Feeding following microinjection of cholinergic substances into substantia nigra.

Microinjections of acetylcholine and eserine localised within the substantia nigra of the rat elicited a dose-dependent increase in feeding, but not drinking when both food and water were freely available. When required to perform an operant response for food, microinjections of carbachol into substantia nigra caused a dose-dependant increase in lever pressing for food (FR5). High doses of carbachol (1.0 and 5.0 μ1) elicited a behavioural stereotypy characterised by chewing, gnawing and biting. A significant negative correlation was found between the effectiveness of cholinergic stimulation and the distance from the site of highest feeding which was in the pars compacta region of substantia nigra. These data suggest a functional role for acetylcholine within substantia nigra and provide indirect support for the concept of an interaction between cholinergic and dopaminergic neurons within this structure.     

Cytophotometric analyses of thalamic neuronal RNA in soman intoxicated rats

Quantitative azure B-RNA cytophotometry was used to monitor metabolic responses of individual neurons within the ventrobasal nuclear complex (VBC) and nucleus reticularis (NR) of the rat thalamus following administration of soman (0.5, 0.9 or 1.5 LD₅₀, sc). A dose-dependent depression in brain acetylcholinesterase (AChE) was evidenced. With respect to thalamic RNA responses, a complex pattern of RNA alterations was evidenced, with these two regions generally exhibiting opposite patterns of dose-related RNA changes. With sub-lethal dosages of soman, RNA accumulation was evidenced in the acetylcholine (ACh) mediated excitatory VBC region and RNA depletion in the ACh mediated inhibitory NR neurons. With a lethal dose, an opposite RNA response pattern observed in both thalamic regions. It is postulated that the observed RNA response pattern with sub-lethal dosages of soman is what one would anticipate with cholinergic brainstem reticular formation activation. The absence of such a response with lethal doses strongly suggests some disruption of functional excitatory cholinergic activity and perhaps also an impairment of inhibitory cholinergic synaptic activity.     

GABAA Receptors in the Pars Compacta and GABAB Receptors in the Pars Reticulata of Rat Substantia Nigra Modulate the Striatal Dopamine Release

The nigral GABAergic regulation of striatal dopamine release was investigated using voltammetry in freely moving rats. The local administration of muscimol (1 nM) in the substantia nigra pars compacta, but not in the substantia nigra pars reticulata, increased the striatal dopamine release. In contrast, the administration of baclofen (10 nM) in the substantia nigra pars reticulata, but not in the substantia nigra pars compacta, produced a decrease of the striatal dopamine release. Opposite effects were respectively observed after administration of GABA_A and GABA_B antagonists. These data lead us to suggest a differential presynaptic GABAergic control of the dopaminergic neurotransmission through GABA_A receptors in the substantia nigra pars compacta, and GABA_B receptors in the substantia nigra pars reticulata.     

Pathological changes in dendrites of substantia nigra neurons in Parkinson's disease: a Golgi study.

Neurons of the substantia nigra show severe morphological changes in Parkinson's disease. Pathological alterations of cell bodies have been described, whereas those of neuronal processes have hardly been investigated. Golgi impregnation has been the chosen method for demonstrating neuronal processes and dendritic and somatic spines. We therefore used the Golgi-Braitenberg method to qualitatively and semi-quantitatively study the substantia nigra of eight patients with Parkinson's disease compared with eight control cases. Golgi impregnation of substantia nigra neurons was good in all control cases. In full agreement with the analysis of Braak and Braak (1986) three neuronal types within the substantia nigra were found. In cases of Parkinson's disease, severe pathological changes such as decrease of dendritic length, loss of dendritic spines and several types of dendritic varicosities were found only in the melanin-containing pars compacta neurons. Pars reticulata nerve cells were intact. These findings support the predominant role played by the dopaminergic efferent pathway in the degenerative process. The afferent pathway was not affected. This suggests that the substantia nigra lesion is primary in Parkinson's disease. Loss of neurons found in H & E sections corresponded to a lesser amount of impregnated pars compacta neurons in cases with Parkinson's disease when compared to controls. Evidences exist that the duration of the disease may be related to the extent of pathologically altered Golgi-impregnated pars compacta cells. The amount of Lewy bodies in H & E sections corresponded to the quantity of round varicosities in impregnated pars compacta neurons. These round dendritic varicosities were considered to be Lewy body inclusions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Brain neuronal RNA metabolism during sustained low-level soman toxication

