Ultrastructural immunocytochemical localization of tyrosine hydroxylase in the neostriatum

The morphology and synaptic associations of dopaminergic axons in the n. caudate-putamen (neostriatum) of the adult rat brain are examined. Identification of dopaminergic axons is based upon the electron microscopic immunocytochemical localization of the catecholamine synthesizing enzyme, tyrosine hydroxylase. Immunoreactivity for the enzyme is detected in unmyelinated axons and axon terminals in serial sections collected throughout the neostriatum. The labeled terminals range from 0.1 to 1.5 micron in diameter and have peroxidase reaction product located around closely packed, round vesicles with a diameter of 40-60 nm. The tyrosine hydroxylase containing axon terminals constitute approximately 21% of the total number of terminals in the n. caudatus-putamen and include 3 types which differ in size and synaptic specializations. The most prevalent (82% of total), type I, is small (0.15-0.39 micron in diameter) and forms symmetric junctions with dendrites and dendritic spines. The other two terminal types (II and III) have a medium to large diameter (0.4-1.5 micron) and show either no membrane specializations or asymmetric junctions with dendrites. The axon terminals without observable membrane densities are occasionally oriented so as to suggest an association with dopaminergic and non-dopaminergic axon terminals. These findings indicate that while the dopaminergic terminals may form axoaxonic connections, the primary synaptic contacts are with dendrites of intrinsic neurons in all regions of n. caudatus-putamen.     

Postnatal development of tyrosine hydroxylase mRNA-expressing neurons in mouse neostriatum

The striatum harbors a small number of tyrosine hydroxylase (TH) mRNA‐containing GABAergic neurons that express TH immunoreactivity after dopamine depletion, some of which reportedly resembled striatal medium spiny projection neurons (MSNs). To clarify whether the TH mRNA‐expressing neurons were a subset of MSNs, we characterized their postnatal development of electrophysiological and morphological properties using a transgenic mouse strain expressing enhanced green fluorescent protein (EGFP) under the control of the rat TH gene promoter. At postnatal day (P)1, EGFP‐TH⁺ neurons were present as clusters in the striatum and, thereafter, gradually scattered ventromedially by P18 without regard to the striatal compartments. They were immunonegative for calbindin, but immunopositive for enkephalin (54.5%) and dynorphin (80.0%). Whole‐cell patch‐clamp recordings revealed at least two distinct neuronal types, termed EGFP‐TH⁺ Type A and B. Whereas Type B neurons were aspiny and negative for the MSN marker dopamine‐ and cyclic AMP‐regulated phosphoprotein of 32 kDa (DARPP‐32), Type A neurons constituted 75% of the EGFP⁺ cells, had dendritic spines (24.6%), contained DARPP‐32 (73.6%) and a proportion acquired TH immunoreactivity after injections of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine and 3‐nitropropionic acid. The membrane properties and N‐methyl‐d ‐aspartate : non‐N‐methyl‐d ‐aspartate excitatory postsynaptic current ratio of Type A neurons were very similar to MSNs at P18. However, their resting membrane potentials and spike widths were statistically different from those of MSNs. In addition, the calbindin‐like, DARPP‐32‐like and dynorphin B‐like immunoreactivity of Type A neurons developed differently from that of MSNs in the matrix. Thus, Type A neurons closely resemble MSNs, but constitute a cell type distinct from classical MSNs.     

Neuropeptide Y-immunoreactive neurons receive synaptic inputs from dopaminergic axon terminals in the rat neostriatum

Double immunocytochemistry using peroxidase-antiperoxidase and protein A-gold was performed to determine whether neuropeptide Y (NPY) immunoreactive neurons receive synaptic inputs from catecholaminergic axon terminals in the rat neostriatum. Tyrosine hydroxylase-immunoreactive axons were found to be in synaptic contact with the somas and proximal dendrites of NPY-immunoreactive neostriatal neurons. These latter neurons were medium-sized and had indented nuclei, and thus thought to be medium aspiny interneurons. Thus nigrostriatal dopaminergic neurons may monosynaptically influence striatal NPY neurons.     

