Catecholamine-synthesizing Enzyme Tyrosine Hydroxylase Research Articles

Immunohistochemical and Ultrastructural Evidence of Adrenal Chromaffin Cell Subtypes in Sea BassDicentrarchus labrax(L.)

Histology demonstrated chromaffin cells in the head kidney and opisthonephros of the sea bass. Immunohistochemistry showed the catecholamine-synthesizing enzymes tyrosine hydroxylase and dopamine β-hydroxylase in these cells. PhenylethanolamineN-methyltransferase was detected in a fraction of the chromaffin cells in the head kidney (59%) and in the opisthonephros (54%). Distinct noradrenaline- and adrenaline-synthesizing cells are therefore suggested. Ultrastructural analysis confirmed the existence of two main chromaffin cell types, distinguished by different types of the secretory granules. Cells that contain vesicles with a round, electron-dense core were interpreted as noradrenaline cells, while cells with electron-lucent vesicles were identified as adrenaline cells. The ultrastructure of these chromaffin cell subtypes does not differ in the head kidney and opisthonephros. A minor population of chromaffin cells was identified, which typically show smaller vesicles with an electron-dense core. This population may account for a limited number of not fully differentiated chromaffin cells.

The dopamine transporter is localized to dendritic and axonal plasma membranes of nigrostriatal dopaminergic neurons

Nigrostriatal dopaminergic neurons play an essential role in the central regulation of motor functions. These functions are initiated through the release of dopamine from axon terminals in the striatum or from dendrites in the substantia nigra (SN) and are terminated by the reuptake of dopamine by the sodium- and chloride-dependent dopamine transporter (DAT). DAT also can transport dopamine neurotoxins and has been implicated in the selective vulnerability of nigrostriatal dopaminergic neurons in major models of Parkinson's disease. We have used electron microscopic immunocytochemistry with an N-terminal domain anti-peptide antibody to examine the subcellular distribution of DAT in the rat SN and dorsolateral striatum. In the SN, immunogold labeling for DAT was localized to cytoplasmic surfaces of plasma membranes and smooth endoplasmic reticulum of dendrites and dendritic spines, few of which contained synaptic vesicles. Neuronal perikarya in the SN contained immunogold-labeled pleomorphic electron-lucent tubulovesicles but showed immunolabeling of plasma membranes only rarely. Axon terminals in the striatum contained extensive immunogold labeling of cytoplasmic surfaces of plasma membranes near aggregates of synaptic vesicles and less frequent labeling of intervaricose segments of plasma membrane or small electron-lucent vesicles. In sections dually labeled for DAT and the catecholamine-synthesizing enzyme tyrosine hydroxylase, both markers were colocalized in most profiles in the SN and striatum. These findings support the proposed topological model for DAT and suggest that this transporter is strategically located to facilitate uptake of dopamine and neurotoxins into distal dendritic and axonal processes of nigrostriatal dopaminergic neurons.

Corticotropin-releasing factor-containing axon terminals synapse onto catecholamine dendrites and may presynaptically modulate other afferents in the rostral pole of the nucleus locus coeruleus in the rat brain.

Physiological and immunohistochemical studies have suggested that corticotropin-releasing factor (CRF), the hypophysiotropic peptide that initiates endocrine responses to stress, may serve as a neurotransmitter to activate noradrenergic neurons in the nucleus locus coeruleus (LC). We combined immunoperoxidase labeling for CRF and immunogold-silver localization of the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH) in single sections through the rat LC to determine potential substrates for interactions between these two transmitters. Light microscopic analysis indicated that CRF processes are dense and highly varicose in the rostral LC region in the vicinity of noradrenergic dendrites. Electron microscopy of this rostral region revealed that immunoperoxidase labeling for CRF was mainly restricted to axons and axon terminals and was rarely seen in somata or dendrites. Axon terminals containing CRF immunoreactivity varied in size, content of synaptic vesicles, and formation of synaptic specializations. The postsynaptic targets of the CRF-labeled axon terminals consisted of both TH-labeled dendrites and dendrites lacking detectable TH-immunoreactivity. Of 113 CRF-immunoreactive axon terminals, approximately 70% were in direct contact with TH-labeled and unlabeled dendrites. Of the CRF-labeled axon terminals forming synapses with TH-labeled and unlabeled dendrites, they were either of the asymmetric (excitatory type; 19%) or symmetric (inhibitory type; 11%) variety or did not form identifiable contacts in the plane of section analyzed. Unlabeled axon terminals and glial processes were also commonly located adjacent to the plasma membranes of CRF-labeled axon terminals. These results provide the first direct ultrastructural evidence that axon terminals containing CRF-immunoreactivity 1) directly contact catecholamine-containing dendrites within the rostral pole of the LC, 2) may presynaptically modulate other afferents, and 3) are often enveloped by astrocytic processes.