Quantitative azure B-RNA cytophotometry was used to monitor metabolic responses of cholinergic elements of the rat brain during sustained low-level administration of soman (0.25-0.50 LD50, sc). RNA contents of caudate and cerebrocortical (Layers III and V) neurons were measured 60 min following 1-5 soman dosages given at 24 h intervals. Marked and progressive RNA depletion was evidenced after 1-4 soman injections, whereas partial or complete restitution of RNA levels was observed following the fifth injection. These data indicate that repetitive soman toxication is associated with metabolic correlates of impaired rather than accentuated activation of CNS cholinergic systems, and that tolerance is developed to CNS actions of the agent. It is postulated that impaired neuronal activation is related to soman or ACh-induced transmission block, and that the same adaptive processes responsible for recovery during acute poisoning may underlie the development of tolerance during repetitive administration of organophosphates.     

Glycine Receptors in the Human Substantia Nigra as Defined by [3H]Strychnine Binding

Abstract: Specific [³H]strychnine binding was used to identify the glycine receptor macromolecular complex in human spinal cord, substantia nigra, inferior olivary nucleus, and cerebral cortex. In material from control patients a high-affinity K d (3–8 n m ) was observed in the spinal cord and the substantia nigra, both the pars compacta and the pars reticulata. This is very similar to the values observed in the rat and bovine spinal cord (8 and 3 n m , respectively) and rat substantia nigra (12 n m ). In the human brain the distribution of [³H]strychnine binding (at 10 n m ) was: spinal cord – substantia nigra, pars compacta > substantia nigra, pars reticulata = inferior olivary nucleus > cerebral cortex. The binding capacity ( B _max) of the rat brain (substantia nigra or spinal cord) was approximately 10-fold that of the human brain. [ ³ H]Strychnine binding was significantly decreased in the substantia nigra from Parkinson's disease patients, both in the pars compacta (67% of control) and the pars reticulata (50% of control), but not in the inferior olivary nucleus. The results were reproduced in a preliminary experiment in rats with unilateral 6-hydroxydopamine lesions of the medial forebrain bundle. In the substantia nigra from patients who died with Huntington's disease, [³H]strychnine binding tended to be high (150% of control, NS) in both the pars compacta and the reticulata. [³H]Strychnine binding was unaltered in the substantia nigra of patients with senile dementia. Together with previous neurophysiological and neuropharmacological findings, those results support the hypothesis of glycine receptors occurring on dopamine cell bodies and/or dendrites in the substantia nigra.     

Cytophotometric analyses of mesencephalic reticular formation neuronal RNA in soman intoxicated rats

Quantitative azure B cytophotometry was used to monitor ribonucleic acid (RNA) responses of individual neurons within the nucleus cuneiformis (NC) and ventrotegmental nucleus (VTN) of the rat mesencephalic reticular formation following single subcutaneous soman (pinacolyl methylphosphonofluoridate) injections (0.5, 0.9 or 1.5 LD₅₀). The sub-lethal (0.5 LD₅₀) dosage of soman produced RNA accumulation in NC neurons, but VTN-RNA levels were not significantly altered. In contrast, both reticular nuclei exhibited prominent RNA depletion with higher soman dosages, the severity of which was greater with lethal (1.5 LD₅₀) than near-lethal (0.9 LD₅₀) dosages. These data indicate that metabolic correlates of enhanced activation of cholinergic reticular nuclei are present only with sub-lethal dosages, and that higher dosages produce characteristics of impaired activation of ascending cholinergic pathways. At present, mechanisms underlying soman-induced metabolic and neurologic deficits remain speculative.     

