Tyrosine Hydroxylase-like Immunoreactivity Research Articles

Postnatal development and aging of the rat ventral root L5: Electron microscopic and immunohistochemical studies

The ventral root L5 of neonatal and adult rats has been used in many experimental studies on ganglionic C-fibers. Since the normal presence of such axons in L5 roots from animals of different ages is unknown, the results of these studies cannot be appropriately interpreted. In the present study we examine L5 ventral roots from developing and aging rats in this respect. Electron microscopic examination revealed that C-fibers occur in neonatal roots. The adult proportion has been established at day 30. Immunohistochemical analysis showed that thin ganglionic fibers with substance P/calcitonin gene-related peptide- or tyrosine hydroxylase-like immunoreactivity in the L5 root and the spinal pia mater seem to increase postnatally from low levels at birth. In roots from aged rats, myelinated fibers with a variety of aberrant features occur in normal numbers. The occurrence of unmyelinated axons is elevated. The increased presence of fibers with calcitonin gene-related peptide- or tyrosine hydroxylase-like immunoreactivity in aged roots indicates that the extra unmyelinated fibers may represent motor sprouts and sympathetic fibers, respectively. We conclude that the rat ventral root L5 contains a variable number of putative sensory and sympathetic axons at all ages.

Localization of serotonin-4 receptors in the striatonigral pathway in rat brain.

Rats were injected unilaterally with 6-hydroxydopamine either in the medial forebrain bundle or in the dorsolateral substantia nigra. Another group was injected unilaterally with kainate in the striatum. The loss of neurons was assessed by a reduction in tyrosine hydroxylase-like immunoreactivity for dopaminergic neurons, and choline acetyltransferase-like and glutamate decarboxylase-like immunoreactivities for cholinergic and GABAergic neurons, respectively. Brain sections also were analysed by autoradiography on 20 micron sections with the radio-iodinated serotonin-4 receptor antagonist [125I]SB 207710 [Brown A. M. et al. (1993) Br. J. Pharmac. 110, 10P]. Kainate injections in the striatum resulted in loss of choline acetyltransferase- and glutamate decarboxylase-like immunoreactive cell bodies in this area. There was also a decrease in glutamate decarboxylase-like immunoreactivity on the ipsilateral side in the substantia nigra and entopeduncular nucleus. These changes were accompanied by substantial (> 50%) decreases in [125I]SB 207710 binding in both the ipsilateral striatum (confined to the lesioned area) and substantia nigra, with no change in either the nucleus accumbens or the globus pallidus. There was also significant loss of [125I]SB 207710 binding in the ipsilateral entopeduncular nucleus. 6-Hydroxydopamine lesions placed either in the medial forebrain bundle or in the substantia nigra failed to decrease [125I]SB 207710 binding in any of these areas, although there was total loss of tyrosine hydroxylase-like immunoreactive terminals in the striatum and cell bodies in the nigra. We conclude that serotonin-4 receptors are present on projection neurons, both on their perikarya in the striatum and terminals in the nigra and entopeduncular nucleus. It is likely that these receptors are located on the GABAergic projection neurons and possibly on cholinergic and GABAergic interneurons. However, serotonin-4 receptors are not located on dopaminergic neurons, either on their cell bodies in the substantia nigra or terminals in the striatum.

Transient local presence of nerve fibers at onset of secondary ossification in the rat knee joint.

