Adrenal medulla and substantia nigra co-grafts in peripheral nerve: chromaffin cells survive for long time periods and prevent degeneration of nigral neurons.

A series of degenerative cytoskeletal changes characterize grafts of embryonic substantia nigra when isolated for long time periods in the peripheral nervous system. This study was designed to determine whether the adrenal medulla could modulate the cytoskeletal changes in the substantia nigra when co-grafted within peripheral nerves. The sciatic nerves of young adult rats received single transplants of embryonic day 15 substantia nigra or co-transplants of substantia nigra plus young adult adrenal medulla in close proximity. Immunocytochemistry was used to examine the expression of tyrosine hydroxylase, phosphorylated (RT97) and non-phosphorylated (SMI-32) neurofilament proteins in the grafts. Single substantia nigra grafts, examined after 1 month, consisted of numerous neurons and fibers that expressed the epitope for tyrosine hydroxylase. Normal spatial distributions of the phosphorylated and non-phosphorylated neurofilament subunits were observed. In contrast, single 1-year-old nigral transplants were shrunken, contained significantly fewer tyrosine hydroxylase positive neurons and displayed abnormal neurofilaments staining patterns including swollen axons, a reduction in the density of labeled axons and perikaryal accumulation of the phosphorylated neurofilament subunit. When co-grafted with the adrenal medulla, the nigral transplants did not show the degenerative cytoskeletal aspects evident in the single 1-year-old grafts. The loss of neurons was prevented and the neurofilament immunolabeling was indistinguishable from the young substantia nigra preparations. In addition, all the 1-year-old adrenal medulla grafts were viable within the peripheral nerves, consisting of hundreds of cells identified by immunoreactivity to tyrosine hydroxylase and the rat beta-nerve growth factor receptor (192-IgG). The experiments illustrate a strong protective effect by the adrenal medulla on neurons of the substantia nigra in the peripheral nerve environment. It is suggested that a catecholaminergic trophic source and/or neural interactions with the adrenal medulla may be important factors in the long-term survival of neurons and maintenance of normal cytoskeletal characteristics in the grafted substantia nigra neurons under these experimental circumstances.     

A catecholaminergic projection from the ventral tegmental area to the diagonal band of broca: Modulation by neurotensin

The ventral tegmental area contains a high density of dopaminergic perikarya having ascending projections to a number of limbic forebrain regions. In this study, we use combined retrograde labeling with horseradish peroxidase (HRP) and immunohistochemical staining for tyrosine hydroxylase to examine the catecholaminergic projection from the ventral tegmental area to the diagonal band of Broca. When injection of HRP was restricted to the diagonal band, only neurons in the nucleus linearis, nucleus interfascicularis and ventromedial portion of the nucleus paranigralis were labeled. In contrast, HRP injection into the adjacent nucleus accumbens labeled neurons throughout these nuclei, plus the nucleus parabrachialis pigmentosus, nucleus retroruber and substantia nigra, pars compacta. Approximately 60% of neurons in the ventral tegmental area labeled from the diagonal band contained tyrosine hydroxylase, compared with 79% of the neurons labeled from the nucleus accumbens. Neurotensin is a tridecapeptide found in the ventral tegmental area which has been shown to activate dopamine neurons projecting to the nucleus accumbens. In this study, microinjection of neurotensin into ventral tegmental nuclei which contained neurons retrogradely labeled from the diagonal band significantly elevated the levels of dopamine metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid, in the diagonal band. The results of this study demonstrate that a catecholaminergic projection exists from the ventral tegmental area to the diagonal band of Broca, and that this pathway can be stimulated by intra-ventral tegmental injection with neurotensin.     

