Histochemistry of sympathetic neurons allotransplanted from young and aged mice to the submandibular gland.

Sympathetic ganglion tissue of young (3 months) and aged (24 months) NMRI mice was allotransplanted into the submandibular gland to study the influence of aging on the survival of grafted neurons. The submandibular gland (SMG) was chosen as a host tissue because of its high concentration of NGF and good blood supply. Four weeks postgrafting the viability of transplants was evaluated using the formaldehyde-induced fluorescence technique, tyrosine hydroxylase (TH) immunohistochemistry, and morphometry. The density of neurons, catecholamine fluorescence and TH immunoreactivity (TH-IR) appeared to be almost unchanged when the transplant was completely surrounded by the SMG tissue, whereas transplants located within the interlobular septum and capsule, or even outside the capsule, showed significantly reduced neuronal survival. The shape of most of the transplanted neurons was not different from those in the intact ganglia. The average diameter of the transplanted young neurons was significantly decreased; this was not the case with the aged neurons. The histograms of grouped diameter values showed a shift to smaller cells in ganglion transplants in both age groups. The transplants in mice treated with 6-OH-dopamine showed considerable regrowth of adrenergic nerve fibers. There seemed to be no marked difference in the survival of transplanted neurons between aged and young animals. The results indicate that the sympathetic neurons from both young adult and aged animals survive the allotransplantation procedure. The neurotrophic factors together with dense vascularization present in the mouse submandibular gland may be beneficial for the restoration of the integrity of mature and aged adrenergic neurons.     

Selective catecholamine depletion of structures along the ventral lamina terminalis: effects on experimentally-induced drinking and pressor responses

Ablation of the periventricular tissue of the anteroventral third ventricle (AV3V) or injection of the chemical neurotoxin, 6-hydroxydopamine (6-OHDA), into the structures along the ventral lamina terminalis will produce deficits in drinking and pressor responses to exogenous angiotensin II (ANG II). Centrally-applied 6-OHDA has been shown to result in widespread depletions of both adrenergic (i.e. both noradrenaline and adrenaline-containing) and dopaminergic neurons. Questions arise, therefore, as to whether a dopaminergic or adrenergic depletion is critical and the locus where reductions must occur. The present experiment was designed to investigate the specificity of the effects of 6-OHDA administration into lamina terminalis-associated structures on ANG II-induced drinking and pressor responses. The nature of the depletion was manipulated with desmethylimipramine (DMI), a drug which blocks the uptake of 6-OHDA into adrenergic but not dopaminergic nerve terminals and thereby spares adrenergic elements. The experimental results indicate that 6-OHDA administration into structures of the ventral lamina terminalis produced ANG II response deficits and marked reductions in catecholamine histofluorescence in the regions of the injection sites. In contrast, pretreatment with DMI protected against the 6-OHDA-produced functional deficits and minimized the effects on histofluorescence. These findings are consistent with the interpretation that adrenergic but not dopaminergic neurons must be present in the structures of the ventral lamina terminalis in order to elicit normal angiotensin-induced drinking and pressor responses.     

Principal neurons and small intensely fluorescent (SIF) cells in the rat superior cervical ganglion have distinct developmental histories.