In view of recent evidence that nerves may be involved in bone formation, the present study examines the local occurrence of axons at the onset of secondary ossification center formation in the knee region of developing rats. Radiographic and histological examination showed that secondary ossification center formation commenced at day 10. At day 15 the epiphyseal ossification had reached a relatively mature state. As seen by light microscopy, cartilage canals first appeared at day 5, reaching the epiphyseal center by day 9. Axons exhibiting a neurofilament-like immunoreactivity emerged from the perichondrial plexa into the cartilage canals. Many calcitonin gene-related peptide (CGRP)-immunoreactive axons were found in the canals, as well as in the perichondrium. Axons with tyrosine hydroxylase-like immunoreactivity were not found in the canals, but such fibers occurred in relation to blood vessels at other sites. The canal-related axons disappeared between days 13 and 15, and the canals themselves did not persist beyond bone formation. As seen in the electron microscope, an individual canal contained 3-10 unmyelinated Schwann cell-enclosed axons with diameters of 0.1-2.0 microM. These observations show that putative sensory unmyelinated axons with CGRP-and SP-like immunoreactivity are transiently present during initiation of bone formation in developing epiphyses. Whether there is a causal relation between transient innervation and osteogenesis remains to be determined.

Nitric oxide synthase and chemical coding in cat sympathetic postganglionic neurons.

Nitric oxide synthase-like immunoreactivity was found in a subpopulation of sympathetic postganglionic neurons in the cat stellate and lower lumbar ganglia. In the ganglia of other segments such cells were rare. Double staining for tyrosine hydroxylase-like immunoreactivity and nitric oxide synthase-like immunoreactivity or the reduced nicotinamide adenine dinucleotide phosphate diaphorase reaction indicated that nitric oxide synthase-like immunoreactivity and reduced nicotinamide adenine dinucleotide phosphate diaphorase reactivity was always co-localized and was confined to tyrosine hydroxylase-negative (presumably cholinergic) ganglion cells, and was present in most of them. The occurrence of nitric oxide synthase in two subpopulations of cholinergic postganglionic neurons was investigated in triple staining experiments. Presumptive sudomotor neurons have been previously defined as scattered cells containing calcitonin gene-related peptide-like immunoreactivity, usually accompanied by vasoactive intestinal peptide-like immunoreactivity: 99% of these contained nitric oxide synthase. Presumptive muscle vasodilator neurons have been previously identified as clumped cells with strong vasoactive intestinal peptide-like immunoreactivity but no calcitonin gene-related peptide-like immunoreactivity: 70% of these contained nitric oxide synthase. Sweat glands were found in the paw pad skin surrounded by varicose fibres showing calcitonin gene-related peptide-like immunoreactivity and vasoactive intestinal peptide-like immunoreactivity, confirming previous work. Such fibres also stained for nitric oxide synthase-like immunoreactivity and reduced nicotinamide adenine dinucleotide phosphate diaphorase reactivity, although their staining was relatively weaker than in the corresponding cell bodies. Varicose fibres with the same chemical coding were also found around all large and most medium and small arteries in the paw skin as well as around arteriovenous anastomoses. Fibres with the muscle vasodilator coding (vasoactive intestinal peptide-like immunoreactivity without calcitonin gene-related peptide-like immunoreactivity) were not seen in paw skin. These results suggest that nitric oxide may act as a co-transmitter (with acetylcholine, substance P, vasoactive intestinal peptide and calcitonin gene-related peptide) in sudomotor neurons and (with acetylcholine and vasoactive intestinal peptide) in vasodilator neurons. Collateral branches of sudomotor neurons may innervate skin vessels, and release vasodilator transmitters including nitric oxide to cause the vasodilatation which provides the fluid supply for sweat formation. Alternatively, separate vasodilator neurons to skin may share the same chemical code as sudomotor neurons.

Regulation of rat sympathetic nerve density by target tissues and NGF in maturity and old age.