Effects of chronic intermittent cold stress on pituitary adrenocortical and sympathetic adrenomedullary functioning

Basal and stress-induced pituitary-adrenocortical (PA) and sympathetic adrenomedullary (SAM) function was investigated in rats exposed to chronic intermittent cold stress (4°C for 4 h a day for 21 days; CHR). We found that basal plasma levels of corticosterone (B), corticosteroid-binding-globulin, ACTH, epinephrine (E) and norepinephrine (NE) were similar in CHR and control (CTL) animals. In contrast, activity of the adrenal catecholamine-synthesizing enzyme tyrosine hydroxylase, but not phenylethanolamine-N-methyl transferase, was significantly elevated in CHR compared to CTL. Following exposure to a heterotypic stressor (20 min restraint), plasma levels of B were significantly higher in CHR than CTL, but the stress-induced levels of E and NE were not different between groups. These data suggest that, although basal PA function is not altered by exposure to chronic intermittent cold stress, components of the SAM system are affected by this paradigm, and that co-ordinate facilitation of both PA and SAM responses to a novel stressor is not a necessary consequence of exposure to chronic intermittent stress.

Colocalization of tyrosine hydroxylase with oxytocin or vasopressin in neurons of the human paraventricular and supraoptic nucleus

In the developing and adult human paraventricular (PVN) and supraoptic (SON) nucleus, a large proportion of neurons contains the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH). In the present study we investigated the possible colocalization of TH with oxytocin (OXT) or vasopressin (VP) in the adult and neonatal PVN and SON. Adjacent paraffin sections were incubated simultaneously with two antibodies: a polyclonal against TH and a monoclonal against OXT or VP and stained with a double peroxidase-antiperoxidase/alkaline phosphatase method. We observed that TH-immunoreactive(IR) perikarya in the human PVN and SON were also positive for OXT or VP. A clear difference between the neonates and adult cases of our sample was observed in the proportion of TH-IR neurons that colocalize OXT or VP. In the neonates the majority of the TH-IR perikarya was also stained for VP, while only few TH-IR neurons were also positive for OXT. The opposite was observed in the adults, where the majority of the double-stained TH-IR neurons colocalizes OXT while only few TH-IR perikarya appear to contain VP. Our study establishes the colocalization of TH with OXT or VP in the adult and neonatal PVN and SON and indicates that antemortem factors such as perinatal hypoxia might increase TH-immunoreactivity of the VP neurons in man.

Colocalization of Nitric Oxide Synthase with Vasoactive Intestinal Peptide, Neuropeptide Y, and Tyrosine Hydroxylase in Nerves Supplying the Human Ureter

The distribution and patterns of colocalization of nitric oxide synthase (NOS), vasoactive intestinal peptide (VIP), neuropeptide Y (NPY) and the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH) were examined in nerve fibers supplying the human lower ureter using double label immunofluorescence. Many nerve fibers immunoreactive for NOS were observed within the ureter. Positive varicose fibers were seen running longitudinally within the smooth muscle bundles, particularly those of the inner layers of the ureter. Immunoreactive axons were also prominent within the subepithelium, and as plexi surrounding many blood vessels. The colocalization studies indicated that NOS was never present in presumptive sympathetic nerve fibers expressing TH. All fibers containing VIP, however, were also immunoreactive for NOS. In addition, a minor population of NOS fibers did not contain VIP. Neuropeptide Y coexisted with NOS in a significant number of nerve terminals, although fibers expressing only NPY were equally common. Several immunochemically distinct nerve populations can therefore be distinguished in the human ureter: (1) nerves containing NOS either with or without VIP; (2) NOS-immunoreactive fibers with NPY; and (3) those fibers expressing TH or NPY which do not contain NOS. The results indicate that some non-noradrenergic peptide-containing nerves in the human ureter have the capacity to synthesize nitric oxide (NO), and that NO may be involved in the regulation of ureteric motility.

Synaptic structure and connectivity of serotonin terminals in the ventral tegmental area: potential sites for modulation of mesolimbic dopamine neurons