Tyrosine hydroxylase and methionine-enkephalin in the human mesencephalon. Immunocytochemical localization and relationships

The immunocytochemical localization of tyrosine hydroxylase (TH) and methionine-enkephalin (met-enkephalin) was determined at two representative caudal and rostral levels of the human mesencephalon. Four main groups of catecholaminergic neurons were delineated, situated in the substantia nigra and the lateral, ventromedial and dorsomedial tegmentum, extending over several cytoarchitectonic divisions. They matched fairly well the dopaminergic cell groups described in monkey midbrain. TH-like immunoreactivity and neuromelanin were closely related in neurons of substantia nigra, but less so in the other groups. A widespread met-enkephalinergic innervation was observed in most areas containing catecholaminergic neurons. It followed a characteristic pattern: homogeneous and very dense in the lateral and posterior portions of substantia nigra; patchy and less dense in the other areas, the medio-ventral and periaqueductal gray being only sparsely innervated, in contrast to observations in rodents. Dopaminergic cell bodies surrounded by met-enkephalinergic varicosities were seen in most groups, particularly in the lateral substantia nigra and medioventral tegmentum. The topography of met-enkephali-like immunoreactive terminals in the substantia nigra was reminiscent of the distribution of neostriatal and pallidal afferents.     

Nigrotectal projection to the inferior colliculus: horseradish peroxidase transport and tyrosine hydroxylase immunohistochemical studies in rats, cats, and bats

The present study investigated descending projections from the substantia nigra to the auditory tectum. Small (0.02-0.05 microliters) injections of a 30-60% aqueous solution of horseradish peroxidase (HRP) were made unilaterally into the inferior colliculus in rats, cats, and bats (Eptesicus fuscus). Tissue blocks including the substantia nigra, superior colliculus, and inferior colliculus were removed, sectioned, and processed for visualization of HRP. Results show that the substantia nigra, pars lateralis, projects to the inferior colliculus ipsilaterally. In addition, retrogradely labeled cells are found dorsal to the pars lateralis, in a column within the lateral tegmental area of the midbrain. Analysis of injection sites suggests that the principal target of this nigral projection is the dorsal and rostral pericentral region of the inferior colliculus. Immunohistochemical studies with an antibody to tyrosine hydroxylase demonstrate catecholaminergic neurons within the pars lateralis and lateral tegmentum that are similar in location and morphology to one class of HRP retrogradely labeled cells within these structures. These immunohistochemical studies also demonstrate a plexus of fine, varicose tyrosine hydroxylase-positive axons in the rostral pericentral region of the colliculus. The presence of this nigrotectal projection to the inferior colliculus is discussed in relation to its possible role in the control of acousticomotor behavior.     

Comparative distribution of mRNAs for glutamic acid decarboxylase, tyrosine hydroxylase, and tachykinins in the basal ganglia: an in situ hybridization study in the rodent brain

Neurotransmitter-related messenger RNAs were detected by in situ hybridization in sections of rat and mouse brains by using 35S-radiolabelled RNA probes transcribed from cDNAs cloned in SP6 promoter-containing vectors. The distribution of messenger RNAs for glutamic acid decarboxylase, tachykinins (substance P and K), and tyrosine hydroxylase was examined in the striatum, pallidum, and substantia nigra. Dense clusters of silver grains were observed with the RNA probe complementary of the cellular messenger RNA for glutamic acid decarboxylase (antisense RNA) over most large neurons in the substantia nigra pars reticulata and medium-sized to large neurons in all pallidal subdivisions. A few very densely and numerous lightly labelled medium-sized neurons were present in the striatum. Among the areas examined, only the striatum contained neurons labelled with the antisense tachykinin RNA. Most of these neurons were of medium size, and a few were large. With the antisense tyrosine hydroxylase RNA, silver grains were found over neurons of the substantia nigra pars compacta and adjacent A10 and A8 dopaminergic cell groups. No signal was observed with RNAs identical to the cellular messenger RNA for glutamic acid decarboxylase or tachykinin (sense RNA). These results show a good correlation with immunohistochemical studies, suggesting that documented differences in the distribution and the level of glutamic acid decarboxylase, tyrosine hydroxylase, and substance P immunoreactivities in neurons of the basal ganglia are related to differences in the level of expression of the corresponding genes rather than to translation accessibility, stability, or transport of the gene products.     

The GABA and substance P input to dopaminergic neurones in the substantia nigra of the rat.

