Persistent loss of tyrosine hydroxylase immunoreactivity in the substantia nigra after neuroleptic withdrawal

A 37 year woman developed neuroleptic induced parkinsonism thatpersisted long after the drug had been discontinued. This prompted astudy of the effect of an eight week course of haloperidol (HAL)followed by two week withdrawal, on dopaminergic neurons of thesubstantia nigra in rats. Animals treated with HAL showed a highlysignificant 32%-46% loss of tyrosine hydroxylase (TH) immunoreactiveneurons in the substantia nigra, and 20% contraction of the TH staineddendritic arbour. Neuroleptic drug induced downregulation of nigraldopaminergic neurons may help to explain the persistent parkinsonismfound in many patients after withdrawal of medication.

     

Brain-derived neurotrophic factor and substantia nigra dopaminergic neurons in Parkinson''s disease

BACKGROUND:Parkinson's disease (PD) is a chronic, progressive neurodegenerative central nervous system disease which occurs in the substantia nigra-corpus striatum system. The main pathological feature of PD is selective dopaminergic neuronal loss with distinctive Lewy bodies in populations of surviving dopaminergic neurons. In the clinical and neuropathological diagnosis of PD, brain-derived neurotrophic factor mRNA expression in the substantia nigra pars compacta is reduced by 70%, and surviving dopaminergic neurons in the PD substantia nigra pars compacta express less brain-derived neurotrophic factor (BDNF) mRNA (20%) than their normal counterparts. In recent years, knowledge surrounding the relationship between neurotrophic factors and PD has increased, and detailed pathogenesis of the role of neurotrophic factors in PD becomes more important.     

Loss of insulin receptor immunoreactivity from the substantia nigra pars compacta neurons in Parkinson's disease

Immunohistochemistry using both a newly developed polyclonal, and a commercially available monoclonal, anti-insulin receptor antibody was done on the midbrain from cases of idiopathic Parkinson's disease (PD), Alzheimer's disease, amyotrophic lateral sclerosis, vascular parkinsonism and non-neurological controls. Both antibodies gave indentical patterns of neuronal staining. The neurons of the oculomotor nucleus were immunopositive in all the brains. However, the neurons in the pars compacta of the substantia nigra, paranigral nucleus, parabrachial pigmental nucleus, tegmental pedunculopontine nucleus, supratrocheal nucleus, cuneiform nucleus, subcuneiform nucleus and lemniscus medialis, which were positive in other diseases and in non-neurological controls, were not stained by these antibodies in PD brains. These results suggest that, in PD, a dysfunction of the insulin/insulin receptor system may precede death of the dopaminergic neurons.The work in the Kinsmen Laboratory was supported by the MRC of Canada and the Parkinson Society of Canada     

Neurotensin terminals form synapses primarily with neurons lacking detectable tyrosine hydroxylase immunoreactivity in the rat substantia nigra and ventral tegmental area.

A light and electron microscopic double antigen localization technique was employed to examine the fine structural relationship between neurotensin-containing axon terminals and dopaminergic neurons in the substantia nigra and ventral tegmental area of the rat. At the light microscopic level, neurotensin-immunoreactive terminals were densely distributed throughout the substantia nigra pars compacta and ventral tegmental area in close proximity to tyrosine hydroxylase-immunoreactive somata and dendrites. On electron microscopic examination, direct synaptic connections were identified between neurotensin-immunoreactive axon terminals and tyrosine hydroxylase-immunopositive perikarya and dendrites. However, only 8.2% and 8.8% of the neurotensin-immunoreactive axonal profiles detected in the substantia nigra and ventral tegmental area, respectively, were found in direct apposition with tyrosine hydroxylase-immunostained elements. In turn, only 9.3% and 10.0% of tyrosine hydroxylase immunoreactive dendrites sampled from the substantia nigra and ventral tegmental area, respectively, were seen in contact with neurotensin immunopositive axon terminals. However, neurotensin-immunoreactive and tyrosine hydroxylase-immunolabelled elements were frequently identified in close anatomical proximity (less than 5 microns) to one another. These results are interpreted in light of the selective association of neurotensin receptors with dopaminergic neurons in the substantia nigra and ventral tegmental area to suggest a predominantly parasynaptic mechanism of action for neurotensin in the ventral 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.     

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.     

