly increased (37%). These results lead to the suggestion that the glutamatergic phenotype of a VTA DA neurons is highly plastic, repressed toward the end of normal embryonic development, and derepressed postnatally following injury. They also support the hypothesis of co-release of glutamate and DA by mesencephalic neurons in vivo, at least in the developing brain.

Fine structural survey of tyrosine hydroxylase immunoreactive terminals in the myenteric ganglion of the rat duodenum

We have examined whether the noradrenergic neurons have direct synaptic projections to the myenteric ganglion neurons of the duodenum and the ultrastructure of their terminals by using immunogold–silver labeling for tyrosine hydroxylase. In the neuropil of the myenteric ganglia, about half of the axon terminals contained round clear vesicles and the rest of them contained pleomorphic clear vesicles. The sizes of axon terminals contacting the dendrites as a whole were 1.62 ± 0.07 μm. All axon terminals formed asymmetric synaptic contacts with dendrites or somata. Immunohistochemical study revealed that the tyrosine hydroxylase-immunoreactive nerve terminals were distributed throughout the ganglia and contained exclusively pleomorphic clear synaptic vesicles (about 20–80 nm long). The tyrosine hydroxylase-immunoreactive terminals were generally large (1.99 ± 0.07 μm). A considerable number of the tyrosine hydroxylase-immunoreactive terminals made asymmetric synaptic contacts with small dendrites, spines or somata of the myenteric ganglion neurons. Serial ultrathin sections through the myenteric neurons revealed that about 16% of the total number of axosomatic terminals showed tyrosine hydroxylase immunoreactivity. These results indicated that the myenteric ganglion neurons of the duodenum receive direct synaptic projection of sympathetic noradrenergic neurons and that their terminals contain pleomorphic vesicles and form asymmetric synaptic contacts.

Ultrastructural analysis of prefrontal cortical inputs to the rat amygdala: spatial relationships to presumed dopamine axons and D1 and D2 receptors

Projections from the prefrontal cortex (PFC) to the amygdala (AMG) regulate affective behaviors in a manner that is modulated by dopamine (DA). Although PFC and DA inputs overlap within the basolateral nucleus (BLA) and intercalated cell masses (ICMs), the spatial relationship between these afferents has not been investigated, nor is it known how DA D1 (D1R) and D2 (D2R) receptors are localized in relationship to PFC terminals. We therefore combined tract-tracing from the rat PFC to the AMG with immunocytochemical labeling of tyrosine hydroxylase (TH) to identify presumed DA axons or D1R and D2R. In both the ICMs and BLA, PFC terminals formed asymmetric synapses onto spines that typically did not receive secondary synaptic inputs. TH-immunoreactive (-ir) fibers in the adjacent neuropil typically contacted different structures. Although PFC and TH-ir axons were sometimes apposed to the same dendrites or to each other, PFC terminals only rarely synapsed onto dendrites that also received synapses from TH-ir axons. D1R-ir spines and dendrites were observed commonly within the ICMs but less frequently within the BLA, and PFC axons in the ICMs occasionally synapsed onto D1R-ir spines. Within both regions, D2R-ir spines, dendrites, and axons were observed. PFC terminals occasionally contained presynaptic labeling for D2R but were not observed to synapse onto D2R-ir targets. The infrequent observation of synaptic convergence between PFC and presumed DA terminals within the AMG suggests that DA modulates PFC inputs primarily via extrasynaptic mechanisms, a conclusion supported by the localization of D2R within and D1R postsynaptic to PFC terminals.

Synaptic Characteristics Between Cortical Cells in the Rat Prefrontal Cortex and Axon Terminals from the Ventral Tegmental Area that Utilize Different Neurotransmitters