Microinfusion of serotonin (5-hydroxytryptamine; 5-HT) into the ventral tegmental area enhances the release of dopamine in the nucleus accumbens, a major target of midbrain dopamine neurons. We examined the synaptic basis for 5-HT modulation of neurons in the ventral tegmental area which either (i) project to the nucleus accumbens or (ii) contain the catecholamine synthesizing enzyme tyrosine hydroxylase, a marker of dopamine neurons in this brain region. In the first study, immunoperoxidase labeling of 5-HT in the ventral tegmental area was combined with retrograde transport of gold particles following unilateral injections of the tracer into the nucleus accumbens of adult rats. The gold particles had been previously coupled to wheat germ agglutinin conjugated to inactive horseradish peroxidase. Gold particles were enlarged for visualization using a silver enhancement procedure. By brightfield microscopy, retrogradely labeled neurons contained black punctate granules within their perikarya and proximal processes. The labeled cells were scattered ipsilateral to the injection within the paranigral and parabrachial subdivisions of the ventral tegmental area. Both regions also contained 5-HT immunoreactive varicosities. By electron microscopy, irrespective of the ventral tegmental subdivision, 5-HT labeling was seen primarily in unmyelinated axons and axon terminals. The terminals contained small, clear and large dense core vesicles and ranged from 0.3 μm to 1.4 μm in cross-sectional diameter. 22% ( n = 250) of the axon terminals containing 5-HT immunoreactivity formed synaptic contacts with neurons containing the retrograde label. Of these 5-HT terminals, 16% formed asymmetric type contacts and 6% formed symmetric junctions on the retrogradely labeled neurons. The remaining 5-HT terminals were either apposed to (but lacked recognized synapses on) perikarya and large dendrites containing the retrogradely transported protein-gold tracer or contacted unlabeled neurons. In the second set of experiments combining immunoperoxidase of 5-HT and immunogold silver for tyrosine hydroxylase, 32% ( n = 250) of the 5-HT-labeled terminals formed synaptic junctions with perikarya or dendrites containing tyrosine hydroxylase immunoreactivity. Of these 5-HT terminals, 23% formed asymmetric type junctions. The remainder were either symmetric or lacked recognized membrane dendrites. The prominence of asymmetric junctions formed by 5-HT-labeled terminals on neurons projecting to the nucleus accumbens and those containing tyrosine hydroxylase in the ventral tegmental area suggests a cellular basis for serotonergic excitation of mesoaccumbens dopamine neurons. Additionally, the multiplicity of junctions formed by 5-HT terminals on targets with or without retrograde labeling or tyrosine hydroxylase immunoreactivity is consistent with known diverse physiological actions of 5-HT in the ventral tegmental area.

Immunocytochemical evidence for chromogranin A and B in neuronal elements in human gut

Chromogranins are a family of acidic proteins known to occur in the secretory granules of endocrine cells and neurons in mammals. The aim of the present study was to investigate the possible neuronal localisation of chromogranins in human intestine and their relationship with other neuronal constituents using one antibody recognizing both chromogranin A and B (CAB) and others recognizing only chromogranin A. Immunoreactive CAB was found to occur in mucosal endocrine cells and in neuronal elements of all layers throughout human gut. A few to large number of CAB-immunoreactive nerve fibers were seen in smooth muscles as well as in submucous and myenteric ganglia and they were regularly found around blood vessels. Occasional CAB immunoreactive nerve cell bodies could be demonstrated in both submucous and myenteric ganglia indicating a local origin of some of the immunoreactive fibers. Most of these neurons seemed to lack acetylcholine esterase, and could be classified as non-cholinergic. CAB coexisted with the catecholamine-synthesizing enzyme tyrosine hydroxylase indicating the presence of chromogranins in noradrenergic nerve fibers. The presence of CAB-immunoreactive nerve cell bodies indicates that some CAB-containing fibers are intrinsic in origin. The distribution of chromogranin A and B immunoreactive nerve fibers in all layers of the gut wall suggests multiple targets for neuronal CAB, and/or their processing products.

Dopaminergic neurons in rat ventral midbrain express brain‐derived neurotrophic factor and neurotrophin‐3 mRNAs

Studies of the trophic activities of brain-derived neurotrophic factor and neurotrophin-3 indicate that both molecules support the survival of a number of different embryonic cell types in culture. We have shown that mRNAs for brain-derived neurotrophic factor and neurotrophin-3 are localized to specific ventral mesencephalic regions containing dopaminergic cell bodies, including the substantia nigra and ventral tegmental area. In the present study, in situ hybridization with 35S-labeled cRNA probes for the neurotrophin mRNAs was combined with neurotoxin lesions or with immunocytochemistry for the catecholamine-synthesizing enzyme tyrosine hydroxylase to determine whether the dopaminergic neurons, themselves, synthesize the neurotrophins in adult rat midbrain. Following unilateral destruction of the midbrain dopamine cells with 6-hydroxydopamine, a substantial, but incomplete, depletion of brain-derived neurotrophic factor and neurotrophin-3 mRNA-containing cells was observed in the ipsilateral substantia nigra pars compacta and ventral tegmental area. In other rats, combined in situ hybridization and tyrosine hydroxylase immunocytochemistry demonstrated that the vast majority of the neurotrophin mRNA-containing neurons in the substantia nigra and ventral tegmental area were tyrosine hydroxylase immunoreactive. Of the total population of tyrosine hydroxylase-positive cells, double-labeled neurons constituted 25-50% in the ventral tegmental area and 10-30% in the substantia nigra pars compacta, with the proportion being greater in medial pars compacta. In addition, tyrosine hydroxylase/neurotrophin mRNA coexistence was observed in neurons in other mesencephalic regions including the retrorubral field, interfascicular nucleus, rostral and central linear nuclei, dorsal raphe nucleus, and supramammillary region. The present results demonstrate brain-derived neurotrophic factor and neurotrophin-3 expression by adult midbrain dopamine neurons and support the suggestion that these neurotrophins influence dopamine neurons via autocrine or paracrine mechanisms. These data raise the additional possibility that inappropriate expression of the neurotrophins by dopaminergic neurons could contribute to the neuropathology of disease states such as Parkinson's disease and schizophrenia.