Soman toxication in hypoxia acclimated rats: Alterations in brain neuronal RNA and survival

Effects of prior hypoxia acclimation (14-day at 380 mm Hg) on soman (pinacolyl methylphosphonofluoridate) induced brain neuronal RNA and acetylcholinesterase (AChE) depletion and lethality were monitored in rats following their return to ambient oxygenation. Quantitative cytochemical techniques were used to measure RNA and AChE changes in individual cerebrocortical (Layer III) and striatal (caudate plus putamen) neurons. In ambient P_{O 2} controls, soman eventuated in a moderate diminution of neuronal RNA in both brain regions and severe, dosedependent suppression of AChE activity. Hypoxia acclimation per se induced RNA alterations as manifested in cortical RNA depletion and increased variability of striatal neuron RNA contents. In hypoxia acclimated rats, the extent of neuronal RNA depletion following soman injection was attenuated in both brain regions, yet there were no discernible differences in saline control AChE levels or in the extent of soman-induced AChE inhibition in ambient control versus hypoxia acclimated treatment groups. Hypoxia acclimated rats, however, were found to be even more susceptible to lethal actions of soman as assessed using 24- and 48-hour survival following a three-point treatment regimen. These data indicate that while compensatory systemic and central metabolic adjustments associated with 14d acclimation to reduced oxygen availability may retard soman-induced neuronal RNA depletion, resistance to lethal or near-lethal soman exposure is not enhanced. It is postulated that hypoxia acclimation is associated with complex adaptive and maladaptive neurophysiological alterations influencing CNS responsiveness to soman toxication, and that detrimental consequences exceed protection afforded by metabolic adaptation.     

Detrimental deletions: mitochondria, aging and Parkinson's disease

As individuals enter their 80s, they are inevitably confronted with the problem of neuronal loss in the brain. The incidence of the common movement disorder 'mild parkinsonian signs' (MPS) is approximately 50% over the age of 85 years. It has long been known that the loss of dopaminergic neurons in the substantia nigra pars compacta is a neuropathological hallmark of Parkinson's disease (PD). Recently, two papers present clear evidence for a high burden of mitochondrial DNA deletions within substantia nigra neurons in aged individuals and individuals with PD, pointing towards a common pathway inevitably leading to neuronal dysfunction and death.     

Soman intoxication in hypothyroid rats: alterations in brain neuronal RNA,acetylcholinesterase and survival

Studies were conducted to investigate relationships among soman (pinacolyl methylphosphonofluoridate) induced seizure activity, central metabolic impairments and lethality in normal vs thyroid-deficient rats. Quantitative cytophotometric measurements of individual cerebrocortical (layer V) and striatal neuron RNA contents were made following dosages of 0.5, 0.9 and 1.5 LD₅₀ soman (LD₅₀ = 135 μg/kg, sc). Hypothyroidism was associated with a marked diminution of overt convulsive activity and reduced susceptibility to lethal actions of soman as indicated by enhanced 24- and 48-h survival rates at 0.9, 1.2 and 1.5 LD₅₀. Hypothyroidism per se produced RNA depletion in both cortical and striatal neurons. Soman treatment diminished cortical RNA to essentially the same extent in thyroid-deficient rats as in euthyroids, whereas there was no further reduction of striatal neuron RNA. It was found that amelioration of convulsive activity and lethal- ity in hypothyroid rats was accompanied by reduced cerebral acetylcholinesterase (AChE, EC 3.1.1.7) inactivation, and that plasma cholinesterase (EC 3.1.1.8) and aliesterase (EC 3.1.1.1) levels were significantly higher in hypothyroid than in euthyroid saline-control rats. The overall data indicate that soman- induced central metabolic impairments can occur independent of paroxysmal neural activity and lethal actions of the agent. Resistance to soman observed with thyroid deficiency may be due in large part to increased binding to plasma enzymes and diminished delivery of soman to AChE in vital cholinergic sites.     