Sympathetic ganglia contain 2 adrenergic derivatives of the neural crest: principal neurons and small, intensely fluorescent (SIF) cells. The developmental mechanisms responsible for the generation of these 2 cell classes in vivo are not well understood. To examine the possible developmental and lineage relationships between differentiating principal neurons and SIF cells, a fluorescence microscopic study utilizing antibodies against tyrosine hydroxylase (TH) and catecholamine histofluorescence has been combined with the ultrastructural examination of embryonic and postnatal rat superior cervical ganglia (SCG). On embryonic day 12.5, before neuroblasts had become postmitotic, the cells in the SCG possessed intense TH immunoreactivity and had weak to bright catecholamine histofluorescence, but no cells displayed the fine structure of mature SIF cells or neurons. At embryonic days 16.5 and 18.5, postmitotic principal neurons expressed more moderate levels of TH and catecholamines characteristic of the late embryonic and postnatal SCG. By contrast, a small number of cells containing intense TH or catecholamine fluorescence were present in embryonic day 16.5 and older ganglia. Almost all of the intensely fluorescent cells observed were found apposed to capillaries within the ganglion. These embryonic intensely fluorescent cells were larger than SIF cells seen postnatally. Ultrastructural examination of developing ganglia confirmed that cells containing numerous large, dense-cored vesicles (LDCVs) were a prominent feature of ganglia that also contained intensely fluorescent cells. In addition, some embryonic cells containing LDCVs were mitotic. From these and other studies, it seems likely that during development, neuron precursors, in response to differentiation factors such as fibroblast growth factor (FGF) and/or NGF, acquire overt neuronal traits and become postmitotic. Subsequently, cells resembling mature SIF cells appear next to blood vessels, where they may have received other instructional signals such as glucocorticoids. This developmental scheme suggests that the differentiation of principal neurons and SIF cells is independently regulated, and that the ability of SIF cells to convert into principal neurons observed in vitro cannot account for the generation of neurons in vivo.     

Evidence for a role of endogenous nerve growth factor in the functional sex difference in the adrenergic innervation of the male and female mouse submandibular gland

In view of the remarkable sex difference in nerve growth factor (NGF) content in mouse submandibular gland (SMG), a direct comparison of the adrenergic innervation as represented by noradrenaline (NA) content in SMG, rate of losing NA after superior cervical ganglionectomy, post-reserpine recovery of NA and survival and growth of implanted neurons of superior cervical ganglion into SMG was carried out. The results showed that the NA contents in the intact male SMG and in the male SMG during the recovery from reserpinization were higher than those in the female. There were marked sex differences in patterns of the time-course of post-ganglionectomy loss of NA in SMG and in the ability of SMG to support survival and growth of the implanted SCG neurons. These results indicate that NGF in the mouse SMG may play a functional role in the adrenergic neurons innervating the gland.     

Nerve stimulation in vivo acutely increases tyrosine hydroxylase activity in the superior cervical ganglion and its end organs

Previous studies have shown that preganglionic nerve stimulation in vitro increases acutely the activity of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, in sympathetic neuronal cell bodies in the rat superior cervical ganglion (SCG). In the present study, we have examined whether a similar increase occurs after nerve stimulation in vivo, and whether this enzyme activity also increases in sympathetic nerve terminals in autonomic end organs. Immediately following stimulation at 10 Hz for 15 min in vivo, TH activity was found to have increased 4- to 8-fold in the SCG and in 3 of its end organs: the iris, the pineal gland and the submaxillary gland. These results indicate that increases in sympathetic nerve activity in vivo can lead to increases in TH activity both in adrenergic cell bodies/dendrites in the SCG and in adrenergic nerve terminals/fibers in various autonomic end organs.     

Transplantation of the superior cervical ganglion into the brain--a new experimental therapy of Parkinson disease

To supplement catecholamine deficit in the brain with Parkinson disease, we have aimed to transplant the superior cervical ganglion (SCG), which contains norepinephrine and dopamine, into the brain. 1. Transplantation of SCG into rat cerebral cortex SCG was transplanted into the same rat's parietal cortex. Three weeks after the transplantation, catecholamine histofluorescence revealed many transplanted catecholamine cells in the cortex. However, no fibers extended from the transplanted tissue to the cerebral cortex. Some catecholamine fibers extended to the cerebral cortex where 6-OHDA (a specific neurotoxin to the catecholamine neuron) had been pretreated. 2. Transplantation of SCG into the caudate nucleus of MPTP-induced Parkinson monkey For animal model of Parkinson disease, MPTP (1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine) was administered to 5 monkeys. Tow weeks after MPTP administration, dopamine terminals disappeared in the caudate nucleus. After transplantation of SCG in the same animal, many transplanted SCG cells extended their axons to the caudate nucleus. The present results showed that transplanted SCG cells were well survived in the brain. Under a special circumstance such as shortage of catecholamine in the brain, transplanted SCG cells extended their axons into the brain. It is suggested that the transplantation of SCG can be a new therapy for Parkinson disease.     