Substance P-Containing terminals in synaptic contact with cholinergic neurons in the neostriatum and basal forebrain: a double immunocytochemical study in the rat

Antibodies against substance P and choline acetyltransferase (ChAT) have been used in a sequential double-immunocytochemical ultrastructural study of the rat forebrain. The peroxidase-anti-peroxidase procedure was used for both antigens, however, two different substrates for the peroxidase reactions were used. The substance P-immunoreactive sites were first localized using 3,3'-diaminobenzidine as the substrate, then the ChAT-immunoreactive sites were localized using benzidine dihydrochloride. The reaction product formed by the two substrates was distinguishable in both the light and electron microscopes. Using this procedure, the cell bodies and proximal dendrites of identified cholinergic neurons in the neostriatum were found to receive symmetrical synaptic input from substance P-immunoreactive boutons. A similar pattern of substance P-immunoreactive synaptic input was observed onto magnocellular basal forebrain cholinergic neurons in the ventral pallidum and ventromedial globus pallidus. In both the striatum and basal forebrain substance P-immunoreactive boutons were also seen in contact with structures that did not display ChAT immunoreactivity.     

The dopaminergic innervation of monkey prefrontal cortex: a tyrosine hydroxylase immunohistochemical study

The distribution of tyrosine hydroxylase (TH)-immunoreactive fibers was characterized immunohistochemically in the prefrontal cortical regions of both Old World cynomolgus monkeys (Macaca fascicularis) and New World squirrel monkeys (Saimiri sciureus). In both species, differences in the density and/or laminar distribution of TH-labeled fibers were detected both across and within almost every prefrontal cytoarchitectonic region. In cynomolgus monkeys, areas 9 and 24 had the greatest density of TH-labeled fibers, areas 11, 12, 13 and 25 were of intermediate density, and areas 10 and 46 had the lowest density of immunoreactive fibers. Differences in fiber density within many of these regions were also consistently observed. On a laminar basis, the distribution of labeled fibers in a given area of cynomolgus prefrontal cortex was systematically related to the overall fiber density of that area. For example, in the lightly innervated fundus of the principal sulcus (area 46), labeled fibers were primarily present in layer I and layers V-VI, whereas in area 9, the most densely innervated region, TH-labeled fibers were present in all cortical layers. Similar regional differences in the density and laminar distribution of TH-immunoreactive fibers were also present in squirrel monkey prefrontal cortex. In previous studies, we have analyzed the regional and laminar distributions of fibers immunoreactive for TH and dopamine-beta-hydroxylase (DBH), a specific marker for noradrenergic cortical fibers, in multiple areas of cortex from both normal and locus ceruleus-lesioned animals. These comparisons, which have been confirmed in the present report, indicate that anti-TH and anti-DBH label distinct populations of axons in monkey neocortex, which presumably are dopaminergic and noradrenergic, respectively. Thus, the distribution of TH immunoreactivity described in the present report suggests that dopaminergic fibers are distributed in a very heterogeneous fashion in monkey prefrontal cortex. The distinctive innervation patterns exhibited by these fibers reveal the regions and layers that may be the principle sites of action of dopamine in exerting its effects on prefrontal cortical function.     

Postmitotic, postmigrational expression of tyrosine hydroxylase in olfactory bulb dopaminergic neurons

The developmental expression of tyrosine hydroxylase (TOH) was studied in a large, specific population of dopaminergic (DA) neurons in the main olfactory bulb (MOB) of the rat. These DA neurons comprise an anatomically distinctive population that has been well characterized in the adult hamster (Davis and Macrides, 1983) and rat (Halasz et al., 1981; Baker et al., 1983, 1984). We addressed a basic question in developmental neurobiology: What factors regulate the expression of neuronal transmitter phenotype during development? Olfactory bulb DA neurons are born in the ventricular and subependymal zones and migrate through all intervening layers to the most superficial layer in the bulb (Altman, 1969; Bayer, 1983). The time of TOH expression in these neurons was determined using immunohistochemistry and light microscopic image-analysis techniques. The results indicate that TOH phenotype is not expressed when the cells are born in the subependymal zone nor during their migration to the periglomerular region but only after they reached their final destination, the glomerular layer. This suggests that epigenetic factors associated with the glomeruli initiate the expression of the key transmitter synthesizing enzyme in these neurons. Primary olfactory neurons in the nasal epithelium project exclusively to glomeruli of the MOB; removal of this input in adult rats (Kawano and Margolis, 1982; Baker et al., 1983, 1984), mice (Nadi et al., 1981; Baker et al., 1983), dogs (Nadi et al., 1981), and hamsters (Kream et al., 1984) appears to down-regulate the expression of the TOH in periglomerular cells. The present results suggested that the input from the primary olfactory nerve is also necessary for the initial expression of the TOH phenotype. In support of this notion, we found that lesions of the olfactory nerve during the first postnatal week caused a significant reduction in the number of TOH-positive juxtaglomerular neurons in the following weeks. Thus, the olfactory nerve appears to be necessary for both the initiation and maintenance of TOH expression in olfactory bulb neurons. These findings suggest that specific cell-cell interactions play a key role in CNS neuronal transmitter phenotype regulation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Increased cell suspension concentration augments the survival rate of grafted tyrosine hydroxylase immunoreactive neurons

