Salsolinol facilitates glutamatergic transmission to dopamine neurons in the posterior ventral tegmental area of rats

Although in vivo evidence indicates that salsolinol, the condensation product of acetaldehyde and dopamine, has properties that may contribute to alcohol abuse, the underlying mechanisms have not been fully elucidated. We have reported previously that salsolinol stimulates dopamine neurons in the posterior ventral tegmental area (p-VTA) partly by reducing inhibitory GABAergic transmission, and that ethanol increases glutamatergic transmission to VTA-dopamine neurons via the activation of dopamine D(1) receptors (D(1)Rs). In this study, we tested the hypothesis that salsolinol stimulates dopamine neurons involving activation of D(1)Rs. By using whole-cell recordings on p-VTA-dopamine neurons in acute brain slices of rats, we found that salsolinol-induced increase in spike frequency of dopamine neurons was substantially attenuated by DL-2-amino-5-phosphono-valeric acid and 6, 7-dinitroquinoxaline-2, 3-dione, the antagonists of glutamatergic N-Methyl-D-aspartic acid and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors. Moreover, salsolinol increased the amplitude of evoked excitatory postsynaptic currents (EPSCs) and the frequency but not the amplitude of spontaneous EPSCs. Additionally, SKF83566, a D(1)R antagonist attenuated the salsolinol-induced facilitation of EPSCs and of spontaneous firing of dopamine neurons. Our data reveal that salsolinol enhances glutamatergic transmission onto dopamine neurons via activation of D(1)Rs at the glutamatergic afferents in dopamine neurons, which contributes to salsolinol's stimulating effect on p-VTA dopamine neurons. This appears to be a novel mechanism which contributes toward rewarding properties of salsolinol.     

Salsolinol, a tetrahydroisoquinoline-derived neurotoxin, induces oxidative modification of neurofilament-L protection by histidyl dipeptides

Salsolinol (1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline) is a compound derived from dopamine metabolism and is capable of causing dopaminergic neurodegeneration. Oxidative modification of neurofilament proteins has been implicated in the pathogenesis of neurodegenerative disorders. In this study, oxidative modification of neurofilament-L (NF-L) by salsolinol and the inhibitory effects of histidyl dipeptides on NF-L modification were investigated. When NF-L was incubated with 0.5 mM salsolinol, the aggregation of protein was increased in a time-dependent manner. We also found that the generation of hydroxyl radicals (?OH) was linear with respect to the concentrations of salsolinol as a function of incubation time. NF-L exposure to salsolinol produced losses of glutamate, lysine and proline residues. These results suggest that the aggregation of NF-L by salsolinol may be due to oxidative damage resulting from free radicals. Carnosine, histidyl dipeptide, is involved in many cellular defense processes, including free radical detoxification. Carnosine, and anserine were shown to significantly prevent salsolinol- mediated NF-L aggregation. Both compounds also inhibited the generation of ?OH induced by salsolinol. The results indicated that carnosine and related compounds may prevent salsolinol-mediated NF-L modification via free radical scavenging.     

Some parasympathetic neurons in the guinea-pig heart express aspects of the catecholaminergic phenotype in vivo

Summary In a histochemical study of intrinsic cardiac ganglia of the guinea-pig in whole-mount preparations, it was found that some 70–80% of the neurons express aspects of the catecholaminergic phenotype. These neurons have an uptake mechanism for L-DOPA, and contain the enzymes for converting L-DOPA, (but not D-DOPA) to dopamine and noradrenaline, i.e. aromatic L-aminoacid decarboxylase and dopamine -hydroxylase. Monoamine oxidase is also present within some of the neurons. In these respects, the neurons resemble noradrenergic neurons of sympathetic ganglia, so we refer to them as intrinsic cardiac amine-handling neurons. However, these neurons do not contain tyrosine hydroxylase and show little or no histochemically detectable uptake of -methyldopa, dopamine or noradrenaline, even after depletion of endogenous stores of amines by pre-treatment with reserpine. Noradrenergic fibres from the sympathetic chain form pericellular baskets around nerve cell bodies. The uptake of L-DOPA into nerve cell bodies is not prevented by treatment with 6-hydroxydopamine sufficient to cause transmitter-depletion or degeneration of the extrinsic noradrenergic fibres. Such degeneration experiments suggest that axons of the amine-handling neurons project to cardiac muscle, blood vessels and other intrinsic neurons. The cardiac neurons do not show any immunohistochemically detectable serotonergic characteristics; there is no evidence for uptake of the precursors L-tryptophan and 5-hydroxytryptophan or 5-HT itself, whereas the extrinsic noradrer ergic nerve fibres within the ganglia can take up 5-HT when it is applied in high concentrations.Abbreviations AChE acetylcholinesterase - DBH-IR dopamine -hydroxylase-like immunoreactivity - L-DOPA L-dihydroxyphenylalamine - 5-HT-IR 5-hydroxytryptamine-like immunoreactivity - 6-OHDA 6-hydroxydopamine - methyldopa L--methyl-dihydroxyphenylalanine - MAO monoamine oxidase - NPY neuropeptide Y - SIF small intensely fluorescent cells - TH-IR tyrosine hydroxylase-like immunoreactivity - VIP vasoactive intestinal polypeptide     