Neuroprotective actions of selegiline

Selegiline, a selective inhibitor of monoamine oxidase-B (MAO-B), was one of the first adjunct therapies in clinical neurology. A retrospective analysis of data from patients with Parkinson's disease found a significant increase in survival in those treated with selegiline plus L-dopa compared with L-dopa alone. The mechanism of action of selegiline is complex and cannot be explained solely by its MAO-B inhibitory action. Pretreatment with selegiline can protect neurons against a variety of neurotoxins, such as 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP), 6-hydroxydopamine, N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4), methyl-beta-acetoxyethyl-2-chloroethylamine (AF64A), and 5,6-dihydroxyserotonin, which damage dopaminergic, adrenergic, cholinergic, and sertoninergic neurons, respectively. Selegiline produces an amphetamine-like effect, enhances the release of dopamine, and blocks the reuptake of dopamine. It stimulates gene expression of L-aromatic amino acid decarboxylase, increases striatal phenylethylamine levels, and activates dopamine receptors. Selegiline reduces the production of oxidative radicals, up-regulates superoxide dismutase and catalase, and suppresses nonenzymatic and iron-catalyzed autooxidation of dopamine. Selegiline compensates for loss of target-derived trophic support, delays apoptosis in serum-deprived cells, and blocks apoptosis-related fall in the mitochondrial membrane potential. Most of the aforementioned properties occur independently of selegiline's efficacy to inhibit MAO-B.     

Immunohistochemical study of catecholamine enzymes and neuropeptide Y (NPY) in the rostral ventrolateral medulla and bulbospinal projection

The purpose of this study was to determine whether neuropeptide Y (NPY) terminals in the intermediolateral spinal cord originate from the rostral ventrolateral medulla (RVLM). Immunohistochemical staining of tyrosine hydroxylase (TH), dopamine-β-hydroxylase (DBH), phenylethanolamine-N-methyltransferase (PNMT), and NPY in the rat brainstem and spinal cord were performed in this study in order to examine consequences of lesions of the RVLM and of intracisternal injections of 6-hydroxydopamine (6-OHDA) on catecholamine and NPY immunoreactivity in the intermediolateral column (IML) of rats. In addition, ricin, a retrograde neurotoxin, was applied in the superior cervical ganglion (SCG) to determine its effect on catecholamine and NPY immunoreactivity in the IML. Computer-aided image analysis was used to quantify the immunohistochemical changes in the RVLM and spinal cord. The results demosntrated that many catecholamine- and NPY-containing neurons and/or fibers existed in the RVLM and their teminals were found in the IML. After administration of 6-OHDA intracisternally, the catecholamine and NPY immunoreactivities were decreased both in the brainstem and IML of the spinal cord. Following unilateral microinjection of 6-OHDA into the RVLM, the number of NPY- and catecholamine-containing neurons decreased and there was a reduction in neuron terminals on the ipsilateral side. After injection of ricin into the SCG, the catecholamine and NPY neurons of the medulla were not affected, whereas their terminals in the IML decreased ipsilaterally. These results indicate that most of the catecholamine- and NPY-immunoreactive terminals found in the IML originated in the RVLM. These terminals appear to project towards the superior cervical ganglia. © 1993 Wiley-Liss, Inc.     