Immunohistochemistry on tyrosine hydroxylase in the substantia nigra of human autopsied cases

Despite the numerous data on the distribution of catecholamine (CA) neurons in a wide variety of species, data on that of human are rare because of technical difficulty in applying fluorescent histochemical methods. On the other hand, immunohistochemistry on tyrosine hydroxylase (TH) which was essential for synthesis of CA, was assumed to be applicable to human autopsied brain. We performed indirect immunoperoxidase study on TH using 10% formalin or buffered formalin fixed, paraffin embedded sections of the substantia nigra from 40 autopsied cases, ranging in age 7 to 70 years. TH was purified from the bovine adrenal medulla using the procedure described by Oka et al. with some modification. Purified TH was injected with Freund's complete adjuvant into rabbits. The specificity of the antiserum was demonstrated by the inhibition study of CA relating enzymes, double immunodiffusion and immunoelectrophoresis against tissue homogenate of bovine adrenal medulla. TH immunoreactive (TH-IR) products were restricted to the neuronal soma, axons and dendrities in almost all of the case except for the "dead brain" cases. TH-IR neurons were of large polygonal or multipolar cells, and closely correspond to melanin-pigmented neurons, located in the compact zone and scattered in the reticular zone in the substantia nigra. On the other hand, melanin free neurons in the reticular and compact zone, were of medium or small bipolar cells and were not stained. It was of interest that the non TH-IR melanin-pigmented neurons firstly appeared in the substantia nigra at third decade and increased their number with age.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neuroprotective effects of subthalamic nucleus high-frequency stimulation on substantia nigra neurons in a Parkinson''s disease rat model

BACKGROUND: Deep-brain stimulation has proven to be beneficial in the treatment of Parkinson's disease (PD) patients. OBJECTIVE: To investigate the effects of high-frequency stimulation (HFS) to the subthalamic nucleus (STN) on neuronal apoptosis and apoptosis-related gene expression in the substantia nigra pars compacta, and to analyze the neuroprotective effect of HFS-STN. DESIGN, TIME AND SETTING: Neuronal morphology experiments were performed in the Beijing Nearosurgical Institute from May to December in 2005. MATERIALS: Forty healthy, adult, Sprague Dawley rats were used to establish a PD model with a unilateral microinjection of 6-hydroxydopamine into two target areas of the right medial forebrain bundle. 6-hydroxydopamine was purchased from Sigma (USA); high-frequency electrical stimulator was produced by World Precision Instruments (USA); Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) kit was a product of Nanjing Jiancheng Technology Co., Ltd. (China); and Bcl-2 and Bax protein assay kit were purchased from Wuhan Boster Bioengineering Co., Ltd. (China). METHODS: Forty rats were randomly divided into three groups. The stimulation group (n = 15) received HFS-STN on the day of PD modeling. The PD model group (n = 15) was used to establish the PD model. The control group (n = 1 0) was injected with normal saline containing 0.2 g/L ascorbic acid into two areas of the right medial forebrain bundle. MAIN OUTCOME MEASURES: Survival of dopaminergic neurons in the substantia nigra pars compacta was determined using Nissl staining. Apoptosis of dopaminergic neurons was detected using TUNEL techniques. Expression of anti-apoptotic protein, Bcl-2, and pro-apoptotic protein, Bax, were assayed by immunohistochemistry. RESULTS: Following 6-hydroxydopamine injection, the number of substantia nigra pars compacta neurons was reduced in the stimulation and PD model groups, compared to the control group. At 2 and 4 weeks post-surgery, the grey value of Nissl stained images was significantly less in the PD model and stimulation groups (P < 0.05), and the stimulation group exhibited greater grey values compared to the model group (P < 0.05). At 2 and 4 weeks post-surgery, the number of apoptotic neurons was significantly less in the stimulation group compared to the model group (P < 0.05). In addition, Bcl-2 and Bax expression, as well as the Bcl-2/Bax ratio, was much higher in the stimulation group compared to the model group (P < 0.05). CONCLUSION: HFS-STN has a neuroprotective effect on dopaminergic neurons in the substantia nigra pars eompacta of PD rats by promoting Bcl-2 expression, inhibiting Bax expression, and reducing the number of apoptotic dopaminergic neurons.     

Substance K (NKA) increases tyrosine hydroxylase mRNA in cultured substantia nigra

Cultured explants of the mouse substantia nigra were used to analyze mechanisms underlying the depolarization-induced increase in tyrosine hydroxylase activity. Steady-state levels of messenger RNA encoding tyrosine hydroxylase were detected using an antisense riboprobe in an RNase protection assay. Explants exposed to the depolarizing agent, veratridine, exhibited an approximate 2-fold increase in tyrosine hydroxylase messenger RNA. Moreover, the native presynaptic excitatory agonist substance K also elicited a significant increase in tyrosine hydroxylase message. We conclude that depolarizing influences induce tyrosine hydroxylase in the cultured substantia nigra in association with an elevation of enzyme messenger RNA.     