Immunohistochemical characterisation of sympathetic and parasympathetic pelvic neurons projecting to the distal colon in the male rat

The pelvic ganglia are mixed ganglia containing both sympathetic and parasympathetic neurons that receive spinal input via the hypogastric (lumbar cord) and pelvic nerves (sacral cord), respectively. A recent study has utilised immunohistochemistry against synaptophysin (a protein associated with small vesicles) to visualise the preganglionic terminals in these ganglia. By selectively cutting the hypogastric or pelvic nerves and allowing subsequent terminal degeneration, the populations of parasympathetic and sympathetic preganglionic terminals, respectively, can be visualised. The present study has used this method in conjunction with retrograde labelling of pelvic neurons from the distal colon and double label immunofluorescence against tyrosine hydroxylase and vasoactive intestinal polypeptide (VIP) to identify and characterise the sympathetic and parasympathetic neurons projecting to the distal colon from the major pelvic ganglia of the male rat. Approximately equal numbers of distal colonic-projecting pelvic neurons are sympathetic and parasympathetic. Almost all noradrenergic neurons are sympathetic. Of the VIP neurons that project to the distal colon approximately one third are sympathetic, one third parasympathetic and the remaining third are possibly innervated by both the lumbar and sacral cord. Extrapolation from our results also suggests that the majority of non-noradrenergic neuropeptide Y neurons (which are known to comprise the remainder of the neurons) are parasympathetic. These studies have demonstrated that the pelvic ganglia are a major source of sympathetic innervation to the distal bowel and have further shown that the distal colon is another target for the non-noradrenergic sympathetic neurons of the pelvic ganglia.     

Regional distribution of tetrahydroisoquinoline derivatives in rodent,human, and parkinson’s disease brain

Several members of the tetrahydroisoquinoline (TIQ) family of monoamine alkaloids can be formed from dopamine or its oxidized metabolites and may be involved in the pathogenesis of monoaminergic cell death in Parkinson’s disease (PD). Using enantiomeric‐selective high‐performance liquid chromatography with electrochemical detection and liquid chromatography with tandem mass spectroscopy, the regional concentrations of several TIQ derivatives, including salsolinols, were determined in mouse, rat, normal human, and PD brain. TIQ derivatives were detected in all regions subjected to analysis. In general, salsolinols were present at higher concentrations than TIQ and its benzyl and methyl derivatives, especially in human brain. Moreover, salsolinols were concentrated in areas with increased dopamine synthesis and turnover such as the ventral midbrain and striatum, respectively. A possible consequence of nigrostriatal dopaminergic cell death, significantly lower levels of (R)salsolinol, (S)salsolinol, N‐methyl‐(R)salsolinol and N‐methyl‐(S)salsolinol were found in the caudate nuclei of PD in comparison with normal human brain. Our data support the hypothesis of endogenous synthesis of salsolinols and provide evidence for their accumulation in catecholaminergic neurons.     