Structure, afferent innervation, and transmitter content of ganglia of the guinea pig gallbladder: relationship to the enteric nervous system

Although a well-developed plexus of nerves and ganglia is known to be present in the wall of the gallbladder, little has previously been learned about the function or organization of this innervation. The current study was undertaken in order to evaluate the hypothesis that the ganglionated plexus of the gallbladder is analogous to elements of the enteric nervous system (ENS). The ganglionated plexus of the gallbladder was found to resemble closely the submucosal plexus of the small intestine in its organization into two irregular anastomosing and interwoven networks of ganglia, in the numbers of neurons per ganglion, and in the manifestation of histochemically demonstrable acetylcholinesterase activity in virtually all ganglion cells. In common with enteric ganglia, laminin immunoreactivity was observed to be excluded from the interiors of gallbladder ganglia, which were surrounded by a periganglionic laminin-immunoreactive sheath. As in the submucosal plexus, intrinsic substance P-, vasoactive intestinal polypeptide (VIP)-, and neuropeptide Y (NPY)-immunoreactive neurons were seen in the ganglionated plexus of the gallbladder. Extrinsic nerves in the gallbladder that degenerated following chemical sympathectomy with 6-hydroxydopamine (6-OHDA), and which contained NPY, tyrosine hydroxylase (TH), and dopamine-beta-hydroxylase (DBH) immunoreactivities, formed a perivascular plexus closely associated with blood vessels. Endogenous catecholamines could also be demonstrated in these perivascular nerves by aldehyde-induced histofluorescence. In addition to perivascular nerves, paravascular nerve bundles were observed that were loosely associated with vessels, did not degenerate following administration of 6-OHDA, and contained NPY immunoreactivity. Other paravascular nerves, probably visceral sensory axons, coexpressed substance P and calcitonin-gene-related peptide (CGRP) immunoreactivities. The ganglionated plexus of the gallbladder resembled enteric ganglia in having intrinsic 5-hydroxytryptamine (5-HT)-immunoreactive cells and highly varicose nerve fibers. The 5-HT-immunoreactive gallbladder axons were, like those of the gut, resistant to 6-OHDA, and separate from fibers that expressed TH immunoreactivity. Differences between the ganglionated plexus of the gallbladder and enteric ganglia of the small intestine included in the gallbladder are 1) the presence of TH-immunoreactive cells that contain an endogenous catecholamine, but not DBH; 2) DBH-immunoreactive neurons, some of which coexpress substance P immunoreactivity, but which contain neither a catecholamine nor TH immunoreactivity; 3) an apparent absence of CGRP-immunoreactive cell bodies.(ABSTRACT TRUNCATED AT 400 WORDS)     

Biological importance of retrograde axonal transport of nerve growth factor in adrenergic neurons.

Previous studies have shown that nerve growth factor (NGF) produces a selective induction of tyrosine hydroxylase (TH) in peripheral adrenergic neurons and that NGF is transported retrogradely with a high selectivity from the adrenergic nerve terminals to the perikaryon. In order to investigate the biological importance of retrograde NGF transport, the following experiments have been performed; (a) effect of NGF on TH activity in superior cervical ganglia (SCG) after unilateral injection into the anterior eye chamber and the submaxillary gland; and (b) effect of systemic injection of NGF on TH activity in SCG after blockage of retrograde axonal transport by axotomy. After unilateral injection of NGF into the anterior eye chamber and submaxillary gland of both 8-10-day-old rats and adult mice, the increase in TH activity in the SCG was considerably larger on the injected than on the non-injected side although the adrenergic neurons supplying the two organs do not account for more than 25% of the total number of adrenergic neurons in the SCG. A direct diffusion mechanism could be excluded by the fact that unilateral local injection of [125 I] produced no significant side difference in the accumulation of radioactivity in the SCG 2 after injection whereas after 14 h there was a several-fold difference between the injected and non-injected side. Moreover, the nodose ganglia which are located very close to the SCG exhibited no statistically significant difference in the accumulation of radioactivity at any time. Forty-eight hours after subcutaneous injections of 10 mg/kg of NGF the increase in TH activity of the SCG amounted to 154% on the intact side and to 92% on the axotomized side. However, these experiments do not permit decisions about the extent the axotomy, as such, impaired the response to NGF. It is concluded that the biological effect of NGF results to a considerable extent, from the moiety which reaches the cell body by retrograde transport from the nerve terminals.     