Previous studies in our laboratory using a transplantation model have shown that target tissues of some autonomic neurons, including cerebral blood vessels, exert a controlling influence on nerve fibre loss in old age. The present study was undertaken in order to discover whether the influence of targets extends to controlling age changes in specific populations of nerves. In old rats, we have demonstrated a significant decrease of approximately 50% in the sympathetic innervation of middle cerebral arteries, using tyrosine hydroxylase-like immunoreactivity. Following transplantation, tyrosine hydroxylase-like immunoreactive nerve density on both young and old implanted middle cerebral arteries mirrored the nerve densities seen in normal, non-transplanted vessels. Furthermore, implanted tissue from old donors became reinnervated with a nerve density approximately 50% less than that of young implanted vessels. Treatment of transplants with nerve growth factor, however, was able to reverse these age changes and restore the sympathetic innervation of aged middle cerebral arteries to levels above those seen in young middle cerebral arteries. These results suggest that the pattern and density of sympathetic innervation that the middle cerebral artery receives is determined by the target rather than by the neurons supplying the tissue. The ability of nerve growth factor to induce regrowth in sympathetic neurons innervating ageing target tissues implies that age-related neuronal atrophy may be due to reduced synthesis or availability of target-derived neurotrophic factors.

Localization of glutamatergic neurons in the dorsolateral pontine tegmentum projecting to the spinal cord of the cat with a proposed role of glutamate on lumbar motoneuron activity

Glutamate is considered to be a major excitatory neurotransmitter in the central nervous system. The presence of glutamate-like immunoreactive neurons in the rodent locus coeruleus has been reported previously. In this study we used both immunohistochemical and electrophysiological techniques to answer two major questions: (1) Is there any glutamate-like immunoreactivity in the catecholaminergic coeruleospinal system of the cat? (2) What is the physiological role, if any, of glutamate in descending locus coeruleus control of spinal motoneurons? Following injections of rhodamine-labeled latex microspheres or Fast Blue into the seventh lumbar segment of the spinal cord of the cat, retrogradely labeled cells were found throughout the rostrocaudal extent of the dorsolateral pontine tegmentum. They were primarily observed in the nucleus locus coeruleus and the Kolliker-Fuse nucleus. Some labeled cells were also present in the nucleus subcoeruleus and, to a lesser extent, in the parabrachial nuclei. Data from immunohistochemical studies indicate that 86% of all dorsolateral pontine tegmentum neurons that project to the spinal cord contain glutamate-like immunoreactivity, and 77% co-contain both glutamate- and tyrosine hydroxylase-like immunoreactivity. Electrical stimulation (four pulses of 500 microseconds duration at 500 Hz; intensity = 50-200 microA) of the locus coeruleus, in decerebrate cats, consistently induced lumbar motoneuron discharges recordable ipsilaterally as ventral root responses. These motoneuronal responses were reversibly antagonized following chemical inactivation of noradrenergic locus coeruleus neurons by local infusion of the alpha 2-adrenergic agonist clonidine, suggesting the locus coeruleus neurons to be the main source of evoked ventral root responses. Additionally, the evoked ventral root responses were reversibly reduced by 34.20 +/- 4.45% (mean +/- S.E.M.) upon intraspinal injections of the non-N-methyl-D-aspartate receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione, into the ventral horn of seventh lumbar spinal cord segment (three to four injections, 20 nmol in 0.2 microliter of 0.1 M Tris-buffered saline for each injection). Similar volumes of vehicle injections had no significant effect on the locus coeruleus-evoked ventral root responses. These ventral root responses were also partially blocked (62.30 +/- 11.76%) by intravenous administration of the alpha 1-adrenergic receptor antagonist prazosin (20 micrograms/kg). In the light of several anatomical reports of noradrenergic and glutamatergic terminals in close contact with spinal motoneurons, our present findings suggest that the locus coeruleus-evoked ventral root response probably involves the synaptic release of both norepinephrine and glutamate onto lumbar motoneurons.(ABSTRACT TRUNCATED AT 400 WORDS)

Induction of uterine activity with oxytocin in late pregnant rats replicates the expression of c-fos in neuroendocrine and brain stem neurons as seen during parturition.