A possible mechanism of dopamine-induced synergistic disinhibition of thalamic cells via "direct" and "indirect" pathways in basal ganglia

On the basis of the mechanism of synaptic plasticity that we have earlier suggested for striatal spiny neurons and with regard to the known data about the predominance of dopamine-sensitive D1/D2 receptors on the striatonigral/striatopallidal cells it is hypothesized that the induction of the long-term potentiation/depression of the efficacy of excitatory cortical inputs to these cells can underlie the excitatory/inhibitory effect of dopamine on the activity of neurons that originate the "direct"/"indirect" pathways through the basal ganglia. Both these effects will lead to an enhancement of the activity of thalamic cells and activity of the efferent neocortical neurons excited by thalamic cells. The long-term potentiation of corticostriatal inputs to striosomal neurons, where, predominantly, D1 receptors are located, can also be induced by dopamine. This effect can be responsible of a rise of inhibition of dopaminergic cells and decrease in dopamine release by these cells. Such an event sequence can provide a stable dopamine concentration in the loop neocortex-basal ganglia-thalamus-neocortex.     

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.     

Catecholamines and catecholamine-synthesizing enzymes in guinea-pig sensory ganglia

Summary Cranial and spinal sensory ganglia of the guinea-pig were investigated by means of histochemistry and biochemistry for the presence of catecholamines and catecholamine-synthesizing enzymes. Sensory neurons exhibiting immunoreactivity to the rate-limiting enzyme of catecholamine synthesis, tyrosine nydroxylase (TH), were detected by immunohistochemistry in lumbo-sacral dorsal root ganglia, the nodose ganglion and the petrosal/jugular ganglion complex. The carotid body was identified as a target of TH-like-immunoreactive (TH-LI) neurons by the use of combined retrograde tracing and immunohistochemistry. Double-labelling immunofluorescence revealed that most TH-LI neurons also contained somatostatin-LI, but TH-LI did not coexist with either calcitonin gene-related peptide- or substance P-LI. TH-LI neurons did not react with antibodies to other enzymes involved in catecholamine synthesis, i.e., aromatic amino acid decarboxylase (AADC), dopamine--hydroxylase (DH), and phenylethanolamine-N-methyltransferase (PNMT). Petrosal neurons as well as their endings in the carotid body lacked dopamine- and L-DOPA-LI. Sensory neurons did not display glyoxylic acid-induced catecholamine fluorescence. Ganglia containing TH-LI neurons were kept in short-term organ culture after crushing their roots and the exiting nerve in order to enrich intra-axonal transmitter content at the ganglionic side of the crush. However, even under these conditions, catecholamine fluorescence was not detected in axons projecting peripherally or centrally from the ganglia. Sympathetic noradrenergic nerves entered the ganglia and terminated within them. Accordingly, biochemical analyses of guinea-pig sensory ganglia revealed noradrenaline but no dopamine. In conclusion, catecholamines within guinea-pig sensory ganglia are confined to sympathetic nerves, which fulfill presently unknown functions. The TH-LI neurons themselves, however, lack any additional sign of catecholamine synthesis, and the presence of enzymatically active TH within these neurons is questionable.     

Dopamine-containing small intensely fluorescent cells and sympathetic ganglion function

This article reviews some of the neuropharmacology of the dopamine (DA)-containing small intensely fluorescent cells of sympathetic ganglia. The major metabolite of DA found in the ganglia is 3,4-dihydroxyphenylacetic acid (DOPAC). DOPAC content appears to be a direct reflection of DA synthesis. DA synthesis can be enhanced by muscarinic agonists and diminished by muscarinic antagonists. Neuroleptic drugs stimulate DA synthesis in the ganglion, which suggests that a local negative neuronal feedback loop might operate within the ganglion. There may be a correlation between deficient DA synthesis in spontaneous hypertensive rats and the development of hypertension. It is possible that some of the peripheral side effects of drugs that act on dopaminergic neurons in the brain might originate from the drugs' action on peripheral dopaminergic neuronal systems such as the sympathetic ganglion.     

Leptin receptors,NPY, and tyrosine hydroxylase in autonomic neurons supplying fat depots in a pig

The goal of this study was to determine the immunohistochemical characteristics of peripheral adrenergic OBR-immunoreactive (OBR-IR) neurons innervating adipose tissue in a pig. The retrograde tracer, Fast Blue (FB), was injected into either the subcutaneous, perirenal, or mesentery fat tissue depots of three male and three female pigs each with approximately 50 kg body weight. Sections containing FB(+) neurons were stained for OBR, tyrosine hydroxylase (TH) or neuropeptide Y (NPY) using a double labeling immunofluorescence method. OBR, TH, and NPY immunoreactivities were present in the thoracic (T) and lumbar (L) ganglia of the sympathetic chain, as well as in the coeliac superior mesenteric ganglion (CSMG), inferior mesenteric ganglion (IMG), intermesenteric ganglia (adrenal-ADG, aorticorenal-ARG, and ovarian-OG or testicular-TG ganglion). These results indicate that, in addition to neuroendocrine functions, leptin may affect peripheral tissues by acting on receptors located in sympathetic ganglion neurons.     