In order to examine the synaptic input to dopaminergic neurones in the substantia nigra from GABAergic terminals and terminals that contain substance P, double and triple immunocytochemical studies were carried out at the light and electron microscopic levels in the rat. In a first series of experiments sections of the substantia nigra were incubated to reveal axon terminals containing either substance P or glutamate decarboxylase and then incubated to reveal dopaminergic neurones using tyrosine hydroxylase immunocytochemistry. Examination of this material in the light microscope revealed that many substance P- and glutamate decarboxylase-immunoreactive boutons were associated with the dopaminergic cells. In the electron microscope it was found that the perikarya and dendrites of the dopaminergic neurons received symmetrical synaptic input from terminals that displayed immunoreactivity for substance P or glutamate decarboxylase. A small proportion of the substance P-positive boutons formed asymmetrical synapses. In a second series of experiments sections of the substantia nigra were processed by the pre-embedding immunocytochemical technique for tyrosine hydroxylase and then the post-embedding immunogold technique for gamma-aminobutyric acid (GABA). Examination in the electron microscope revealed that the tyrosine hydroxylase-positive neurons received symmetrical synaptic input from many GABA-positive terminals. Quantitative analyses demonstrated that a minimum of 50-70% of all boutons afferent to the dopaminergic neurones display glutamate decarboxylase or GABA immunoreactivity. Triple immunocytochemical studies i.e. pre-embedding immunocytochemistry for tyrosine hydroxylase and substance P, combined with post-embedding immunogold staining for GABA, revealed that some of the substance P-immunoreactive boutons that were in contact with the dopaminergic neurones also displayed GABA immunoreactivity. In a third series of experiments the combination of anterograde transport of lectin-conjugated horseradish peroxidase or biocytin with post-embedding GABA immunocytochemistry demonstrated that at least one of the sources of GABA-containing terminals in the substantia nigra is the striatum. The results of the present study: (1) demonstrate that dopaminergic neurones in the substantia nigra receive symmetrical synaptic input from GABAergic and substance P-containing terminals, (2) show that a proportion of these terminals contain both substance P and GABA and (3) suggest that the major synaptic input to dopaminergic neurones is from GABAergic terminals and that a part of this innervation is derived from the striatum.     

Subpopulations of mesencephalic dopaminergic neurons express different levels of tyrosine hydroxylase messenger RNA.

Subpopulations of mesencephalic dopamine containing neurons possess different electrophysiological, pharmacological, biochemical, and anatomical properties. In order to determine whether such differences are related to the regulation of tyrosine hydroxylase, the rate limiting enzyme in the synthesis of catecholamines, the regional distribution of tyrosine hydroxylase messenger RNA in these neurons was examined using in situ hybridization histochemistry. In the mouse, labelling for tyrosine hydroxylase messenger RNA associated with individual neurons was significantly less in the lateral substantia nigra pars compacta than in the medial substantia nigra pars compacta and the ventral tegmental area. A similar pattern of labelling was observed in the rat. Labelling for tyrosine hydroxylase messenger RNA was significantly less in the lateral substantia nigra pars compacta than in medial pars compacta (a densely cellular region), the area dorsal to the medial substantia nigra pars compacta (a less cell dense region), and the ventral tegmental area. Differences in levels of labelling for messenger RNA in mesencephalic dopamine neurons were not related to differences in cell size as measured in sections processed for tyrosine hydroxylase immunohistochemistry. The results suggest that tyrosine hydroxylase messenger RNA is differentially regulated in subpopulations of mesencephalic dopamine neurons, supporting the view that these neurons are physiologically distinct.     

Overlap in the distribution of cholinergic and catecholaminergic neurons in the upper brainstem of the ferret