We investigated whether delivery could be induced with pulsatile oxytocin and whether such treatment activated neurons in the supraoptic nucleus (SON) and putative afferent neurons in the nucleus tractus solitarii and ventrolateral medulla, as seen during spontaneous parturition. Rats were implanted with a jugular venous cannula 1 day before the expected term and on the next morning were given a pulse of oxytocin or saline every 10 min for 4 h. Pulses of 10 and 20 mU oxytocin induced delivery in 77% (14 of 18) of rats, whereas none of the control animals (0 of 9) gave birth during the treatment. Lower doses of oxytocin were not effective at this time in inducing delivery. Animals were killed either before (prepartum groups) or during (parturient groups) delivery, and the brains were processed for immunocytochemistry. Oxytocin treatment induced Fos expression in SON and brain stem neurons in both parturient rats and rats in which parturition was not induced. Fos expression in all sites was significantly higher than that in control prepartum rats, but was similar in extent and distribution to that in spontaneous parturient rats. In the brain stem, a substantial proportion of Fos-immunoreactive cells contained tyrosine hydroxylase-like immunoreactivity, and the number of these cells was increased in response to oxytocin treatment. As only very few Fos-immunoreactive nuclei in either the SON or the nucleus tractus solitarii were observed in virgin rats injected with oxytocin, we suggest that intermittent oxytocin injections in late pregnant rats induce strong uterine activity, which can stimulate magnocellular and putative afferent neurons even before the expulsion of pups.

Invasion of the rat ventral root L5 by putative sympathetic C-fibers after neonatal sciatic nerve crush

The present study examines the occurence of C-fibers in lumbar ventral roots after sciatic nerve crush in neonatal and adult rats. Electron microscopic analysis showed that the number of C-fibers in the ventral root L5 increased significantly on the lesion side after neonatal but not adult sciatic nerve crush and that the number of C-fibers was higher in the ventral root L5 on the unoperated side compared to this root in normal control rats. In order to determine whether the new C-fibers in the L5 root on the lesion side are sensory or sympathetic we made immunohistochemical studies on roots from neonatally crushed rats. We found that there was no obvious lesion side/contralateral side or operated rat/control rat difference with respect to the occurence and general configuration of axons with substance P-, calcitonin gene-related peptide- or vasoactive intestinal polypeptide-like immunoreactivity. However, the occurrence of axons with tyrosine hydroxylase-like immunoreactivity appeared clearly higher in the ventral root L5 on the lesion side compared to the unoperated side in neonatally crushed rats. Moreover, these axons seemed to be more numerous also in the ventral root L5 on the unoperated side compared to normal control rats. No lesion side/contralateral side or operated rat/control rat differences were seen in the ventral root L4. We propose that the ventral root L5 is invaded by putative sympathetic C-fibers after sciatic nerve crush lesions in newborn rats.

Photomechanical movements of cultured embryonic photoreceptors: regulation by exogenous neuromodulators and by a regulable source of endogenous dopamine

In the retina of nonmammalian vertebrates, light regulates photoreceptor morphology by causing rod photoreceptor elongation and cone photoreceptor contraction. The opposite photomechanical movements occur in the dark, and proceed with a circadian rhythm in many species in vivo. Using dissociated cultures of embryonic chick retina cells, we have recently demonstrated that photoreceptor cells that differentiate in vitro acquire the capacity of responding to light/dark cycles with photomechanical movements (Stenkamp and Adler, 1993). Here we report that the putative neuromodulators melatonin and dopamine can mimic the effects of darkness and light, respectively, on in vitro photomechanical movement. Pharmacological studies showed that dopamine appears to function by means of a D2-type receptor negatively coupled to adenylate cyclase. The effects of light on the cultured photoreceptors were inhibited by dopamine D2 receptor antagonists, and were attenuated by the dopaminergic neurotoxin 6-hydroxydopamine and by the dopamine synthesis inhibitor alpha-methyl-p-tyrosine. The possible existence of an endogenous source of dopamine in the cultures was also suggested by the presence of tyrosine hydroxylase-like immunoreactivity, and of an Na(+)-dependent mechanism for the accumulation of 3H-dopamine, which was predominantly associated with nonphotoreceptor cells. Additionally, 3H-dopamine release occurred in vitro through a Ca(2+)-dependent mechanism, as well as through reverse function of a nomifensine-sensitive dopamine transporter. Both of these putative release mechanisms appeared to be regulated by light and by melatonin, suggesting a mechanism whereby the putative dopaminergic cells may interact with other cells present in the cultures. These studies suggest that complex paracrine neuromodulatory mechanisms can differentiate in low-density embryonic cell culture, that dopaminergic activities exist in vitro, and that they are important for mediating photomechanical movements.