Projections from the ventral tegmental area (VTA) have been demonstrated to terminate in the prefrontal cortex (PFC) and to be dopaminergic and/or γ-aminobutyric acidergic (GABAergic), forming a neural circuit implicated in certain memory and cognitive processes. However, it has not been determined whether γ-aminobutyric acid (GABA) and dopamine (DA) are localized in certain types of axon terminals from the VTA to the PFC. To determine the synaptic characteristics made by postsynaptic prefrontal cortical structures and mesoprefrontal fibers utilizing either GABA or DA, we performed a double-labeling method for electron microscopy, in which we combined peroxidase markers for anterograde tract-tracing with postembedding immunogold labeling for tyrosine hydroxylase, DA, and GABAin rats. The anterograde tract-tracing studies showed that tegmentocortical fibers from the VTA terminated as both symmetric and asymmetric axon terminals with the predominantly symmetric synaptic type in the prelimbic cortex of the rat. Furthermore, a study using the combination of anterograde tract-tracing and postembedding immunocytochemistry indicated that tegmentocortical axon terminals forming symmetric synapses were either GABAergic or dopaminergic, whereas a small fraction of tegmentocortical terminals ending as asymmetric synapses were not immunopositive for DA or GABA. These findings indicate that the mesocortical projections to the PFC exert an inhibitory effect on the spontaneous activity of PFC cells via symmetric synapses that use DA and GABA as neurotransmitters and that these projections also have as yet unknown effects via asymmetric synapses using other neurotransmitters.

Up-regulation of IL-1 receptor type I and tyrosine hydroxylase in the rat carotid body following intraperitoneal injection of IL-1β

It is well established that reciprocal modulation exists between the central nervous system and immune system. Interleukin (IL)-1beta, a proinflammatory cytokine secreted at early stage of immune challenge, has been recognized as one of the informational molecules in immune-to-brain communication. However, how this large molecule is transmitted to the brain is still unknown. In recent years it has been reported that the cranial nerves, especially the vagus, may play a pivotal role in this regard. It is proposed that IL-1beta may bind to its corresponding receptors located in the glomus cells of the vagal paraganglia and then elicit action potentials in the nerve. The existence of IL-1 receptor type I (IL-1RI) in the vagal paraganglia has been shown. The carotid body, which is the largest peripheral chemoreceptive organ, is also a paraganglion. We hypothesize that the carotid body might play a role similar to the vagal paraganglia because they are architectonically similar. Recently we verified the presence of IL-1RI in the rat carotid body and observed increase firing in the carotid sinus nerve following IL-1beta stimulation. The aim of this study was to observe the changes in expression of IL-1RI and tyrosine hydroxylase (TH), a rate-limiting enzyme for catecholamine synthesis, in the glomus cells of the rat carotid body following intraperitoneal injection of IL-1beta. The radioimmunoassay result showed that the blood IL-1beta level was increased after the intraperitoneal injection of rmIL-1beta (750 ng/kg) from 0.48+/-0.08 to 0.78+/-0.07 ng/ml (P<0.05). Immunofluorescence and Western blot analysis showed that the expression of IL-1RI and TH in the rat carotid body was increased significantly following peritoneal IL-1beta stimulation. In addition, double immunofluorescence labeling for TH and PGP9.5, a marker for glomus cells, or TH immunofluoresence with hematoxylin-eosin (HE) counterstaining revealed that a considerable number of glomus cells did not display TH immunoreactivity. These data provide morphological evidence for the response of the carotid body to proinflammatory cytokine stimulation. The results also indicate that not all of the glomus cells express detectable TH levels either in normal or in some abnormal conditions.

Dynorphin-containing axons directly innervate noradrenergic neurons in the rat nucleus locus coeruleus

Stress causes increased dynorphin (DYN) expression in limbic brain regions and antagonism of kappa-opioid receptors may offer therapeutic potential for the treatment of depression. A potential site of DYN action relevant to stress and related neuropsychiatric disorders is the locus coeruleus (LC), the primary source of forebrain norepinephrine. Therefore, using immunofluorescence and immunoelectron microscopic analyses, we characterized the cellular substrates for interactions between DYN and tyrosine hydroxylase (TH), a catecholamine synthesizing enzyme in single sections through the rat LC. Light microscopic analysis of DYN immunoreactivity indicated that DYN fibers are distributed within the core and pericoerulear subregions of the LC. Using electron microscopy, immunoperoxidase labeling for DYN was primarily found in axon terminals, although in some cases was diffusely localized to somatodendritic processes. When DYN-containing axons formed synaptic contacts, they typically (89%) exhibited an asymmetric morphology. Almost a third (28%) of the postsynaptic targets of DYN-containing axons contained immunogold labeling for TH. These findings reveal some diversity as to the localization of DYN in the LC within axons that contact both TH and non-TH containing dendrites. However, the present data provide the first ultrastructural evidence that DYN-containing axon terminals directly innervate catecholaminergic LC dendrites. Moreover, DYN axon terminals targeting catecholaminergic LC dendrites via asymmetric synapses are consistent with localization within excitatory type afferents to the LC. Therefore, direct modulation of catacholaminergic LC neurons maybe an important site of action for DYN relevant to stress and stress-related disorders.