The poor survival rate (5-20%) of grafted embryonic dopamine (DA) neurons is one of the primary factors preventing cell replacement from becoming a viable treatment for Parkinson's disease. Previous studies have demonstrated that graft volume impacts grafted DA neuron survival, indicating that transplant parameters influence survival rates. However, the effects of mesencephalic cell concentration on grafted DA neuron survival have not been investigated. The current study compares the survival rates of DA neurons in grafts of varying concentrations. Mesencephalic cell suspensions derived from E14 Fisher 344 rat pups were concentrated to 25,000, 50,000, 100,000 and 200,000 cells/microl and transplanted into two 0.5 microl sites in the 6-OHDA-denervated rat striatum. Animals were sacrificed 10 days and 6 weeks post-transplantation for histochemical analysis of striatal grafts. The absolute number of DA neurons per graft increased proportionally to the total number of cells transplanted. However, our results show that the 200,000 cells/microl group exhibited significantly higher survival rates (5.48+/-0.83%) compared to the 25,000 cells/microl (2.81+/-0.39%) and 50,000 cells/microl (3.36+/-0.51%) groups (p=0.02 and 0.03, respectively). Soma size of grafted DA neurons in the 200,000 cells/microl group was significantly larger than that of the 25,000 cells/microl (p<0.0001) and 50,000 cells/microl groups (p=0.004). In conclusion, increasing the concentration of mesencephalic cells prior to transplantation, augments the survival and functionality of grafted DA neurons. These data have the potential to identify optimal transplantation parameters that can be applied to procedures utilizing stem cells, neural progenitors, and primary mesencephalic cells.     

Substance P synaptic interactions with GABAergic and dopaminergic neurons in rat substantia nigra: An ultrastructural doublelabeling immunocytochemical study

The distribution of substance P (SP), tyrosine hydroxylase (TH), and glutamic acid decarboxylase (GAD) immunoreactivity in the substantia nigra of the rat was studied by means of an ultrastructural double-labeling immunocytochemical method. Direct synaptic contact between SP-immunoreactive terminals and GAD-positive nigral neurons was more often observed in the pars lateralis than the pars reticularis and was rarely observed in the pars compacta. Substance P-positive terminals also formed synapses with cell bodies and dendrites of TH-positive, dopaminergic neurons in the pars compacta and pars reticulata. Multiple SP-immunoreactive terminals were often observed with symmetrical and, less frequently, asymmetrical synapses on individual TH-containing dendrites. Evidence of SP-containing terminals contacting both GABAergic and dopaminergic neurons in the substantia nigra suggests a direct excitatory action upon nigral projection neurons.     

Chronic lesions differentially decrease tyrosine hydroxylase messenger RNA in dopaminergic neurons of the substantia nigra

Long-term effects of lesions were analyzed in terms of gene expression. Nine months after unilateral 6-hydroxydopamine (6-OHDA) lesions of the substantia nigra pars compacta (s. nigra), the remaining dopaminergic (DAergic) neurons (tyrosine hydroxylase (TH) cells determined by immunocytochemistry (ICC] on the lesioned side were atrophic with smaller nucleoli. By in situ hybridization, the DAergic neurons on the lesioned side had a 50% smaller TH-mRNA concentration than on the contralateral non-lesioned side. However, beta-tubulin mRNA concentration in DAergic neurons was unaffected by the lesion. The lesions did not alter TH-mRNA concentration in the contralateral non-lesioned side by comparison with unoperated controls. We propose that chronic lesions have long-term effects on gene expression because of damage sustained during compensatory hyperactivity after the lesion, or because of decreased trophic support from other neurons.     