The distribution of catecholaminergic and cholinergic neurons in the upper brainstem of the ferret were mapped by staining immunohistochemically two adjacent series of sections of brainstem for tyrosine hydroxylase and choline acetyltransferase, respectively. As in other species, large numbers of tyrosine-hydroxylase-positive neurons are localized in the ventral tegmental area (A10), the substantia nigra (A9), and in A8. Tyrosine-hydroxylase-positive neurons in the dorsolateral pontine tegmentum (A4, A6, and A7--the locus coeruleus complex) of the ferret are rather diffusely distributed, as has been observed in other carnivore species such as the cat and the dog, but unlike the cat, these cells in the ferret display a relative uniformity in size and morphology. Choline-acetyltransferase-positive neurons which extend in the ferret's pedunculopontine tegmental nucleus and ventral parabrachial area (Ch5) are relatively large cells that stain intensely for choline acetyltransferase, and their dendrites form prominent bundles in regions where unstained fibre tracts are prevalent. Choline-acetyltransferase-positive neurons distributed in the laterodorsal tegmental nucleus (Ch6) are smaller than the cholinergic cells of Ch5, and they stain less intensely for choline acetyltransferase. Rostrally, there is little overlap between the catecholaminergic cell groups A8, A9, and A10 and the cholinergic cell groups of Ch5 and Ch6. Caudally, the Ch5 neurons extend some considerable extent into the locus coeruleus complex. In the region of overlap, no cells with staining for both tyrosine hydroxylase and choline acetyltransferase were observed, as was ascertained with a double staining method employing a combination of tyrosine hydroxylase immunofluorescence and choline acetyltransferase peroxidase-antiperoxidase immunohistochemistry. In conclusion, the ferret has a typically carnivore pattern for the distribution of catecholaminergic cells in the upper brainstem, and there is a significant overlap between the catecholaminergic and cholinergic cell groups in the dorsolateral pontine tegmentum.     

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

Studies of the trophic activities of brain-derived neurotrophic factor and neurotrophin-3 indicate that both molecules support the survival of a number of different embryonic cell types in culture. We have shown that mRNAs for brain-derived neurotrophic factor and neurotrophin-3 are localized to specific ventral mesencephalic regions containing dopaminergic cell bodies, including the substantia nigra and ventral tegmental area. In the present study, in situ hybridization with ³⁵S-labeled cRNA probes for the neurotrophin mRNAs was combined with neurotoxin lesions or with immunocytochemistry for the catecholamine-synthesizing enzyme tyrosine hydroxylase to determine whether the dopaminergic neurons, themselves, synthesize the neurotrophins in adult rat midbrain. Following unilateral destruction of the midbrain dopamine cells with 6-hydroxydopamine, a substantial, but incomplete, depletion of brain-derived neurotrophic factor and neurotrophin-3 mRNA-containing cells was observed in the ipsilateral substantia nigra pars compacta and ventral tegmental area. In other rats, combined in situ hybridization and tyrosine hydroxylase immunocytochemistry demonstrated that the vast majority of the neurotrophin mRNA-containing neurons in the substantia nigra and ventral tegmental area were tyrosine hydroxylase immunoreactive. Of the total population of tyrosine hydroxylase-positive cells, double-labeled neurons constituted 25–50% in the ventral tegmental area and 10–30% in the substantia nigra pars compacta, with the proportion being greater in medial pars compacta. In addition, tyrosine hydroxylase/neurotrophin mRNA coexistence was observed in neurons in other mesencephalic regions including the retrorubral field, interfascicular nucleus, rostral and central linear nuclei, dorsal raphe nucleus, and supramammillary region. The present results demonstrate brain-derived neurotrophic factor and neurotrophin-3 expression by adult midbrain dopamine neurons and support the suggestion that these neurotrophins influence dopamine neurons via autocrine or paracrine mechanisms. These data raise the additional possibility that inappropriate expression of the neurotrophins by dopaminergic neurons could contribute to the neuropathology of disease states such as Parkinson's disease and schizophrenia. © 1994 Wiley-Liss, Inc.     

Decreased tyrosine hydroxylase content in the dopaminergic neurons of MPTP–intoxicated monkeys: Effect of levodopa and GM1 ganglioside therapy