Pro‐opiomelanocortin colocalizes with corticotropin‐ releasing factor in axon terminals of the noradrenergic nucleus locus coeruleus

We previously demonstrated that the opioid peptide enkephalin and corticotropin-releasing factor (CRF) are occasionally colocalized in individual axon terminals but more frequently converge on common dendrites in the locus coeruleus (LC). To further examine potential opioid cotransmitters in CRF afferents we investigated the distribution of pro-opiomelanocortin (POMC), the precursor that yields the potent bioactive peptide beta-endorphin, with respect to CRF immunoreactivity using immunofluorescence and immunoelectron microscopic analyses of the LC. Coronal sections were collected through the dorsal pontine tegmentum of rat brain and processed for immunocytochemical detection of POMC and CRF or tyrosine hydroxylase (TH). POMC-immunoreactive processes exhibited a distinct distribution within the LC as compared to the enkephalin family of opioid peptides. Specifically, POMC fibers were enriched in the ventromedial aspect of the LC with fewer fibers present dorsolaterally. Immunofluorescence microscopy showed frequent coexistence of POMC and CRF in varicose processes that overlapped TH-containing somatodendritic processes in the LC. Ultrastructural analysis showed POMC immunoreactivity in unmyelinated axons and axon terminals. Axon terminals containing POMC were filled with numerous large dense-core vesicles. In sections processed for POMC and TH, approximately 29% of POMC-containing axon terminals (n = 405) targeted dendrites that exhibited immunogold-silver labeling for TH. In contrast, sections processed for POMC and CRF showed that 27% of POMC-labeled axon terminals (n = 657) also exhibited CRF immunoreactivity. Taken together, these data indicate that a subset of CRF afferents targeting the LC contain POMC and may be positioned to dually impact LC activity.

Decreased susceptibility to oxidative stress underlies the resistance of specific dopaminergic cell populations to paraquat-induced degeneration

The vulnerability of different dopaminergic cell populations to damage caused by the herbicide paraquat was assessed by stereological counts of tyrosine hydroxylase-positive and calbindin-D 28k-immunoreactive neurons in A9 (substantia nigra pars compacta) and A10 (ventral tegmental area and other cell groups). In saline-treated control mice, tyrosine hydroxylase-immunoreactive neurons represented 80% and 45% of the total neuronal population in A9 and A10, respectively, and the number of calbindin-D 28k-positive neurons was five times greater in A10 than A9. Sequential injections with paraquat resulted in a significant loss of dopaminergic neurons in A9. In contrast, tyrosine hydroxylase-positive cells in A10 were spared from paraquat-induced degeneration. Furthermore, expression of calbindin-D 28k was consistently associated with neuronal resistance to the herbicide in both A9 and A10. Paraquat exposure also induced oxidative stress as indicated by an increase in the number of midbrain cells positive for 4-hydroxy-2-nonenal, a marker of lipid peroxidation. Co-localization studies revealed that calbindin-D 28k immunoreactivity overlapped with tyrosine hydroxylase labeling and that, after paraquat administration, (i) the vast majority of midbrain 4-hydroxy-2-nonenal-immunoreactive cells were dopaminergic (tyrosine hydroxylase-immunoreactive), (ii) tyrosine hydroxylase/4-hydroxy-2-nonenal-positive neurons were much more prevalent in A9 than A10, and (iii) all calbindin-D 28k-containing neurons were characterized by lack of lipid peroxidation (4-hydroxy-2-nonenal immunoreactivity). Results in this paraquat model emphasize that, despite sharing a similar dopaminergic phenotype, different groups of midbrain neurons vary dramatically in their vulnerability to injury. Data also indicate that these differences are attributable, at least in part, to a varying susceptibility of dopaminergic cell populations to oxidative stress.