Reversible reduction of tyrosine hydroxylase enzyme protein during the retrograde reaction in mesolimbic dopaminergic neurons

Changes in the activity and amount of the neurotransmitter synthesizing enzyme tyrosine hydroxylase (TH) were measured in dopaminergic (DA) neurons of the A10-mesolimbic system of the rat following electrolytic lesions of their axons. Unilateral hypothalamic lesions in close proximity to the cell bodies resulted, within 24--48 h, in a permanent anterograde reduction of TH to 10--20% of control in the ipsilateral olfactory tubercle and nucleus accumbens. The retrograde reaction in the A10-DA nerve cell bodies was characterized by an initial increase in TH activity to 133% by 24--48 h followed by a gradual and permanent fall to 50% of control by day 14 due to retrograde cell death of DA neurons. In contrast, lesions of DA axon terminals in the olfactory tubercle resulted in a reversible retrograde reduction of TH activity of the A10. The enzyme activity declined during the first 7 days to 70% of control and then gradually recovered, reaching control levels by 28 days after the operation. The reduction in TH activity in the A10 was demonstrated by immunochemical titration with a specific antibody to TH to be entirely due to reduced amounts of enzyme protein. We conclude that in mesolimbic DA neurons; (a) the anterograde reaction is characterized by a rapid and permanent decline of TH in degenerating terminals; (b) the retrograde reaction is dependent upon the proximity of the lesion to the nerve cell body, and (c) a reversible reduced accumulation of TH characterizes the retrograde reaction in response to lesions of distal axons. The reaction of DA neurons of the mesolimbic system to axonal injury is comparable to that of the nigrostriatal system.     

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.     

The immunocytochemical localization of tyrosine hydroxylase within rat sympathetic neurons that release acetylcholine in culture

In vitro populations of neurons dissociated from the superior cervical ganglion of the rat embryo can acquire many of the properties of cholinergic neurons and yet retain some of the characteristics of adrenergic neurons. To determine whether one neuron can stably express properties of two transmitter systems, identified neurons were characterized by both immunocytochemical and electrophysiological methods. It was found that more than 90% of the neurons that formed functional cholinergic synapses had tyrosine hydroxylase within their cytoplasm. Furthermore, this mixed phenotypic expression persisted for more than 1 month. It is concluded that, under certain conditions in vitro, most neurons from the superior cervical ganglion have at least the potential for dual function.     

Coexistence of oxytocin and tyrosine hydroxylase in the rat hypothalamus,an immunocytochemical study

Summary Immunocytochemical double labelling was used to determine the structural relationship of oxytocin (OT) and tyrosine hydroxylase (TH) containing perikarya and processes in the rat hypothalamus. Extrahypothalamic TH fibers, as well as parvocellular TH neurons were found to form contacts with OT cells. A fraction of the OT neurons contained TH immunoreactivity. It is likely that in addition to the classical mesencephalic afferences also hypothalamic interneurons and magnocellular dopaminergic neurons control the hypothalamo neurohypophysial system.     

Ontogeny of tyrosine hydroxylase mRNA signal levels in central dopaminergic neurons: development of a gender difference in the arcuate nuclei.

Using in situ hybridization and immunocytochemistry, this study examined the tyrosine hydroxylase (TH) mRNA signal levels and immunostaining in the arcuate nuclei of the hypothalamus, zona incerta and substantia nigra of male and female rats during neonatal, peripubertal and adult life. The catalytic activity of TH in the stalk-median eminence was also investigated using the in vitro accumulation of 3,4 dihydroxyphenylalanine (DOPA) after inhibiting aromatic amino acid decarboxylase activity. In the arcuate nuclei, TH mRNA levels increased 3.5-fold between 5 and 15 days of age and remained at a steady level between 15 and 35 days of age in both male and female rats. Similar to TH mRNA levels in the arcuate nuclei, TH activity in the stalk-median eminence increased 2-3 fold between days 10 and 15 of age and remained at a steady level between 15 and 35 days of age in both sexes. A later increase in TH mRNA levels in the arcuate nuclei and catalytic activity in the stalk-median eminence was observed between 35 and 40 days in females, but not males. During adulthood, TH mRNA levels and enzyme activity were 2.7-fold higher in the arcuate nuclei and stalk-median eminence, respectively, of diestrous females vs males. These data suggest that the changes in TH mRNA in the arcuate nuclei may contribute to the developmental alterations, as well as the adult gender differences, in enzyme activity in the stalk-median eminence. Circulating progesterone levels were low (1-10 ng/ml) between days 5 and 25 of age and increased 6-fold between 25 and 35 days of age in both males and females. Progesterone levels increased 2-fold in females, but not males, between days 35 and 40 and were 4-fold higher in diestrous females as compared to adult males. Circulating prolactin levels were low (2-3 ng/ml) between days 5 and 15, increased 15-fold between days 15 and 25 and increased an additional 2- to 3-fold between days 35 and 70 in both males and females. TH mRNA signal levels increased between days 5 and 15 of age in dopaminergic perikarya in the zona incerta and the substantia nigra of both female and male rats. The TH mRNA levels remained constant between days 15 and 70 in the zona incerta, whereas TH mRNA levels declined with age in the substantia nigra.(ABSTRACT TRUNCATED AT 400 WORDS)     