Non-neuronal cells inhibit catecholaminergic differentiation of primary sensory neurons: role of leukemia inhibitory factor

Although some sensory ganglion cells in mature animals are catecholaminergic, most mammalian sensory neurons that express the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH) do so only transiently during early gangliogenesis in vivo. The lack of TH expression at later stages appears to be due to modulation of this catecholaminergic potential. A previous study showed that the phenotype reappears, for example, when E16.5 and older sensory ganglia are dissociated in culture into single cells, suggesting that extracellular influences can modulate TH expression. Moreover, TH expression in dissociate cultures is cell-density dependent, as a four-fold increase in plating density led to a 30% decrease in the percentage of TH neurons. The present study demonstrates that inhibition of TH expression in high density cultures is mediated by ganglionic non-neuronal cells (NNC), as removal of NNC abolished density-dependent inhibition. Moreover, plating E16.5 trigeminal neurons at low density on top of NNC monolayers resulted in an 85% decrease in the percentage of TH neurons. Treatment of cultures with non-neuronal cell conditioned medium (NNC-CM) reproduced the effect of coculture with NNC, suggesting that diffusible factors from NNC were involved in the inhibition of TH. The inhibitory effect of NNC-CM was mimicked by treatment of dissociate cultures with ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF). However, immunoprecipitation of NNC-CM with antibodies against LIF or CNTF showed that only anti-LIF antibodies were able partially to remove the TH inhibitory activity of NNC-CM. Therefore, LIF is one, but not the only, factor mediating NNC inhibition of TH expression in cultured sensory neurons. In summary, these data indicate that ganglionic NNC can regulate sensory transmitter phenotype in culture by inhibiting expression of specific molecular traits. The finding that LIF can partially account for the inhibitory effect of ganglionic NNC on TH expression suggests a novel role for this cytokine in regulating differentiation of catecholaminergic properties in sensory neurons.

Distribution of dopamine-containing neurons and fibres in the feline medulla oblongata: A comparative study using catecholaminesynthesizing enzyme and dopamine immunohistochemistry

The distribution of dopamine-immunoreactive neurons and fibres in the feline medulla oblongata was examined by immunocytochemistry with antisera to the catecholamine-synthesizing enzymes tyrosine hydroxylase, dopamine-β-hydroxylase andphenylethanolamine- N-methyltransferase, and with antisera to the catecholamines dopamine and l-dihydroxyphenylalanine. Neurons immunoreactive for the catecholamine-synthesizing enzymes were found in two regions of the medulla, the ventrolateral A1 region and the dorsomedial A2 region. Double-staining studies with antisera to the enzymes indicated that a population of neurons within both regions were immunoreactive for tyrosine hydroxylase but not dopamine-β -hydroxylase orphenylethanolamine- N-methyltransferase, implying that they synthesize dopamine. Studies using the dopamine antisera demonstrated the presence of dopamine-immunoreactive neurons in both the ventrolateral and dorsomedial regions of the medulla; in the dorsomedial region, they were found in the area postrema, nucleus tractus solitarius and dorsal motor vagal nucleus, mainly at levels caudal to the obex. Dopamine-immunoreactive fibres were found in several areas of the medulla including the nucleus tractus solitarius, inferior olive, dorsal motor vagal, spinal trigeminal, hypoglossal, cuneate, gracile, and raphe nuclei. Double-staining studies with antisera to dopamine and dopamine-β-hydroxylase revealed a population of cells immunoreactive for dopamine alone. The presence of some double-stained neurons, however, implies some cross-reactivity of the dopamine antiserum with noradrenaline or adrenaline and/or recognition of dopamine present as a metabolic intermediary in some noradrenergic neurons. No l-dihydroxyphenylalanine-immunoreactive neurons were found in the medulla, although fibres were seen. These data provide evidence for the existence of catecholamine neurons which utilize dopamine as a final synthetic product within the medulla oblongata.