Neuronal hypertrophy in the pars reticulata of the substantia nigra in Parkinson''s disease

The size of neurons in the pars reticulata of the substantia nigra in patients dying with Parkinson's disease has been compared with that in non-parkinsonian control cases. Parkinson's disease is accompanied by a significant (+53%) hypertrophy of reticulata neurons. This is similar to changes seen in experimental rats with ipsilateral damage of the striatum. It is suggested that the enlargement seen in Parkinson's disease, similar to that in the rat, is indicative of plasticity in the GABA-ergic reticulata neurons, and may be associated with increased inhibitory flux in pathways arising from the pars reticulata to the superior colliculus and thalamus.     

Verbascoside promotes the regeneration of tyrosine hydroxylase-immunoreactive neurons in the substantia nigra

Tyrosine hydroxylase is a key enzyme in dopamine biosynthesis. Change in tyrosine hydroxylase expression in the nigrostriatal system is closely related to the occurrence and development of Parkinson’s disease. Verbascoside, an extract from Radix Rehmanniae Praeparata has been shown to be clinically effective in treating Parkinson’s disease. However, the underlying mechanisms remain unclear. It is hypothesized that the effects of verbascoside on Parkinson’s disease are related to tyrosine hydroxylase expression change in the nigrostriatal system. Rat models of Parkinson’s disease were established and verbascoside (60 mg/kg) was administered intraperitoneally once a day. After 6 weeks of verbascoside treatment, rat rotational behavior was alleviated; tyrosine hydroxylase mRNA and protein expression and the number of tyrosine hydroxylase-immunoreactive neurons in the rat right substantia nigra were signiifcantly higher than the Parkinson’s model group. These ifndings suggest that the mechanism by which verbascoside treats Parkinson’s disease is related to the regeneration of tyrosine hy-droxylase-immunoreactive neurons in the substantia nigra.     

A quantitative morphometrical study of neuron degeneration in the substantia nigra in Parkinson''s disease

We studied the pigmented neurons of the substantia nigra (SN) from 8 controls and 20 patients with Parkinson's disease (PD) using a computerized morphometric methodology. On the basis of neuronal topography, several anatomic regions were outlined in the SN. In these subrogions the area, perimeter, diameter of the cell bodies and cell numbers were measured and were counted in the controls and PD patients. The measurements were made at the level of the exit of the third cranial nerve from the brain stem. In PD patients, when the whole SN was considered, the mean area, mean perimeter and diameter of the pigmented cell bodies were significantly reduced by 35%, 20% and 21% respectively from the control mean values. Regionally, the pigmented neuron area in the medial ventral part (VM), medial dorsal part (DM), lateral ventral part (VL), lateral dorsal part (DL) and pars lateralis part (PL) showed a significant reduction of 33–41% as compared to controls. In these subregions, a significant decrease in PD patients from the control mean values was seen both in the pigmented neuron perimeter, by 19–26%, and the diameter by 19–25%. This decrease in cell size suggests that, in PD patients, the remaining pigmented neurons in the SN are in a process of degeneration and atrophy. In PD patients the number of pigmented neurons in the whole SN decreased about 76% from control values. Evaluation of the influence of cell size on the apparent quantity of cells in sections indicates, however, that in PD patients the impact of true loss of pigmented neurons is far more dramatic than the impact of their decrease in size.     

A monoclonal antibody to non-phosphorylated neurofilament protein marks the vulnerable cortical neurons in Alzheimer's disease

Various cytoskeletal proteins have been implicated in the formation of neurofibrillary tangles in Alzheimer's disease. A monoclonal antibody to non-phosphorylated neurofilament protein labels a distinct subset of pyramidal cells in the normal human cortex which have a distribution very similar to that of neurofibrillary tangles in brains from patients with Alzheimer's disease. In addition, regions and layers that normally contain a high density of such cells, in Alzheimer's disease, have large numbers of neurofibrillary tangles and few remaining immunoreactive cells.     

Subpopulations of neurons in rat substantia nigra display GABABR2 receptor immunoreactivity

A functional gamma-aminobutyric acid (GABA) B receptor is the first metabotropic receptor known to be composed of two heteromeric subunits, GABA_BR1 and GABA_BR2. Our previous report [Neuroscience 99 (2000) 65] has demonstrated that subpopulations of neurons in the rat substantia nigra display distinct patterns of distribution of GABA_BR1 receptor immunoreactivity. A robust level of GABA_BR1 receptor is only found in the dopaminergic neurons of the substantia nigra pars compacta (SNc). The objective of the present study was to determine the precise cellular localization of GABA_BR2 subunit in the rat substantia nigra using double immunofluorescence. Neuropilar elements in the SNc and the substantia nigra pars reticulata (SNr) were found to display GABA_BR2 immunoreactivity. In addition, the tyrosine hydroxylase-immunoreactive dopaminergic neurons and the parvalbumin-immunoreactive GABAergic neurons in the SNr were also found to display GABA_BR2 immunoreactivity. The present results thus demonstrate that a functional GABA_B receptor may be expressed by the dopaminergic neurons in the SNc. It is less clear whether neurons in the SNr express a functional GABA_B receptor. The present findings have important functional implications in GABA neurotransmission in the substantia nigra.     