The localization of GABA-Transaminase in the striato-nigral system

The localization of gamma-aminobutyric acid transaminase (GABA-T), the degrading enzyme for γ-aminobutyric acid, was examined in the striatum and substantia nigra using biochemical techniques. Selective destruction of the nigrostriatal dopaminergic system with 6-hydroxydopamine had no effect on the activity of GABA-T in either the striatum or the substantia nigra, although striatal tyrosine hydroxylase activity was reduced by half. Intrastriatal injection of kainic acid in adult rats resulted in a significant dose-dependent decrease in GABA-T activity in both the striatum and the substantia nigra. The decrease in both of these regions was significantly correlated with the decrease in the GABA synthetic enzyme glutamate decarboxylase (GAD). The intrastriatal injection of kainic acid in ten day old rats did not affect striatal GAD or GABA-T activities, although striatal choline acetyl-transferase activity was reduced by half.It is concluded that the GABA-T activity in the striatum is predominantly localized in neuronal elements, although not, apparently, in cholinergic neurons. Some GABA-T activity is also present in the terminals of the striatonigral neurons. However, the dopaminergic nigrostriatal neurons do not appear to contain GABA-T. It is suggested that high GABA-T activity may be characteristic of GABA neurons.     

Possible destruction of cholinoceptive neurons by overstimulation of cholinergic tracts

It is well established that intracerebral injections of kainic acid may cause not only neuronal cell destruction at the injection site, but also losses in some distant regions. The mechanisms are different. The distant, but not the local, destruction can be produced by folic as well as by kainic acid and prevented by pretreatment of the animal with diazepam. Overexcitation of excitatory projections is believed responsible for the distant damage and evidence is presented that in some instances the projections involved are cholinergic. Thus, for example, injections of kainic acid or folic acid into the substantia innominata of rats destroy neurons in areas such as the pyriform cortex and amygdala which receive cholinergic projections from the injected area. Some of the destroyed neurons are GABAergic. That the distant toxicity in these areas can be partially blocked by scopolamine and is accompanied by decreases in the number of muscarinic binding sites is consistent with a cholinergic mechanism. Distant damage also occurs in the thalamus but this appears to be mediated by a noncholinergic projection. Similar injections of folic acid or kainic acid into the rostral pontine tegmentum, another area with cholinergic cells, cause destruction of both dopaminergic and GABAergic neurons in the substantia nigra. The effect on the GABAergic but not that on the dopaminergic cells is blocked by scopolamine. The results are discussed in relation to possible mechanisms of epilepsy and of selective neuronal losses in diseases such as Parkinson's disease.     

Hyperthermia-Induced Seizures Modify the GABA<Subscript><Emphasis Type="Bold">A</Emphasis></Subscript> and Benzodiazepine Receptor Binding in Immature Rat Brain

Summary Effects of hyperthermia-induced seizures (HS) on GABA_A and benzodiazepine (BDZ) receptor binding in immature rat brain were evaluated using in vitro autoradiography. HS were induced in 10-day-old rats by a regulated stream of moderately heated air directed 50 cm above the animals. Rats were killed 30 min, 24 h, or 20 days after HS and their brains were used for in vitro autoradiography experiments to determine GABA_A and BDZ receptor binding. GABA_A binding was significantly enhanced in all brain areas evaluated 30 min after HS, an effect that endures 24 h and 20 days after seizures. Concerning BDZ receptor binding, a significant increase was detected in entorhinal and perirhinal cortices and decreased in basolateral amygdala 30 min following HS. One day after HS, animals demonstrated enhanced BDZ binding in the cingulate, frontal, posterior parietal, entorhinal, temporal, and perirhinal cortices; striatum, accumbens, substantia nigra pars compacta, and amygdala nuclei. Twenty days after HS enhanced BDZ binding was restricted in the cingulated, frontal, anterior and posterior parietal cortices, as well as in substantia nigra pars reticulata, whereas decreased values were found in accumbens nucleus and substantia nigra pars compacta. Our data indicate differential effects of HS in GABA_A and BDZ binding in immature brain. HS-induced GABA_A and BDZ changes are different from those previously described in experimental models of temporal lobe epilepsy in adult animals.     