Supraspinal fiber outgrowth and apparent synaptic remodelling across transected-reconstructed feline spinal cord.

Following complete transection of the spinal cord at T9, 12 cats were separated into two groups: Group 1 received a collagen matrix (CM) treated with a neuroactive agent or with saline to bridge the spinal cord stumps and an omental transposition which was placed on the dorsal surface of the matrix; Group 2 received spinal cord transection only. Two cats received no spinal cord transection. After 90 days, all animals were killed and their brains and spinal cords were removed for immunohistochemical examination. Two weeks prior to sacrifice, spinal cord blood flows (SCBF) were measured and the retrograde axonal tracer Fluoro-Gold was injected below the transection site. Results show that omental transposition to the CM bridge in Group 1 animals increased SCBF an average 59% (assessed by clamping the omental blood supply to the cord). Examination of the brain 90 days after cord transection revealed Fluoro-Gold accumulation in the cytoplasm and processes of neurons located in the brainstem, midbrain, and diencephalic region which are known to contribute pathways to the spinal cord. Immunohistochemical staining with antibodies against the catecholamine synthesizing enzymes tyrosine hydroxylase and dopamine-B-hydroxylase, indicated that only Group I treated cats developed dense bundles of dopaminergic and noradrenergic fibers within the CM bridge and distal spinal cord tissue. These fibers were seen to extend 90 mm below the transection site. In addition, the synaptogenic marker synaptophysin (SYN) was observed in association with dopaminergic and noradrenergic fibers distal to the collagen matrix bridge, an indication that synaptic remodelling (regeneration) by previously denervated supraspinal axons may have occurred. Immunostaining for glial fibrillary acidic protein (GFAP) showed little to none reactive astrocytosis near the transection site of cats treated with the CM and omentum transposition (Group 1). No catecholaminergic fibers or SYN expression below the transection site were observed in Group 2 treated cats. Group 2 treated cats also showed dense immunostaining of GFAP near the transection site indicating significant astrocytic proliferation. These findings indicate that following complete spinal cord transection in cats and reconstruction with a treated collagen matrix and omental transposition, disconnected supraspinal fibers have the ability to regenerate for long anatomic distances and seemingly engage in synaptic remodelling with distal target tissue.

Transient expression of somatostatin peptide is a widespread feature of developing sensory and sympathetic neurons in the embryonic rat.

Previous studies from this and other laboratories demonstrated that many embryonic sensory ganglion cells in the rat transiently express the catecholamine synthesizing enzyme tyrosine hydroxylase (TH), a trait not expressed by most mature sensory neurons. We, therefore, sought to determine whether transient expression was uniquely associated with catecholaminergic traits, or, alternatively, whether embryonic ganglion cells transiently expressed peptidergic properties as well. Of the four peptides examined (somatostatin [somatotropin release inhibiting factor] (SRIF), galanin (Gal), calcitonin gene-related peptide (CGRP), and substance P (SP)), only SRIF was found to be transiently expressed during early stages of sensory gangliogenesis. Surprisingly, SRIF immunoreactivity was observed in virtually all cranial and spinal sensory ganglion cells on embryonic day (E) 12.5. In addition to perikaryal labeling, intense SRIF immunoreactivity was also observed in the central and peripheral processes of E12.5 sensory neurons, suggesting the peptide may be released from nerve endings. The time course of SRIF appearance in cranial ganglion cells paralleled that previously described for TH, and double-labeling studies revealed extensive co-localization of these two phenotypes. By E16.5, however, the number of neurons expressing SRIF had diminished markedly, indicating that SRIF is only transiently expressed by most sensory neurons during early stages of ganglion development. An unexpected finding was that transient expression of SRIF is also a prominent feature of sympathetic ganglion cells; however, the temporal pattern of staining in the sympathetic and sensory ganglia differed substantially. Whereas virtually no SRIF staining was observed in E12.5 sympathetics, the vast majority of cells in the E16.5 superior cervical ganglion (SCG) were labeled. This contrasted sharply with the adult SCG, in which only low levels of SRIF expression were found. These findings demonstrate that SRIF peptide is transiently expressed at high levels in peripheral sensory and sympathetic neurons during embryogenesis. The time course and widespread distribution of SRIF expression indicates that the peptide may play a role in early stages of ganglion cell growth and development. Moreover, these data, in conjunction with previous studies demonstrating SRIF immunoreactivity in developing central neurons, suggest that transient expression of this peptide is a common property of diverse neuronal cell types.