Flunarizine induces a transient loss of tyrosine hydroxylase immunoreactivity in nigrostriatal neurons

Neurotoxic effects of flunarizine (Fz), a selective calcium channel blocker, on the nigrostriatal dopamine system was investigated. Systemic injections of Fz to mice resulted in a transient loss of tyrosine hydroxylase (TH) immunoreactive nigrostriatal neurons without cell loss. TH immunoreactivity in these neurons was greatly reduced as rapidly as one day after drug administration (regardless of dosage used) and thereafter recovered in both dose- and time-dependent manners. Such a novel neurotoxic action of Fz may constitute a morphological substrate for reversible drug-induced parkinsonian signs described in recent clinical case reports.     

Selective colocalization of immunoreactivity for intracellular gonadal hormone receptors and tyrosine hydroxylase in the ventral tegmental area,substantia nigra,and retrorubral fields in the rat

Gonadal hormones influence brain functions, including motor and motivational behaviors, transmitter release, and receptor binding in midbrain dopamine systems. Much of this influence suggests genomic hormone action. To identify which midbrain cells may be targets of genomic influence, double label immunocytochemistry was used to map intracellular estrogen and androgen receptors and tyrosine hydroxylase (TH) in the ventral tegmental area (VTA), substantia nigra (SN), and retrorubral fields (RRF) in intact, adult rats. The distribution of estrogen and androgen receptor immunoreactivity was highly selective, similar in males and females, and largely nonoverlapping. Estrogen receptors were present within subpopulations of cells in the ventrolateral paranigral VTA and rostrolateral RRF; of these, only a few cells in the RRF were immunoreactive for TH. Cells immunoreactive for androgen receptors were numerous in the paranigral and parabrachial VTA, SN pars lateralis and dorsomedial pars compacta, and lateral RRF. Nearly every androgen receptor-bearing cell in the VTA and SN pars compacta, roughly half in the SN pars lateralis, and about one-third in the RRF were TH immunopositive. The localization of estrogen receptors approximates the distribution of subsets of cells labeled following neostriatal injections, whereas androgen receptors tend to occupy regions labeled by injections in cortical or limbic targets. These receptor-specific alignments with origins of nigrostriatal, mesolimbic, and mesocortical projections are consistent with identified estrogen influence over motor behaviors and androgen involvement in motivational functions and may hold clues for understanding hormone action in these and other functions and dysfunctions of midbrain dopamine systems. J. Comp. Neurol. 379:247–260, 1997. © 1997 Wiley-Liss, Inc.     

Interrelationship of the distribution of neuropeptides and tyrosine hydroxylase immunoreactivity in the human substantia Nigra

The relationship in the human substantia nigra of peptidergic fibers with intrinsic dopaminergic neurons was studied in adjacent coronal sections of the mesencephalon immunohistochemically stained for enkephalin (ENK), substance P (SP), and tyrosine (TH) hydroxylase immunoreactivity. TH-positive elements are present in the substantia nigra in at least two different arrangements: 1) a dorsal tier of rather loosely arranged neurons, which is continuous medially with the ventral tegmental area and laterally with the retrorubral area, 2) a ventral tier of more closely packed neurons, clusters of which frequently form finger-like extensions deep into the pars reticulata. This ventral region contains TH-positive dendrites extending ventrally into the pars reticulata. The distribution of ENK is mainly restricted to the medial half of the ventral aspect of the substantia nigra, while SP occupies its entire rostral-caudal and medial-lateral extents. Peptide-positive fibers vary in density from dense to light. There is very little overlap between the dorsal tier of the TH-positive neurons and the ENK or SP staining. The dorsal part of the peptide-immunoreactive area extensively overlaps with the TH-positive neurons of the ventral tier of cells. The ventral part of the peptide-positive area overlaps with the pars reticulata of the substantia nigra in which the TH-positive dendrites extend. The overlap between the neuropeptide fibers and the TH-positive cells of the ventral tier is not complete, with cells found both within and outside peptide-positive fiber networks. Three patterns of overlap emerge. In dorsal regions elongated cell clusters lie partially within and partially outside the dense peptide-positive fiber networks. In the ventral regions TH-positive cells are either completely embedded within peptide fibers or clusters of cells are present in peptide-free zones. These data suggest that specific peptidergic pathways differentially innervate the substantia nigra. TH cells which lie within or outside these fibers may reflect functionally different subsystems in the striatonigral pathways in the human.