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. © 1992 John Wiley & Sons, Inc.     

Nitric oxide synthase-containing neurons in rat parasympathetic, sympathetic and sensory ganglia: a comparative study

Summary In rats, the distribution of nerve structures staining for NADPH-diaphorase, and showing immunoreactivities for nitric oxide synthase (NOS), tyrosine hydroxylase and various neuropeptides was studied in sensory ganglia (dorsal root, nodose and trigeminal ganglia), in sympathetic ganglia (superior cervical, stellate, coeliac-superior and inferior mesenteric ganglia), parasympathetic ganglia (sphenopalatine, submandibular, sublingual and otic ganglia), and in the mixed parasympathetic/ sympathetic ganglia (major pelvic ganglia). The coincidence of neuronal cell bodies with strong NOS-immunoreactivity and strong NADPH diaphorase reactivity was almost total. The relative proportions of NOS-immunoreactive nerve cell bodies were largest in parasympathetic ganglia and major pelvic ganglia followed by sensory ganglia. In sympathetic ganglia no NOS-immunoreactive neuronal cell bodies could be detected. In parasympathetic and major pelvic ganglia, there was a very significant neuronal co-localization of immunoreactivities for NOS and vasoactive intestinal polypeptide (VIP). This was almost total in major pelvic ganglia, in which NOS-/VIP-immunoreactive nerve cell bodies were separate from sympathetic (tyrosine hydroxylase-/neuropeptide Y-immunoreactive), suggesting that NOS-/VIP-immuno-reactive neurons might also be parasympathetic.     

Distribution and ontogeny of SP, CGRP, SOM, and VIP in chick sensory and sympathetic ganglia

The distribution and ontogeny of four neuropeptides in developing chick lumbosacral sensory and sympathetic ganglia were studied using immunohistochemical techniques. Antibodies to two of these peptides, substance P (SP) and calcitonin gene-related peptide (CGRP), stained small neurons in the medial part of the dorsal root ganglia from embryonic Day 5 and Day 10, respectively, whereas neurons in the lateral part of the ganglia were negative; this distribution persisted throughout development. Both sets of neurons apparently send fibers to the dorsal horn of the spinal cord: SP to laminae I and II, and CGRP to lamina I, suggesting that the SP- and CGRP-positive sensory neurons are nociceptive or thermoreceptive. This correlation between the presence of SP or CGRP in a neuron and a particular functional modality thus provides evidence for a functional distinction between the mediodorsal and ventrolateral zones that are apparent during the development of chick dorsal root ganglia. Moreover, this study suggests that the type of neuron that develops within the dorsal root ganglion correlates with its position within the ganglion. In contrast to SP and CGRP, somatostatin (SOM) and vasoactive intestinal polypeptide (VIP) immunoreactivities were not seen in the lumbosacral sensory ganglia at any stage during development. However, both were present in sympathetic ganglia: SOM from embryonic Day 4.5 and VIP from embryonic Day 10. VIP immunoreactivity persisted throughout development in a large number of sympathetic neurons, but the number of cells with SOM immunoreactivity decreased from embryonic Day 10 onward. SOM therefore appears to be present only transiently in most chick lumbosacral sympathetic cells.     

Selective Depletion of Dopamine, Octopamine, and 5-Hydroxytryptamine in the Nervous Tissue of the Cockroach (Periplaneta americana)