Axonal regeneration after spinal cord transection and reconstruction

Following complete transection of the spinal cord, cats were separated into 2 groups to undergo: (i) surgical reconstruction of the disconnected cord using a neuroactive agent mixed into a collagen matrix bridge and omental transposition and (ii) cord transection-only. After 90 days, animals were killed and the brain and spinal cord were removed for immunohistochemistry. Two weeks prior to sacrifice, spinal cord blood flows were measured and the retrograde axonal tracer Fluoro-Gold was injected below the transection site. Gross inspection of the spinal cords at autopsy showed excellent integration and continuity of the collagen matrix bridge with the proximal-distal stumps in the surgical reconstruction group. In the transection-only group, the proximal-distal stumps were connected by a fibrotic, often tapered in the middle, tissue bridge. Results show that omental transposition in the surgical reconstruction group increased spinal cord blood flow by 58% when compared to transection-only animals. Fluoro-Gold was found in mesencephalic and brainstem catecholaminergic and cholinergic neurons known to send axons to the spinal cord. Immunohistochemical staining with antibodies against catecholamine synthesizing enzymes tyrosine hydroxylase (TH) and dopamine-β-hydroxylase (DBH) showed that surgical reconstruction treated cat cords but not transection-only, developed dense bundles of dopaminergic and noradrenergic fibers which were present in the collagen matrix bridge and in the distal spinal cord. Extension of these catecholaminergic fibers in surgical reconstruction treated cats showed maximal outgrowth of 90 mm below the transection site when the neuroactive agent 4-aminopyridine was mixed into the collagen matrix. In addition, the synaptogenic marker synaptophysin (SYN) was observed on preganglionic sympathetic neurons in association with dopaminergic- and noradrenergic-containing varicosities distal to the collagen matrix bridge, an indication that neo-synaptic contacts may have been made on these previously denervated neurons. No TH, DBH or SYN was observed below the transection site in transection-only cats. These findings indicate that surgical reconstruction treated cords can develop dense supraspinal fiber outgrowth across a treated collagen matrix bridge fed by an omental blood supply and that these fibers may have made neo-synaptic contacts with appropriate distal spinal cord target tissue.

Presence of a dynorphin-like peptide in a restricted subpopulation of catecholaminergic neurons in rat nucleus tractus solitarii

Immunofluorescence colocalization techniques were used to examine the extent of coexistence of the endogenous opioid peptide dynorphin with catecholamines and the related opioid peptide enkephalin within neurons of the rat medulla oblongata. Immunoreactivities for dynorphin and the catecholamine-synthesizing enzyme tyrosine hydroxylase were found to coexist within a limited subpopulation of A2 catecholamine cells, localized to the medial nucleus of the nucleus tractus solitarii. Colocalization of the two opioid peptides was found mainly within perikarya situated in the medial and ventrolateral nuclei of the nucleus tractus solitarii. Triple-labeling studies revealed only rare cases of catecholamine/dynorphin/enkephalin coexistence. These data demonstrate that dynorphin is present within a restricted subpopulation of catecholamine neurons in the dorsal medulla oblongata. In addition, the content of either of the opioids enkephalin or dynorphin appears to distinguish subsets of medullary catecholamine neurons.

Dual ultrastructural localization of enkephalin and tyrosine hydroxylase immunoreactivity in the rat ventral tegmental area: multiple substrates for opiate-dopamine interactions

Endogenous opiates modulate activity in the mesocorticolimbic dopaminergic system, and this interaction is thought to underlie major aspects of motoric, reward-seeking, and stress-coping behaviors. We sought to determine the ultrastructural substrate for this modulatory action at the level of dopaminergic perikarya in the rat ventral tegmental area (VTA). Using a dual-labeling, immunoperoxidase and immunogold-silver method, we localized antisera directed against leu5-enkephalin (ENK) and the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH) in acrolein-fixed sections through the VTA. ENK-like immunoreactivity (ENK-LI) was visualized within unmyelinated axons and in axon terminals. In terminals, ENK-LI was densely localized to one or more dense-cored vesicles and either densely or lightly detected surrounding small clear vesicles. Immunoreactive dense-cored vesicles were occasionally associated with the presynaptic specialization but were more frequently detected at distant sites along the plasmalemmal surface, often in apposition to astrocytic processes. ENK-immunoreactive terminals formed both symmetric and asymmetric synapses, most frequently on large proximal dendrites. Direct appositions without glial separation were also detected between terminals containing ENK-LI and other ENK-labeled or unlabeled terminals. In contrast to ENK-LI, immunolabeling for TH was primarily detected within perikarya and dendrites in the VTA. Of the ENK-immunoreactive terminals that formed synaptic contacts in single sections, approximately 50-60% were in association with TH-labeled dendrites. The remainder formed synapses on dendrites lacking detectable immunoreactivity for TH. Multiple ENK-immunoreactive terminals occasionally formed convergent synaptic contacts on single TH-labeled or unlabeled dendrites. Furthermore, individual ENK-labeled terminals sometimes formed divergent contacts on two TH-labeled or unlabeled dendrites. When a single ENK-immunoreactive terminal made dual synaptic contacts on TH-labeled dendrites, the latter were usually in close apposition to one another. These findings represent the first ultrastructural demonstration that opioid peptide-containing terminals provide a direct synaptic input to dopaminergic, as well as nondopaminergic, neurons in the VTA. In addition, the morphological evidence suggests that endogenous opioid peptides (1) may be released from nonsynaptic sites, (2) may modulate the release of transmitters from other terminals, and/or (3) may synchronize the activity of multiple neuronal targets in the VTA. These results provide a number of morphological substrates through which opiates may directly or indirectly regulate activity in mesocorticolimbic dopaminergic pathways.