Co-localization of GABA- and tyrosine hydroxylase-like immunoreactivities in amacrine cells of the rabbit retina

Previous studies have shown that many of the tyrosine hydroxylase-immunoreactive (TH-IR) amacrine cells in the retina of mammals also show GABA-like immunoreactivity (GABA-IR). However such co-localization has yet to be demonstrated in the rabbit retina. In this study the proportion of TH-IR amacrine cells that show GABA-IR has been determined in the retina of the cat, rat and rabbit following fixation with 4% paraformaldehyde plus varying concentrations of glutaraldehyde. In the retina of the cat and rat, most of the TH-IR amacrine cells showed GABA-IR following fixation using low concentrations (0.01 or 0.1%) of glutaraldehyde. However in the rabbit retina very few of the TH-IR amacrine cells showed GABA-IR unless the glutaraldehyde concentration in the fixative was 0.5% or greater. The results suggest that the dopaminergic amacrine cells in the rabbit retina contain only very low levels of GABA, which are close to the detection limit of the antibody.

Demonstration of neuropeptide containing nerves and vasomotor responses to perivascular peptides in human cerebral arteries.

A rich supply of nerve fibers containing neuropeptide Y-like (NPY-LI) and tyrosine hydroxylase-like immunoreactivity was seen in human cerebral arteries, arterioles and veins. Only a sparse supply of vasoactive intestinal polypeptide (VIP-LI), substance P (SP-LI), and calcitonin gene-related peptide (CGRP-LI) was demonstrated in the walls of human cerebral vessels. In isolated ring segments of human cerebral arteries, NPY and noradrenaline caused vasoconstriction but did not potentiate each other. VIP, peptide histidine methionine, SP, neurokinin A, and CGRP relaxed arteries precontracted by prostaglandin F2 alpha. The degree of innervation and the vasomotor responses are discussed in relation to migraine pathophysiology.

Noradrenergic Innervation of the External Granular Layer of the Rat following Low-Level X-Irradiation

The distribution of the noradrenergic system within the rat cerebellar cortex following low-level X-irradiation was studied using tyrosine hydroxylase immunocytochemistry. X-Irradiation treatments consisted of 1, 3, or 5 successive daily dose beginning on postnatal day 1. When studied 24 h after the last exposure, a dense plexus of tyrosine hydroxylase-like immunoreactive (THIR) fibers, not found in age-matched controls, was observed in the reduced external granular layer (EGL) in each treatment group. At 10 days of age, the THIR fibers were found in association with the reconstituted cells of the premigratory zone of the EGL. The rapid and abnormal innervation of the EGL by the THIR fibers suggests that the noradrenergic system could play a role in the regeneration of this layer.

Combination of alkaline phosphatase in situ hybridization with immunohistochemistry: colocalization of calretinin-mRNA with calbindin and tyrosine hydroxylase immunoreactivity in rat substantia nigra neurons