Parkinson's disease is characterized by the degeneration of melanized dopaminergic neurons of the substantia nigra. The functional capacity of the surviving dopaminergic neurons is affected, as suggested by the subnormal levels of tyrosine hydroxylase messenger RNA and protein found in the remaining cells. The reduced expression of tyrosine hydroxylase may be due to either the evolving neurodegenerative process or its downregulation, possibly secondary to chronic levodopa treatment. The cellular content of tyrosine hydroxylase was determined in the mesencephalon from 16 Macaca fascicularis monkeys, using a semiquantitative immunocytochemical method. Thirteen monkeys were rendered parkinsonian by weekly intravenous injections of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) for 2 (subacute treatment) or 20 (chronic treatment) weeks. Three of the monkeys received levodopa and 3 others received GM1 ganglioside. The loss of dopaminergic neurons in the mesencephalon of the MPTP-intoxicated monkeys was severe in the substantia nigra, intermediate in cell groups A8 and A10, and almost undetectable in the central gray substance. After both subacute and chronic treatment, the cellular content of tyrosine hydroxylase was reduced by 40% in the surviving neurons of the lesioned substantia nigra, but by less in the other mesencephalic dopaminergic regions. Neuronal survival and tyrosine hydroxylase content in monkeys that had received levodopa were not significantly different. The cellular content of tyrosine hydroxylase was increased in the substantia nigra of the monkeys that received GM1 ganglioside injections. The results show that the decreased expression of tyrosine hydroxylase found in nigral dopaminergic neurons after partial degeneration of the mesostriatal dopaminergic system is not influenced by levodopa treatment and is partially reversed by GM1 ganglioside administration.     

Histochemical identification of acetylcholinesterase in dopaminergic nigrostriatal neurons

Summary By combining histochemical procedures, evidence was obtained in the rat that acetylcholinesterase (AChE) is contained within perikarya of dopaminergic substantia nigra cells which project to the caudoputamen. Nigrostriatal neurons were identified by retrograde transport of horseradish peroxidase (HRP). Simultaneous incubation of substantia nigra sections for the presence of AChE and HRP demonstrated both reaction products in the same cell bodies. The catecholaminergic nature of these cells was determined by a loss of HRP and AChE reaction products subsequent to intranigral injection of 6-hydroxydopamine.     

Nerve growth factor (NGF) in the rat CNS: Absence of specific retrograde axonal transport and tyrosine hydroxylase induction in locus coeruleus and substantia nigra

Selective, highly efficient uptake of [125I]NGF by nerve terminals followed by retrograde axonal transport, and specific induction of tyrosine hydroxylase by NGF are well known phenomena in peripheral adrenergic neurons of adult rats. In the present study these parameters were used in order to detect possible interactions of NGF with central catecholaminergic neurons. No selective retrograde transport of [125I]NGF could be detected by light microscopic autoradiography from the caudate nucleus to the dopaminergic neurons in the substantia nigra or from the hippocampus to the noradrenergic nerve cells of the locus coeruleus. Biochemically, no change in tyrosine hydroxylase activity could be observed for up to 3 days after injection of either NGF, anti-NGF antibodies, or control proteins close to the nerve cell bodies in the substantia nigra or the locus coeruleus. These data suggest a fundamental difference between central and peripheral adrenergic neurons with regard to their responsiveness of NGF.     

Loss of non-phosphorylated neurofilament immunoreactivity, with preservation of tyrosine hydroxylase, in surviving substantia nigra neurons in Parkinson''s disease.

The distribution of neurofilament immunoreactivity in the substantia nigra was examined by immunohistochemistry in five patients dying with Parkinson's disease and six control patients dying without neurological disease. In controls, pigmented neurons in the substantia nigra were intensively labelled by SMI32, a monoclonal antibody to non-phosphorylated neurofilament protein. In the substantia nigra from patients who had Parkinson's disease, there was a pronounced reduction of SMI32 labelling intensity in surviving pigmented neurons. By contrast, tyrosine hydroxylase immunoreactivity in surviving pigmented neurons was normal. SMI32 labelling was normal in regions of the brainstem not affected by the neuropathological process of Parkinson's disease. Findings with either antibodies to phosphorylated neurofilament, or enzymatic dephosphorylation followed by SMI32 labelling, indicated that loss of SMI32 immunostaining in Parkinson's disease was not due to masking of the neurofilament epitopes by phosphorylation. Our results indicate that neurofilament proteins are particularly likely to be disrupted or destroyed by the neuropathological process of Parkinson's disease. Nevertheless, the normal appearance of tyrosine hydroxylase indicates that protein synthesising systems may be intact in surviving neurons. Loss of neurofilament immunoreactivity may prove a sensitive neuropathological marker for characterisation of degenerating neurons in Parkinson's disease.     