Expression of catecholamine-synthesizing enzymes in paraganglionic and ganglionic cells in the laryngeal nerves of the rat

The rat recurrent and superior laryngeal nerves with adjacent connective tissue were examined by immunohistochemical techniques for localization of the catecholamine-synthesizing enzymes tyrosine hydroxylase, dopamine-beta-hydroxylase and phenylethanolamine-N-methyltransferase. Most of the cells in the paraganglia of the recurrent and superior laryngeal nerves showed an intense tyrosine hydroxylase-like immunoreactivity. A few paraganglionic cells exhibited dopamine-beta-hydroxylase-like immunoreactivity while none of the cells displayed phenylethanolamine-N-methyltransferase-like immunoreactivity. Some of the ganglionic cells in the recurrent and superior laryngeal nerves showed dopamine-beta-hydroxylase-like immunoreactivity whilst these cells never showed tyrosine hydroxylase- or phenylethanolamine-N-methyltransferase-like immunoreactivity. The arterioles were supplied with plexuses of nerve fibres showing tyrosine hydroxylase- and dopamine-beta-hydroxylase-like immunoreactivity. The results indicate that dopamine is the major catecholamine located in the laryngeal nerve paraganglia and show that ganglionic cells in the recurrent and superior laryngeal nerves show immunolabelling for one of the enzymes in the catecholamine synthetic pathway, dopamine-beta-hydroxylase.

Cholinergic neurons in the rat septal complex: Ultrastructural characterization and synaptic relations with catecholaminergic terminals

Physiological and pharmacological studies have suggested that catecholamines modulate cholinergic neurons in the medial septal and diagonal band nuclei (i.e., the septal complex). Thus, the ultrastructural morphology of neurons containing choline acetyltransferase (ChAT), the biosynthetic enzyme for acetylcholine, and their relation to catecholaminergic terminals exhibiting immunoreactivity for the catecholamine synthesizing enzyme tyrosine hydroxylase (TH) were examined in the rat septal complex. Dual immunoautoradiographic and peroxidase anti-peroxidase labeling methods were used to simultaneously localize antibodies raised in rabbits against TH and from rat-mouse hybridomas against ChAT in single sections. At least two types of perikarya with ChAT-immunoreactivity (ChAT-I) were observed. The first type were large (20-30 microns), elongated or round, and contained a small indented nucleus with an abundant cytoplasm and an occasional lamellar body. The second type was also either ovoid or round but was medium-sized (15-20 microns) and contained a larger indented nucleus and a smaller amount of cytoplasm than the first type. Both types of perikarya as well as dendrites with ChAT-I were surrounded by astrocytic processes apposed to most of their plasmalemmal surfaces. The distribution and types of terminal associations (i.e., asymmetric synapses, symmetric synapses and appositions which lacked a membrane specialization in the plane of section analyzed) with ChAT-labeled perikarya and dendrites were quantitatively evaluated. The majority (68% of 197) of the presynaptic terminals were unlabeled; the remaining terminals were immunoreactive for TH (25%) or ChAT (7%). All three types of terminals contacted primarily the shafts of small dendrites and more rarely ChAT-labeled perikarya and large dendrites. ChAT-labeled terminals: (1) formed associations with unlabeled perikarya and dendrites (31% of 176); (2) formed associations with perikarya and dendrites with ChAT-I (7%); (3) contacted the same unlabeled perikarya and dendrite as a TH-containing terminal (21%); (4) were in apposition to TH-labeled terminals (25%); or (5) were either in apposition to unlabeled or ChAT-labeled terminals or lacked associations with any processes. The majority of associations formed by the terminals with ChAT-I were on the shafts of small dendrites. Moreover, most of the associations formed were either symmetric synapses or appositions not separated by astrocytes in the plane of section analyzed. These findings provide cellular substrates in the septal complex (1) for sparse synaptic input relative to astrocytic investment of cholinergic neurons and (2) for direct synaptic modulation of cholinergic and non-cholinergic neurons by catecholamines and/or acetylcholine. These findings have direct relevance to catecholaminergic-cholinergic interactions and to the neuropathological basis for Alzheimer's disease.