We describe a method to combine non-radioactive in situ hybridization using alkaline phosphatase (AP) labelled oligonucleotide-probes with immunohistochemistry on the same thin paraffin section. The simultaneous detection of calretinin-mRNA and calbindin- or tyrosine hydroxylase-like immunoreactivity in neurons of rat substantia nigra, pars compacta, was used as a test system to develop the method. Brains were fixed by perfusion with 4% paraformaldehyde and embedded in paraffin. Five-μm-thick sections were processed for non-radioactive in situ hybridization with a 33-base alkaline phosphatase conjugated synthetic oligonucleotide complementary to calretinin mRNA. After hybridization and colour reaction to visualize calretinin mRNA, sections were incubated with antibodies against calbindin D 28K or tyrosine hydroxylase. Immunoreaction was visualized using the avidin-biotin-complex-technique and diaminobenzidine. As the colour of both reaction products differ markedly, the distribution of calretinin mRNA-containing neurons (purple-blue, alkaline phosphatase product) and calbindin/tyrosine hydroxylase immunopositive cells (brown peroxidase product) could be differentiated easily on the same section. Calbindin- and tyrosine hydroxylase-like immunoreactivity was found in the majority of calretinin mRNA-containing cells within the substantia nigra, pars compacta, indicating that in this nucleus a proportion of the dopaminergic neurons contain both calcium binding proteins calbindin and calretinin. In conclusion, non-radioactive in situ hybridization using alkaline phosphatase labelled oligonucleotide probes can be readily combined with immunohistochemistry.

Noradrenergic innervation of ectopic granule cells following low-level X-irradiation.

The distribution pattern of the noradrenergic system within the cerebellar cortex following low-level X-irradiation was studied using immunocytochemistry. Following one X-irradiation exposure on postnatal day 1, the laminar distribution of tyrosine hydroxylase-like immunoreactive (THIR) fibers was similar to controls at postnatal day 30, but the orientation of the fibers in the molecular layer (ML) was slightly changed. Successive daily doses, however, produced alterations in both the pattern and concentration of THIR fibers within the ML of the cerebellar cortex. In addition, THIR fibers were found among ectopic cell clusters within the ML. This relationship suggests a potential role for the noradrenergic system in the development and/or maintenance of the ectopic cell clusters.

Existence of glutamate in noradrenergic locus coeruleus neurons of rodents

This study distinguished three types of immunolabeled neurons in nucleus locus coeruleus (LC) of the rat and mouse: cells single labeled either for tyrosine hydroxylase-like immunoreactivity (TH-LI) or glutamate (Glu)-LI, and those double labeled for both antigens. Although the double labeled neurons tend to be located in the middle and ventral thirds of the rat LC nucleus, throughout its rostrocaudal extent, such feature was not apparent in the mouse. Quantitatively a majority of neurons cocontaining TH- and Glu-LI were commonly observed in the rat (62%) and mouse (77%) LC. Additional studies utilizing the combined retrograde and immunohistochemical labeling revealed that such a high incidence of coexistence of the TH- and Glu-LI was also represented by coeruleocortical neurons in the rat (69% and 75% of all ipsilateral and contralateral projection cells, respectively). A possible role of coeruleocortical neurons involvement in Glu- and norepinephrine-mediated target neuron dysfunction is discussed.

Chronic nicotine treatment counteracts nigral cell loss induced by a partial mesodiencephalic hemitransection: An analysis of the total number and mean volume of neurons and glia in substantia nigra of the male rat

This study combines immunocytochemical and stereological methods for the first time to obtain unbiased estimates of the number of cells in the entire substantia nigra and their respective mean volume. Nicotine, delivered by subcutaneously implanted osmotic pumps (0.125mg/kg/h, 14 days) to male Sprague-Dawley rats with a partial unilateral mesodiencephalic lesion, caused a significant counteraction of the lesion-induced reduction in total number of nigral tyrosine hydroxylase-like immunoreactive neurons counterstained with Cresyl Violet compared with saline treated control animals. The number of Nissl stained neurons without tyrosine hydroxylase-like immunoreactivity was not affected by the lesion nor by nicotine. The numbers of non-neuronal glial fibrillary acidic protein-like immunoreactive cells counterstained with Cresyl Violet and smaller cells seen after Cresyl Violet staining alone, possibly representing microglia, were increased by the lesion but not affected by nicotine. No nicotine-induced effects were found on the number of nigral cells located contralateral to the lesion. The lesion-induced reduction in the mean volume of the nigral cells showing tyrosine hydroxylase-like immunoreactivity, as determined with the stereological rotator method, was not affected by nicotine. These findings suggest that continuous nicotine infusion exerts protective effects on lesioned nigroneostriatal dopamine systems and that these protective effects are selective for the nigral dopamine neurons not affecting other populations of neurons or non-neuronal cells. This neuroprotective effect might lead to new therapeutic strategies in clinical neurodegenerative disorders such as Parkinson's Disease.