Atlas of catecholamine perikarya, varicosities and pathways in the brainstem of the cat

By application of a modified glyoxylic acid--paraformaldehyde histofluorescence technique, catecholamine perikarya, varicosities, and pathways were delineated within the brainstem of kittens that were either untreated, pretreated pharmacologically, or injected intracerebrally with 6-OHDA. Three principle catecholamine cell groups were identified within the medulla and pons; the dorsomedial medullary cell group, the dorsolateral pontine cell group, and a ventrolateral cell group extending from the medulla into the pons. Induced axonal accumulation of catecholamines with intracerebral 6-OHDA injections revealed a major longitudinal catecholamine bundle which courses in a dorsolateral position through the entire brainstem tegmentum. The dorsomedial medullary and dorsolateral pontine cell groups contribute ascending and descending fibers to this bundle. Axons of the ventrolateral pontomedullary cells also feed into the bundle at successive levels through radially coursing transverse fibers. Via this major dorsolateral conduit and its ventrally and medially coursing tributaries, catecholamine fibers and terminals are distributed to multiple nuclei through the brainstem. The regions of the catecholamine cell groups and the serotonin raphe nuclei all receive a dense catecholamine innervation. Varicosities are also dense in the visceral cranial nerve nuclei, moderately dense in most somatic spinal and cranial nerve motor nuclei, and moderate to light in sensory cranial nerve and relay nuclei. The lateral and ventromedial reticular formation are moderately innervated by varicose catecholamine fibers that traverse these regions. The longitudinal catecholamine bundle continues caudally into the lateral funiculus to descend into and innervate the spinal cord. Rostrally it continues into the tegmental fascicles of the midbrain to ascend into and innervate the diencephalon and there join the medial forebrain bundle to ascend into the telencephalon. Thus, the catecholamine neurons utilize this dorsolateral longitudinal bundle to distribute collaterals to multiple bulbar nuclei and to travel beyond the brainstem to innervate the spinal cord and forebrain.     

Neonatal 6-hydroxydopamine treatment eliminates cholinergic sympathetic innervation and induces sensory sprouting in rat sweat glands

Previous studies of the development of cholinergic sympathetic innervation of sweat glands in rat footpads suggested that these terminals initially exhibit noradrenergic properties which are lost as the glands and their innervation mature. We have treated neonatal and adult rats with 6-hydroxydopamine (6-OHDA), a toxic congener of norepinephrine, and compared its effects on the cholinergic sympathetic innervation of sweat glands and the noradrenergic sympathetic innervation of the iris, salivary gland, and blood vessels. As reported by others, 6-OHDA treatment of neonates caused the destruction of noradrenergic fibers in the iris and salivary gland but did not affect other fibers projecting to these targets that stain for acetylcholinesterase (AChE). We found that 6-OHDA treatment of neonatal animals also caused the destruction of the sympathetic axons in immature sweat glands that possess catecholamine histofluorescence and tyrosine-hydroxylase-like immunoreactivity. Furthermore, when such animals were examined as adults, we found no AChE staining, vasoactive intestinal peptide (VIP)-like immunoreactivity, or characteristic sympathetic axonal varicosities. However, the denervated glands were invested by a plexus of sensory axons, some of which exhibited substance P-like immunoreactivity (SP-IR). An increase in the number of SP-IR fibers also occurred in the sympathetically denervated irides of these animals. Chronic treatment of neonates with guanethidine, another adrenergic sympathetic neurotoxin, resulted in similar loss of cholinergic sweat gland innervation. Treatment of adults rats with doses of 6-OHDA identical to those used to treat neonates caused the loss of noradrenergic fibers from the iris, salivary gland, and many blood vessels but did not noticeably affect AChE and VIP staining or axonal ultrastructure in the sweat glands. However, treatment with higher doses of 6-OHDA did cause significant axonal degeneration. The response of the sympathetic innervation of developing but not mature sweat glands to 6-OHDA provides evidence for a transition from noradrenergic to cholinergic phenotype during the development of sympathetic neurons in vivo similar to the transition observed in cell culture. The sprouting of sensory axons may be caused by NGF-like trophic influences present in some sympathetically denervated tissues.     