Efferent connections of the anterior hypothalamic nucleus: a biocytin study in the cat

The efferent connections of the anterior hypothalamic nucleus (AH) were examined using biocytin as anterograde tracer in the cat. The results provide several new findings in addition to confirming earlier observations. In the hypothalamus, the AH projections terminated mainly in the medial regions which are related to the defensive, reproductive and feeding behaviors, and autonomic functions. Moreover, we found dense patches of the AH terminals in the medial preoptic area and ventromedial hypothalamic nucleus, which suggests the existence of modular connections between sub-regions of each nucleus. In addition, the AH projected to regions which may be related to the emotional and autonomic responses, i.e., such regions in the amygdala, midline thalamus, septum, subthalamus, and midbrain. The data suggest that the AH may play an important role in the autonomic functions and behaviors between animals, and thus may play a key role in the defensive behavior elicited in the medial preoptic area and ventromedial hypothalamic nucleus.     

Slow changes of tyrosine hydroxylase gene expression in dopaminergic brain neurons after neurotoxin lesioning: a model for neuron aging.

Slow neuron regression develops during the adult phase of life in select brain systems of mammals. We describe a model in adult rats that resolves several phases in a slow atrophic process that differentially influences levels of mRNA and protein for tyrosine hydroxylase (TH). Responses of striatal dopaminergic markers to 6-hydroxydopamine (6-OHDA) lesions in rats indicated that the striatal terminals maintained TH protein, despite greater than 3-fold loss of TH mRNA in the substantia nigra pars compacta (SNC) cell bodies whose axons project to the striatum. The loss of TH mRNA/cell was progressive up to 9 months, whereas SNC cell body shrinkage stabilized by 3 months post-lesioning. Consideration of possible mechanisms in protein turnover motivated a search for PEST motifs in the TH of rats and other vertebrates that could be a point of regulation by altering the rate of TH protein turnover.     

Activity of nigral dopaminergic neurons after lesion of the neostriatum in rats

As shown by post-mortem analysis the major neuropathological trait of Huntington's chorea is a degeneration of the intrinsic neurons of the neostriatum (caudate nucleus and putamen). Such a situation can be reproduced by a destruction of the neostriatum by kainic acid. When injected into the caudate nucleus this excitatory amino acid destroys the intrinsic neurons of the neostriatum and spares fairly well the passing fibers.In the present work, we have chosen to examine the influence of neostriatal destruction on the activity of identified dopaminergic cells in the pars compacta of the substantia nigra. As a key element in the nigro-neostriato-nigral loop, this structure is a relevant site for observing the functional effects of neostriatal lesion.Our research hypothesis was based on the generally accepted view that the suppression of the important neostriato-nigral pathway and in particular the inhibitory GABAergic contingent, could generate a hyperactivity of nigral dopaminergic cells. One may therefore consider that the dopaminergic hyperactivity produces abnormal messages which can influence via several pathways the motoneurons, and which participates in the genesis of the hyperkinetic movements characteristic of chorea.After destruction of the neostriatum, we have shown that the pattern of discharge of most identified nigral dopaminergic neurons becomes greatly disorganized. This drastic change in the pattern of activity cannot be interpreted as the simple ‘lift of brake’ on these cells by the suppression of the inhibitory GABAergic striato-nigral tract.     

Efferent connections of the vestibular nuclei in the rat: a neuromorphological study using PHA-L

The efferent connections of the superior, medial, lateral, and descending vestibular nuclei were studied with anterograde tracing methods in rats. The following areas of termination could be discerned: (1) In the diencephalon, labeled terminals were detected in the thalamus. (2) In the mesencephalon, the red nucleus and motor nuclei involved in eye movements were richly supplied by the vestibular nuclei. (3) In the rhombencephalon, extensive intrinsic connections of all vestibular nuclei were demonstrated. Strong commissural connections were found among the medial, superior, and descending vestibular nuclei. The inferior olive received labeled fibers exclusively from the lateral vestibular nuclei. Individual differences were demonstrated in the termination areas in the reticular formation. (4) In the spinal cord, most of the descending vestibular fibers were found in the ipsilateral anterior funiculus.