Sprouting of dopaminergic fibers from spared mesencephalic dopamine neurons in the unilateral partial lesioned rat

A unilateral partially lesioned rat model of Parkinson's disease was developed following selective lesioning of the dopamine neurons of the substantia nigra pars compacta by stereotactic injection of the neurotoxin 6-hydroxydopamine. In this animal model the dopamine neurons of the ventral tegmental area and medial substantia nigra are spared. The neuronal loss in such partial lesioned models mimics more closely that seen in human mid-stage parkinsonism. Cografts of adrenal medullary cells and sciatic nerve to the partially lesioned striatum induced a sprouting response in grafted animals that was confirmed by immunocytochemical staining with antibodies to tyrosine hydroxylase (TH) and by quantification of the high affinity dopamine uptake complex using [³H]GBR 12935 binding. Enhanced TH fiber immunostaining was evident even in the presence of poor cograft survival. The origin of the TH-like immunostained fibers in the striatum was determined using Lucifer yellow retrograde axonal transport. Following discrete tracer injections into the striatum adjacent to a cograft, neurons in the medial substantia nigra and ventral tegmental area (areas A9 and A10, respectively) were labelled with Lucifer yellow. These labelled neurons displayed a morphology characteristics of dopamine neurons and, in double-labelling experiements, also immunostained for TH. These results support the utility of unilateral partially lesioned rat models of Parkinson's disease for studies investigating a host sprouting or upregulation response and confirm that the immunostained striatal fibers originate from spared dopamine neurons in the ventromedial midbrain.     

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.     

Direct demonstration of interactions between substance P immunoreactive terminals and tyrosine hydroxylase immunoreactive neurons in the substantia nigra of the rat: an ultrastructural study

The results of many anatomical, physiological, and pharmacological studies suggest that substance P-containing neurons of the striatum project to the substantia nigra, and that substance P influences the activity of dopaminergic nigrostriatal neurons. The purpose of the present ultrastructural study was to employ dual immunocytochemical labeling to determine the morphological basis for the observed actions of substance P on nigral dopaminergic neurons. Substance P-like and tyrosine hydroxylase-like immunoreactivities were localized simultaneously at the ultrastructural level in the substantia nigra of the rat. A double label method was utilized which relied on a combination of the peroxidase-antiperoxidase method (Sternberger, 1979) for substance P, and immunogold or silver enhanced immunogold labeling for tyrosine hydroxylase. The present results indicate that tyrosine hydroxylase immunoreactive (THLI) dendrites in the substantia nigra receive synaptic input from terminals exhibiting substance P-like immunoreactivity. These findings support the idea that substance P is a major neurotransmitter in the striatonigral loop, and suggest that striatal substance P neurons act directly upon nigral dopaminergic cells.     

The distribution and morphological characteristics of catecholaminergic cells in the diencephalon and midbrain of the bottlenose dolphin (Tursiops truncatus)

The present study describes the distribution and cellular morphology of catecholaminergic neurons in the diencephalon and midbrain of the bottlenose dolphin (Tursiops truncatus). Tyrosine hydroxylase immunohistochemistry was used to visualize these putatively dopaminergic neurons. The standard A1-A17, C1-C3, nomenclature is used for expediency; however, the neuroanatomical names of the various nuclei have also been given. Dolphins exhibit certain tyrosine hydroxylase immunoreactive (TH-ir) catecholaminergic neuronal groups in the midbrain (A8, A9, A10) and diencephalon (A11, A12, A14), however, no neuronal clusters clearly corresponding to the A13 and A15 groups could be identified. The subdivisions of these neuronal groups are in general agreement with those of other mammals, but there is a high degree of species specificity. First, three TH-ir neuronal groups not identified in other species were found: in the ventral lateral peri-aqueductal gray matter, posterior dorsal hypothalamus, and rostral mesencephalic raphe. Second, the normal components of the substantia nigra (A9 or pars compacta, A9 lateral or pars lateralis, A9 ventral or pars reticulata) were extremely cell sparse, but there was a substantial expansion of the A9 medial and A10 lateral subdivisions forming an impressive 'ventral wing' in the posterior substantia nigra. The findings of this and previous studies suggest a distinct evolutionary trend occurring in the neuromodulatory systems in mammals. The results are discussed in relation to motor control, thermoregulation, unihemispheric sleep, and dolphin cognition.     