The aminergic control of locust ( Locusta migratoria) salivary glands: Evidence for dopaminergic and serotonergic innervation

Tyrosine hydroxylase-like immunoreactivity and serotonin-like immunoreactivity of the subesophageal ganglion of the locust, Locusta migratoria, reveals several pairs of cells. One pair of tyrosine hydroxylase-like positive cells (SN1) and one pair of serotonin-like positive cells (SN2) both project through nerve 7b to the salivary glands, where they branch extensively over the acini. Individual acini receive innervation from both SN1 and SN2. Co-localization of immunoreactivity was never observed. Neurons that stain positively for tyrosine hydroxylase-like immunoreactivity are considered to be catecholaminergic since tyrosine hydroxylase is the first and rate limiting enzyme in the pathway for the production of catecholamines. High-performance liquid chromatography of the salivary glands reveals the presence of dopamine and serotonin. Both dopamine and serotonin induce an elevation of cyclic AMP in salivary glands of adult male locusts in a dose-dependent manner. Thus it appears that cyclic AMP is a second messenger for the action of dopamine and serotonin on salivary glands. The presence of distinct dopamine and serotonin receptors on the glands was shown via the ability of the receptor antagonists, SCH-23390 and ketanserin, to inhibit the elevation of cyclic AMP levels induced by dopamine and serotonin, respectively.

Localization of NADPH diaphorase activity in monoaminergic neurons of the rat brain

Nitric oxide has recently been implicated as a neurotransmitter, and may modulate synaptic transmission, cerebral blood flow, and neurotoxicity. NADPH diaphorase histochemistry has been shown to be a reliable marker for nitric oxide synthase, the enzyme that synthesizes nitric oxide, in the nervous system. Because monoaminergic neurons frequently contain co-transmitters, we examined whether these cells also exhibit NADPH diaphorase activity. Frozen sections from postnatal and adult rat brains were stained for NADPH diaphorase activity and either serotonin-like immunoreactivity or tyrosine hydroxylase-like immunoreactivity. Numerous neurons in the mesopontine serotoninergic cell groups (including the caudal linear, dorsal, median, supralemniscal, and pontine raphe nuclei) contained both serotonin-like immunoreactivity and NADPH diaphorase activity. Within the dorsal raphe nucleus, approximately 70% of the serotoninergic neurons in the medial subnuclei displayed NADPH diaphorase activity, while less than 10% of the serotoninergic neurons in the lateral subnuclei were doubly labeled. Retrograde labeling with fluorescent microspheres indicated that many raphe-cortical neurons contained NADPH diaphorase activity. No NADPH diaphorase activity was detected in serotoninergic neurons in the medullary nuclei (including the raphe magnus, raphe pallidum, and raphe obscurus). Only a small proportion of tyrosine hydroxylase-like immunoreactive neurons in the periaqueductal gray, rostral linear nucleus, and rostrodorsal ventral tegmental area contained NADPH diaphorase activity. Tyrosine hydroxylase-like immunoreactive neurons in the substantia nigra, locus coeruleus, hypothalamus, olfactory bulb, and dorsal raphe nucleus did not contain detectable NADPH diaphorase activity. The observation that many mesopontine (but not medullary) serotoninergic neurons contain NADPH diaphorase activity suggests that these neurons may release both serotonin and nitric oxide.