Immunochemical detection of diverse molecular species of horseradish peroxidase recovered from rat superior cervical ganglion following retrograde transport

The sympathetic innervation of the submandibular gland (SMG) by neurons of the superior cervical ganglion (SCG) was used as a model system to characterize the different molecular species of retrogradely transported horseradish peroxidase (HRP). Following injections of 3 mg of HRP into the SMG, enzymatically active HRP was demonstrated by histochemistry in SCG neurons for up to 14 days. Parallel immunochemical studies were conducted on electroblots of SCG homogenates from other rats subjected to similar SMG injections of HRP. This technique allowed the detection of nanogram amounts of HRP. These experiments demonstrated that an immunochemically detectable 40 Kd species of HRP was retrogradely transported and retained in SCG neurons for up to 6 days. Beyond 8 days, only lower molecular weight (15-30 Kd) immunoreactive breakdown products of HRP were observed in SCG homogenates. Similar spontaneous HRP breakdown products displayed enzymatic activity in non-denaturing gels. These studies suggest that retrogradely transported HRP is degraded by neurons to lower molecular weight species which retain enzymatic activity. We conclude that morphological studies of retrogradely transported HRP visualized by histochemical methods reflect the response of neurons to a variety of enzymatically active molecular forms of HRP.     

Prolonged sympathetic innervation of sensory neurons in rat thoracolumbar dorsal root ganglia during chronic colitis

Background Peripheral irritation‐induced sensory plasticity may involve catecholaminergic innervation of sensory neurons in the dorsal root ganglia (DRG). Methods Catecholaminergic fiber outgrowth in the thoracolumbar DRG (T13‐L2) was examined by tyrosine hydroxylase (TH) immunostaining, or by sucrose‐potassium phosphate‐glyoxylic acid histofluorescence method. TH level was examined by Western blot. Colonic afferent neurons were labeled by retrograde neuronal tracing. Colitis was induced by intracolonic instillation of tri‐nitrobenzene sulfonic acid (TNBS). Key Results The catecholaminergic fibers formed ‘basket‐like’ structures around the DRG cells. At 7 days following TNBS treatment, the number of DRG neurons surrounded by TH‐immunoreactive fibers and the protein levels of TH were significantly increased in T13, L1, and L2 DRGs (two‐ to threefold, P < 0.05). The DRG neurons that were surrounded by TH immunoreactivity were 200 kDa neurofilament‐positive, but not isolectin IB4‐positve or calcitonin gene‐related peptide‐positive. The TH‐immunoreactive fibers did not surround but adjoin the specifically labeled colonic afferent neurons, and was co‐localized with glial marker S‐100. Comparison of the level of TH and the severity of colonic inflammation showed that following TNBS treatment, the degree of colonic inflammation was most severe at day 3, subsided at day 7, and significantly recovered by day 21. However, the levels of TH in T13‐L2 DRGs were increased at both 3 days and 7 days post TNBS treatment and persisted up to 21 days (two‐ to fivefold increase, P < 0.05) as examined. Conclusions & Inferences Colonic inflammation induced prolonged catecholaminergic innervation of sensory neurons, which may have relevance to colitis‐induced chronic visceral hypersensitivity and/or referred pain.     

Early appearance of tyrosine hydroxylase immunoreactive cells in the mesencephalon of mouse embryos

The development of mesencephalic catecholaminergic neurons in the embryonic and fetal mouse was analysed in tissues fixed with 5% acrolein using polyclonal rabbit antibodies against tyrosine hydroxylase (TH), the first enzyme in catecholamine synthesis. The first TH positive cells were identified as early as day 8.5-9 of gestation and some expressed TH while apparently still migrating from the proliferative layer. The number of catecholamine cells increased dramatically by embryonic day 9.5-10; at gestation days 10.5-11 numerous TH positive cells bearing many neurites were localized in the ventral part of the mesencephalon but they were not yet separated into two different groups (A9 and A10). After 13 days of gestation two separate catecholaminergic groups could be visualized, although many TH positive cells with long neurites (putative dopaminergic neurons) could still be seen at the edges of the ventricle, and appeared to be moving towards the ventral mesencephalon. On the basis of these results the possibility that catecholamine cells that are produced early during the development of the midbrain may have neurotrophic and/or morphogenetic roles is discussed.     