Regulation of neurotensin-containing neurons in the rat striatum and substantia nigra. Effects of unilateral nigral lesion with 6-hydroxydopamine on neurotensin content and its binding site density

The effect of unilateral lesion of the rat substantia nigra with 6-hydroxydopamine (6-OHDA) was investigated on the endogenous contents of neurotensin (NT) and its binding site densities in the striatum and substantia nigra. Tyrosine hydroxylase (T-OH) activity, γ-aminobutyric acid (GABA) content, binding site densities of dihydrotetrabenazine (TBZOH), a marker of dopaminergic synaptic vesicles, and of iodosulpride, a ligand for dopamine D₂ receptors, were also determined. Fourteen days following nigral lesions, these markers were analyzed by means of radioimmunoassay for NT levels, fluoremetric method for GABA content, radiochemical method for T-OH activity and quantitative autoradiography for NT, TBZOH and iodosulpiride binding site densities. Unilateral nigral lesion with 6-OHDA provoked only ipsilateral modifications in dopamine markers. T-OH activity and TBZOH binding site densities significantly decreased in both the ipsilateral striatum and substantia nigra. Iodosulpiride binding sites decreased in the substantia nigra and increased in the striatum on the ipsilateral side. In contrast to these unilateral changes observed for dopamine markers, dramatic increases in NT contents were found in both the ipsi- and contralateral striata. No change was found in nigral NT levels on either side. On the other hand, NT binding sites decreased in the ipsilateral striatum and substantia nigra, which reflected the destruction of dopaminergic elements in these regions. The present results strongly suggest a dopaminergic control of striatal NT systems and demonstrate that a unilateral loss of this control may lead to strong bilateral alterations in NT levels.     

The effects of nerve growth factor (NGF) and antiserum to NGF on the development of embryonic sympathetic neurons in vivo

The role of nerve growth factor (NGF) in the development of embryonic sympathetic neurons was examined in vivo. Individual mouse embryos received transuterine injections of NGF or antiserum to NGF (anti-NGF), and the effects on the superior cervical ganglion (SCG) were studied. Treatment with NGF at any gestational stage, from the time of ganglion aggregation to birth, increased ganglion tyrosine hydroxylase (T-OH) activity. Both the number of catecholaminergic neurons and T-OH activity per neutron were increased. Choline acetyltransferase (ChAc) activity was increased by NGF at early gestational stages, but not at later stages. These observations suggest that perikarya containing ChAc are responsive to NGF, whereas preganglionic nerve terminals are not. Treatment with anti-NGF rapidly and permanently decreased ganglion T-OH activity. The effects of anti-NGF were more pronounced at later gestational stages, suggesting that ganglia become increasingly dependent on NGF during development. Alteration of maternal levels of NGF had no effect on development of the embryonic SCG, suggesting that local embryonic concentrations of NGF are responsible for modulating sympathetic ontogeny.     

Glial cell line-derived neurotrophic factor augments midbrain dopaminergic circuits in vivo

Recently, a novel glial cell line-derived neurotrophic factor (GDNF) has been identified, cloned, and shown to have potent survival- and growth-promoting activity on fetal rat midbrain dopaminergic neurons in cell culture. In this study, we document marked and long-lasting effects on adult rat midbrain dopaminergic neurons in vivo after intracranial administration. A single injection of this factor into the substantia nigra elicited a dose-dependent increase in both spontaneous and amphetamine-induced motor activity, and a decrease in food consumption, lasting 7–10 days. Using immunocytochemistry, we found sprouting of tyrosine hydroxylase-positive neurites towards the injection site, and increased tyrosine hydroxylase immunoreactivity of the ipsilateral striatum was produced by GDNF. There was also a marked and dose-dependent increase in dopamine turnover in the substantia nigra and striatum, and in ipsilateral dopamine levels in the substantia nigra. Little or no effects of GDNF were seen on norepinephrine or serotonin levels. The neurochemical changes on dopaminergic afferents persist for at least 3 weeks after a single intracranial injection of 10 μg. Taken together, these data suggest that this glial cell line-derived factor has a potent influence on adult rat dopamine neurons and may have a potentially important role as a trophic factor for these neurons.