High-performance liquid chromatography with electrochemical detection was used to measure the concentrations of 3,4-dihydroxyphenylethylamine (dopamine), 5-hydroxytryptamine (5-HT), p-hydroxyphenylethanolamine (octopamine), alpha-methyl-p-tyrosine, and tryptophan in the cerebral ganglia of cockroaches (Periplaneta americana) after peripheral administration of alpha-methyl-p-tyrosine and alpha-methyltryptophan. In addition, the levels of dopamine, 5-HT, octopamine, alpha-methyl-p-tyrosine, and tryptophan were determined after injection of alpha-methyl-p-tyrosine, 6-hydroxydopamine, or 5,7-dihydroxytryptamine directly into the cerebral ganglia by means of microinjection needles. Peripheral administration of alpha-methyl-p-tyrosine (400-1,600 micrograms/insect) caused a reduction in dopamine and 5-HT concentrations in cockroach cerebral ganglia, although the reduction in dopamine concentrations was more pronounced. Peripheral injections of alpha-methyl-p-tyrosine also reduced octopamine levels in the cerebral ganglia. Peripheral injection of alpha-methyltryptophan (400-1,600 micrograms/insect) caused a marked reduction in 5-HT and tryptophan concentrations in cockroach cerebral ganglia without altering dopamine or octopamine concentrations. Central injections of alpha-methyl-p-tyrosine (80 micrograms/insect) reduced dopamine concentrations in the cerebral ganglia. However, neither 6-hydroxydopamine (20 micrograms/insect) nor 5,7-dihydroxytryptamine (20 micrograms/insect) caused reductions in amine levels when applied near or directly into the cerebral ganglia. The results suggest that specific lesions of aminergic neurons in insects by either 6-hydroxydopamine or 5,7-dihydroxytryptamine are impractical. The specific, long-lasting depletion of 5-HT by alpha-methyltryptophan suggests that this chemical may be useful in elucidating the functions of 5-HT in insects.     

Inhibition of Tyrosine Hydroxylase by R and S Enantiomers of Salsolinol, 1-Methyl-6,7-Dihydroxy-1,2,3,4- Tetrahydroisoquinoline

Salsolinol is one of the dopamine-derived tetrahydroisoquinolines and is synthesized from pyruvate or acetaldehyde and dopamine. As it cannot cross the blood-brain barrier, salsolinol as the R enantiomer in the brain is considered to be synthesized in situ in dopaminergic neurons. Effects of R and S enantiomers of salsolinol on kinetic properties of tyrosine hydroxylase [tyrosine, tetrahydrobiopterin:oxygen oxidoreductase (3-hydroxylating); EC 1.14.16.2], the rate-limiting enzyme of catecholamine biosynthesis, were examined. The naturally occurring cofactor of tyrosine hydroxylase, L-erythro-5,6,7,8-tetrahydrobiopterin, was found to induce allostery to the enzyme polymers and to change the affinity to the biopterin itself. Using L-erythro-5,6,7,8-tetrahydrobiopterin, tyrosine hydroxylase recognized the stereochemical structures of the salsolinols differently. The asymmetric center of salsolinol at C-1 played an important role in changing the affinity to L-tyrosine. The allostery of tyrosine hydroxylase toward biopterin cofactors disappeared, and at low concentrations of biopterin such as in brain tissue, the affinity to the cofactor changed markedly. A new type of inhibition of tyrosine hydroxylase, by depleting the allosteric effect of the endogenous biopterin, was found. It is suggested that under physiological conditions, such a conformational change may alter the regulation of DOPA biosynthesis in the brain.     

Development of the cholinergic neurotransmitter phenotype in postganglionic sympathetic neurons

Sympathetic ganglia are composed of noradrenergic neurons and cholinergic neurons that differ in the expression of neurotransmitter-synthesizing enzymes, neurotransmitter transporters and neuropeptides. The analysis of the cholinergic differentiation during development revealed important principles involved in the generation of neuronal diversity, in particular the importance of signals from the innervated target. Some peripheral targets, such as the sweat glands in the mammalian footpads, are purely cholinergically innervated in the adult, whereas skeletal muscle arteries receive both noradrenergic and cholinergic innervation. For sympathetic neurons innervating sweat glands there is convincing evidence that these neurons are initially noradrenergic and that the interaction of innervating fibers and target tissue induces a shift in the neurotransmitter phenotype from noradrenergic to cholinergic. In addition to this target-dependent differentiation, an earlier expression of cholinergic characters was observed in sympathetic ganglia that occurs before target contact. These data raise the possibility that different subpopulations of cholinergic sympathetic neurons, innervating distinct peripheral targets, may develop along distinct schedules. In vitro studies suggest that growth factors of the family of neuropoietic cytokines are involved in the specification of the cholinergic sympathetic phenotype. Recent in vivo studies that interfered with cytokine receptor expression in developing avian sympathetic ganglia indicate that only the late, target-dependent differentiation depends on cytokine signaling. The signals involved in the early, target-independent expression of cholinergic properties remain to be determined, as well as the identity of the target-derived cytokine. Thus, cholinergic sympathetic differentiation seems to be more complex than expected, involving either both target-independent and target-dependent control or only target-induced differentiation, according to the specific neuronal subpopulation and target.     