Ultrastructural localization of neurotensin‐like immunoreactivity within dense core vesicles in perikarya, but not terminals, colocalizing tyrosine hydroxylase in the rat ventral tegmental area

Within the rat ventral tegmental area (VTA), the parabrachial pigmentosus and paranigral subdivisions are known to differ in their functional responses to injected neurotensin. These subdivisions also vary in their connections with other brain regions and in their number of neurotensin-containing perikarya as seen by light microscopy. In both subdivisions, there may be intracellular as well as synaptic relations between dopamine and neurotensin. Dopaminergic neurons are known to be physiologically activated by neurotensin (NT) and may also contain this peptide. To characterize further the cellular relationships in each subdivision, we examined the ultrastructural immunocytochemical localization of a rat antiserum against NT and a rabbit antiserum against the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH) in single sections. The NT antiserum was raised against the entire peptide sequence. Immunoblots showed that the antiserum recognized the original antigen as well as the related peptides neuromedin N and lysine 8- arginine 9- neurotensin 10-13 (LANT-6). In both the parabrachial pigmentosus and paranigral subdivisions, neurotensin-like immunoreactivity (NTLI) was localized predominantly in the large (80-100 nm) dense core vesicles using the peroxidase anti-peroxidase (PAP) method. In tissue labeled for NT by the PAP method and for TH by immunoautoradiography, serial section analysis revealed that all perikarya containing NTLI (n = 19) were also TH-positive. Three times as many perikarya colocalized NTLI and TH in the parabrachial pigmentosus subdivision (n = 15) as in the paranigral subdivision (n = 4). Occasionally, a perikaryon containing TH and NTLI could be found in direct apposition to a TH-labeled perikaryon without glial separation. In contrast to perikarya and dendrites, terminals showing NTLI (38 in parabrachial pigmentosus, 29 in paranigral) lacked detectable TH labeling. Of the terminals containing NTLI whose synaptic junctions could be identified, 48% were symmetric and 10% were asymmetric. The targets of these terminals included perikarya and dendrites lacking detectable immunoreactivity (69% in parabrachial pigmentosus, 55% in paranigral), immunolabeled for TH (26% in parabrachial pigmentosus, 38% in paranigral) or containing both NTLI and TH (5% in parabrachial pigmentosus, 7% in paranigral). Single terminals containing NTLI sometimes contacted more than one neuronal target, some of which were apposed to each other without glial separation. TH-labeled terminals synapsed onto double-labeled perikarya in the paranigral subdivision, but were not observed to do so in the parabrachial pigmentosus subdivision.(ABSTRACT TRUNCATED AT 400 WORDS)

Distribution and origin of noradrenergic and serotonergic fibers in the cochlear nucleus and inferior colliculus of the rat

We examined the monoaminergic innervation of the rat cochlear nucleus (CN) and the inferior colliculus (IC) by using retrograde transport of the fluorescent dye Fluoro-Gold combined with immunohistochemistry. We used antisera against the catecholamine synthesizing enzymes tyrosine hydroxylase (TH), dopamine-β-hydroxylase (DBH) and phenylethanolamine- N-methyltransferase (PNMT), and one against the transmitter serotonin (5-HT). Each substance revealed a distinct pattern of immunoreactive staining in the CN and the IC. In the CN, DBH-immunoreactive (-ir) fibers were present in all subnuclei. The molecular layer of the dorsal CN and the granular layer of the ventral CN, however, were largely devoid of DBH-ir fibers. In contrast, 5-HT-ir fibers were abundant in the molecular layer and the granular cell layer of the CN. In the dorsal CN and the postero- and anteroventral CN, however, this innervation was less dense and evenly distributed across subnuclei. In the IC, the DBH-ir fibers were slightly more numerous in layer 2 of the dorsal cortex than in other subnuclei, while the layer 1 of both the dorsal and the external cortex contained only a few fibers. In contrast, the 5-HT-ir fibers formed a dense network in both the dorsal and external cortices of the IC, while they were less abundant in the remaining subnuclei. PNMT-ir fibers were not found in any of the auditory brainstem nuclei. Following Fluoro-Gold injections into the CN or IC, retrogradely labeled DBH-ir neurons were found in the A6 noradrenergic cell group (locus coeruleus). The CN received additional projections from the A5 noradrenergic cell group, as well as sparse projections from the A4 and A7 cell groups. The serotonergic innervation of the CN and IC originated largely in the B7 serotonergic cell group (dorsal raphe nucleus). Serotonergic neurons in other groups of the raphe nuclei were only occasionally labeled. Our data indicate that both noradrenaline and serotonin may play a role in central auditory processing. Their differential distribution in the IC and CN subnuclei suggests that these transmitter systems might influence different functional circuits.