Regeneration of central catecholamine fibers in young and aged rat brain

Catecholamine histofluorescence patterns were examined in brains of young and aged rats, 1 to 14 days following neurosurgical transection of the medial forebrain bundle. At all ages examined, two phenomena were observed: degeneration of nerve fibers and vigorous regrowth of catecholamine-containing fibers in the lesion site. Regenerated catecholamine fibers invaded the area of scarred tissue. This invasion of the scarred area implies that the robust plasticity of catecholaminergic pathways, known to exist in young animals, persists in aged brain.     

Effect of 6-hydroxydopamine treatment on kynurenine aminotransferase-I (KAT-I) immunoreactivity of neurons and glial cells in the rat substantia nigra

Parkinson’s disease (PD), a progressive neurodegenerative disorder, is characterized by a preferential loss of dopaminergic neurons in the substantia nigra pars compacta (SNPC). Neurons in the SNPC are known to express tyrosine hydroxylase (TH); therefore, in a commonly used PD model, 6-hydroxydopamine (6-OHDA), a selective catecholamine neurotoxin, induces neuronal death in SNPC. We have shown with immunohistochemical techniques that kynurenine aminotransferase-I (KAT-I), the enzyme taking part in the formation of kynurenic acid (KYNA)—the only known endogenous selective NMDA receptor antagonist and a potent neuroprotective agent—is also expressed in the rat SNPC. We found that KAT-I and TH co-exist in the very same neurons of SNPC and that 6-OHDA injected into the lateral ventricle produced loss of the majority of nigral neurons. Densitometric analysis proved that, in consequence of 6-OHDA treatment, not only TH but also KAT-I immunoreactivity diminished considerably in the remaining SNPC neurons. Astrocytes in the substantia nigra were found to express KAT-I under normal conditions; the amount of this enzyme increased after administration of 6-OHDA, whereas microglial cells became KAT-I immunoreactive only after 6-OHDA treatment. Since intrinsic KYNA in SNPC neurons is perceptibly insufficient to protect them from the deleterious effect of 6-OHDA, it is hypothesized that biochemical approaches which increase KYNA content of the central nervous system might prevent the deleterious effect of 6-OHDA and, supposedly, also the neuronal degradation characterizing PD.     

Colocalization of gamma-aminobutyric acid-like immunoreactivity and catecholamines in the feeding network of Aplysia californica

Functional consequences of neurotransmitter coexistence and cotransmission can be readily studied in certain experimentally favorable invertebrate motor systems. In this study, whole-mount histochemical methods were used to identify neurons in which gamma-aminobutyric acid (GABA)-like immunoreactivity (GABAli) was colocalized with catecholamine histofluorescence (CAh; FaGlu method) and tyrosine hydroxylase (TH)-like immunoreactivity (THli) in the feeding motor circuitry (buccal and cerebral ganglia) of the marine mollusc Aplysia californica. In agreement with previous reports, five neurons in the buccal ganglia were found to exhibit CAh. These included the paired B20 buccal-cerebral interneurons (BCIs), the paired B65 buccal interneurons, and an unpaired cell with projections to both cerebral-buccal connectives (CBCs). Experiments in which the FaGlu method was combined with the immunohistochemical detection of GABA revealed double labeling of all five of these neurons. An antibody generated against TH, the rate-limiting enzyme in the biosynthesis of catecholamines, was used to obtain an independent determination of GABA-CA colocalization. Biocytin backfills of the CBC performed in conjunction with TH immunohistochemistry revealed labeling of the rostral B20 cell pair and the unpaired CBI near the caudal surface of the right hemiganglion. THli was also present in a prominent bilateral pair of caudal neurons that were not stained with CBC backfills. On the basis of their position, size, shape, and lack of CBC projections, the lateral THli neurons were identified as B65. Double-labeling immunohistochemical experiments revealed GABAli in all five buccal THli neurons. Finally, GABAli was observed in individual B20 and B65 neurons that were identified using electrophysiological criteria and injected with a marker (neurobiotin). Similar methods were used to demonstrate that a previously identified catecholaminergic cerebral-buccal interneuron (CBI) designated CBI-1 contained THli but did not contain GABAli. Although numerous THli and GABAli neurons and fibers were present in the cerebral and buccal ganglia, additional instances of their colocalization were not observed. These findings indicate that GABA and a catecholamine (probably dopamine) are colocalized in a limited number of interneurons within the central pattern generator circuits that control feeding-related behaviors in Aplysia.     