Gas chromatographic/mass spectrometric methodology for simultaneous assay of salsolinol, dopamine, norepinephrine, dihydroxyphenylacetic acid and dihydroxyphenylethanol

Synthesis of deuterated (2H4)salsolinol from (2H4)dopamine via a Pictet-Spengler condensation is described. This (2H4)salsolinol is an ideal internal standard to determine picomole (ng) amounts of salsolinol (SAL) in a variety of sample types including urine, plasma, beverages and fruits. The deuterated standard is completely free of contamination by the non-deuterated species. The extraction procedure described is fast, highly efficient and does not lead to artifactual salsolinol formation even in the face of high dopamine concentrations. As well as SAL the method described allows simultaneous determination of norepinephrine (NE), dopamine (DA) and its two metabolites dihydroxyphenylacetic acid (DOPAC) and dihydroxyphenylethanol (DOPET). Each of the analytes is measured as its trifluoroacetyl derivative. Using trifluoroacetic anhydride in conjunction with trifluoroethanol allows simultaneous one-step derivatization of the acid function of DOPAC. All compounds were measured in the single ion monitoring (SIM) mode and quantified using appropriate deuterated internal standards. SAL, DA, DOPET, DOPAC and NE have been quantified in a variety of food and beverage sources. Soy sauce and dried banana have been identified as rich dietary sources of SAL. These data suggest diet should be considered a potentially important source of 'mammalian alkaloids' such as SAL, and the presence of SAL in mammalian systems is not necessarily evidence for an in vivo Pictet-Spengler condensation.     

Nerve growth factor-independent development of embryonic mouse sympathetic neurons in dissociated cell culture

Although ganglia from neonatal mouse sympathetic ganglia require nerve growth factor (NGF) for survival in culture, explanted sympathetic ganglia from early embryonic stages do not require added NGF for survival and growth. To determine whether the change in growth factor requirement is due to changes in the neurons themselves, to variations in neuronal populations, or to changes in nonneuronal cells, we examined the response to growth factors by dissociated sympathetic neurons at various stages of development. Results indicate that neurons from the 14-day gestational (E14) superior cervical ganglion (SCG) do not require NGF for initial survival and neurite extension, but do require the conditioned medium neurite extension factor, CMF. By 2 to 3 days thereafter, whether in vivo or in culture, most neurons have developed a requirement for NGF for survival in culture. During the same period, there is a concomitant increase in responsiveness to NGF alone as a trophic agent. Changes in response to NGF are not due to changes in NGF content of ganglia, to interactions in culture with nonneuronal cells, or to age-related differences in NGF requirements for maximum survival. The changes in growth factor requirements may be related to mechanisms regulating specificity of nerve-target connections.     

NRAGE, a p75NTR adaptor protein, is required for developmental apoptosis in vivo

NRAGE (also known as Maged1, Dlxin) is a member of the MAGE gene family that may play a role in the neuronal apoptosis that is regulated by the p75 neurotrophin receptor (p75NTR). To test this hypothesis in vivo, we generated NRAGE knockout mice and found that NRAGE deletion caused a defect in developmental apoptosis of sympathetic neurons of the superior cervical ganglia, similar to that observed in p75NTR knockout mice. Primary sympathetic neurons derived from NRAGE knockout mice were resistant to apoptosis induced by brain-derived neurotrophic factor (BDNF), a pro-apoptotic p75NTR ligand, and NRAGE-deficient sympathetic neurons show attenuated BDNF-dependent JNK activation. Hair follicle catagen is an apoptosis-like process that is dependent on p75NTR signaling; we show that NRAGE and p75NTR show regulated co-expression in the hair follicle and that identical defects in hair follicle catagen are present in NRAGE and p75NTR knockout mice. Interestingly, NRAGE knockout mice have severe defects in motoneuron apoptosis that are not observed in p75NTR knockout animals, raising the possibility that NRAGE may facilitate apoptosis induced by receptors other than p75NTR. Together, these studies demonstrate that NRAGE plays an important role in apoptotic-signaling in vivo.