Tyrosine Hydroxylase Immunoreactivity and Monoamine and Metabolite Levels in Cryopreserved Human Fetal Ventral Mesencephalon

Both fresh and cryopreserved-thawed human fetal ventral mesencephalon have been used for preclinical research and implantation into the brains of patients with Parkinson's disease. Further characterization and an evaluation of the effects of cryopreservation on immunocytochemical and neurochemical markers of monoamine neurons in human fetal ventral mesencephalic tissue are reported here. Fresh and cryopreserved-thawed human fetal mesencephalic tissue of 7-10 weeks fetal age was analyzed for the presence of tyrosine hydroxylase-like immunoreactivity and levels of dopamine (DA), norepinephrine, serotonin, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindole acetic acid. After fixation, cryopreserved-thawed mesencephalic tissue exhibited cellular tyrosine hydroxylase-like immunoreactivity identical to that seen in fresh tissue. The levels of DA, norepinephrine, serotonin, and 5-hydroxyindole acetic acid in the cryopreserved thawed tissue were the same as the levels in fresh tissue. The levels of DOPAC were higher, and those of HVA were lower, in the cryopreserved-thawed tissue compared to the levels in fresh tissue. The changes in the levels of the DA metabolities, DOPAC and HVA, without corresponding change in the levels of the parent monoamine in cryopreserved-thawed tissue, indicate ongoing metabolic activity in DA-containing neurons. These results further suggest that cryopreservation and subsequent thawing does not have a measurable adverse effect on DA biosynthesis in the human fetal mesencephalon. The presence of the monoamines and their metabolites in the ventral mesencephalon at 7-10 weeks of fetal age coincides well with the early presence of immunocytochemical markers of monoamine neurons and reflects the early function of the nuclear groups containing specific monoamine neurotransmitters.

Dopaminergic lateral efferent innervation of the guinea-pig cochlea: Immunoelectron microscopy of catecholamine-synthesizing enzymes and effect of 6-hydroxydopamine

We have used an immunocytochemical approach to gain further data supporting a possible neurotransmitter or neuromodulator function for dopamine at the level of efferent (olivocochlear) innervations of the guinea-pig cochlea. Immunofluorescence screening was first done on cochleas two or seven days after infusion with the neurotoxin 6-hydroxydopamine. Two days after neurotoxin perfusion, the intensity of the tyrosine hydroxylase-like immunoreactivity was decreased in the inner and tunnel spiral bundles of the organ of Corti (and in known noradrenergic sympathetic fibers outside this organ), with respect to the control contralateral cochleas. In cochleas screened seven days after 6-hydroxydopamine infusion, no tyrosine hydroxylase-like immunoreactivity could be found in the organ of Corti. Only occasional faint fluorescence could be detected in sympathetic fibers. In another set of experiments, a pre-embedding immunoperoxidase technique was used to localize tyrosine hydroxylase and aromatic amino acid decar☐ylase, another catecholamine-synthesizing enzyme, at the ultrastructural level. With both types of antibody, the same kind of results were observed. Immunoreactivities were only seen in vesiculated fibers within the inner and tunnel spiral bundles, thus are likely in lateral efferent varicosities. These immunostained fibers accounted for approximately half of the efferent profiles in the inner spiral bundle. Within this bundle, the immunoreactive fibers established axodendritic synapses with the radial afferent processes of type I neurons which contacted the inner hair cells. In no case was immunoreactivity to either enzyme observed in the outer hair cell region, at the level of medial efferent terminals. The synaptic localization of tyrosine hydroxylase- and aromatic amino acid decar☐ylase-like immunoreactivities in the lateral efferent varicosities of the inner spiral bundle, as well as the effect of 6-hydroxydopamine on the tyrosine hydroxylase-like immunoreactivity in this same bundle, further support the hypothesis that a catecholamine could act as a lateral efferent neurotransmitter or neuromodulator. Based on previous data reporting a lack of dopamine-β-hydroxylase-like immunoreactivity within the organ of Corti, and the effectiveness of a D 2 agonist on the cochlear compound action potential of the auditory nerve, this catecholamine could well be dopamine.