A review of the pharmacology of selegiline

Selegiline (1-deprenyl) is an irreversible inhibitor of monoamine oxidase (MAO) type B. Because in the human brain, dopamine is metabolised mainly by MAO-B, selegiline increases dopamine content in the central nervous system. Besides the inhibition of MAO-B, selegiline also inhibits the uptake of dopamine and noradrenaline into presynaptic nerve and increases the turnover of dopamine. Thanks to these properties, selegiline significantly potentiates the pharmacological effects of levodopa. These favourable characteristics have been applied in the treatment of Parkinson's disease using selegiline both with levodopa and alone.
Unlike earlier MAO-inhibitors, selegiline does not potentiate the hypertensive effects of tyramine. This is due to the selectivity to MAO-B, leaving intestinal MAO-A intact, and also due to the fact that selegiline inhibits the uptake of tyramine into neurons.
Selegiline can prevent the parkinsonism caused by MPTP in animals; similar findings have been reported with other toxins like 6-OHDA and DSP-4, that destroys noradrenergic nuclei. Furthermore, selegiline reduces oxidative stress caused by degradation of dopamine and increases free radical elimination by enhancing superoxide dismutase and catalase activity. These findings may be important when considering the possible neuroprotective effects of selegiline.
Besides the basic pharmacology also the interactions and pharmacokinetics of selegiline are reviewed in this article.     

The origin of catecholaminergic nerve fibers in the subdiaphragmatic vagus nerve of rat.

It is known that the vagus nerve contains catecholaminergic fibers. However, the origin of these fibers has not been systematically examined. In this study, we addressed this issue using retrograde tracing from the subdiaphragmatic vagus nerve combined with immunocytochemistry. The cervical and thoracic sympathetic trunk ganglia, the nodose ganglia and the dorsal motor nucleus of the vagus nerve were examined following injection of Fluoro-Gold or cholera toxin horseradish peroxidase conjugate into the trunks of the subdiaphragmatic vagus nerve of rats. Numerous retrogradely labeled neurons were seen in the nodose ganglion and the dorsal motor nucleus of the vagus nerve. Very few labeled neurons were found in the sympathetic ganglia (less than 0.06% of the neurons in either superior cervical ganglion or cervicothoracic ganglion were retrogradely labeled). Double labeling with immunofluoresence for catecholamine synthesizing enzymes revealed that: (1) 92% of all Fluoro-Gold retrogradely labeled tyrosine hydroxylase immunoreactive neurons were found in parasympathetic sources (75% in the dorsal motor nucleus of the vagus nerve and 17% in the nodose ganglia), and only 8% in the cervicothoracic sympathetic ganglia; (2) 12% of the retrogradely labeled catecholaminergic neurons in the dorsal motor nucleus of the vagus nerve were also dopamine-beta-hydroxylase immunopositive neurons; (3) 70% of the retrogradely labeled neurons in the sympathetic ganglia were tyrosine hydroxylase immunopositive and 54% of these catecholaminergic neurons contained dopamine-beta-hydroxylase, while 30% of the retrogradely labeled neurons were non-catecholaminergic neurons. These results indicate that catecholaminergic fibers in the abdominal vagus nerve are primarily dopaminergic and of parasympathetic origin, and that only an extremely small number of these fibers, mostly noradrenergic in nature, arise from postganglionic sympathetic neurons.