The neurotoxicity of DOPAL: behavioral and stereological evidence for its role in Parkinson disease pathogenesis

Background

The etiology of Parkinson disease (PD) has yet to be fully elucidated. We examined the consequences of injections of 3,4-dihydroxyphenylacetaldehyde (DOPAL), a toxic metabolite of dopamine, into the substantia nigra of rats on motor behavior and neuronal survival.

Methods/Principal Findings

A total of 800 nl/rat of DOPAL (1 µg/200 nl) was injected stereotaxically into the substantia nigra over three sites while control animals received similar injections of phosphate buffered saline. Rotational behavior of these rats was analyzed, optical density of striatal tyrosine hydroxylase was calculated, and unbiased stereological counts of the substantia nigra were made. The rats showed significant rotational asymmetry ipsilateral to the lesion, supporting disruption of dopaminergic nigrostriatal projections. Such disruption was verified since the density of striatal tyrosine hydroxylase decreased significantly (p<0.001) on the side ipsilateral to the DOPAL injections when compared to the non-injected side. Stereological counts of neurons stained for Nissl in pars compacta of the substantia nigra significantly decreased (p<0.001) from control values, while counts of those in pars reticulata were unchanged after DOPAL injections. Counts of neurons immunostained for tyrosine hydroxylase also showed a significant (p = 0.032) loss of dopaminergic neurons. In spite of significant loss of dopaminergic neurons, DOPAL injections did not induce significant glial reaction in the substantia nigra.

Conclusions

The present study provides the first in vivo quantification of substantia nigra pars compacta neuronal loss after injection of the endogenous toxin DOPAL. The results demonstrate that injections of DOPAL selectively kills SN DA neurons, suggests loss of striatal DA terminals, spares non-dopaminergic neurons of the pars reticulata, and triggers a behavioral phenotype (rotational asymmetry) consistent with other PD animal models. This study supports the “catecholaldehyde hypothesis” as an important link for the etiology of sporadic PD.     

Hyperthermia-Induced Seizures Modify the GABA<Subscript>A</Subscript> and Benzodiazepine Receptor Binding in Immature Rat Brain

Effects of hyperthermia-induced seizures (HS) on GABA_A and benzodiazepine (BDZ) receptor binding in immature rat brain were evaluated using in vitro autoradiography. HS were induced in 10-days-old rats by a regulated stream of moderately heated air directed 50 cm above the animals. Rats were killed 30 min, 24 h or 20 days after HS and their brains were used for in vitro autoradiography experiments to determine GABA_A and BDZ receptor binding. GABA_A binding was significantly enhanced in all brain areas evaluated 30 min after HS, an effect that endures 24 h and 20 days after seizures. Concerning BDZ receptor binding, a significant increase was detected in entorhinal and perirhinal cortices and decreased in basolateral amygdala 30 min following HS. One day after HS, animals demonstrated enhanced BDZ binding in the cingulate, frontal, posterior parietal, entorhinal, temporal and perirhinal cortices; striatum, accumbens, substantia nigra pars compacta and amygdala nuclei. Twenty days after HS enhanced BDZ binding was restricted in the cingulated, frontal, anterior and posterior parietal cortices, as well as in substantia nigra pars reticulata, whereas decreased values were found in accumbens nucleus and substantia nigra pars compacta. Our data indicate differential effects of HS in GABA_A and BDZ binding in immature brain. HS-induced GABA_A and BDZ changes are different from those previously described in experimental models of temporal lobe epilepsy in adult animals.     

Age, strain, and semi-chronic hydergine treatment effects on motor activity and neuronal nucleic acid-protein metabolism in male mice

This study was designed to examine the effects of Hydergine (DHET), co-dergocrine mesylate, treatment on motor activity and neuronal nucleoprotein metabolism in several motor areas of the aging rodent brain, specifically the caudate-putamen (CP), the substantia nigra (SN), and the cerebral cortex layer V (CX). Three age groups of two different strains of mice were used which represented two different aging rates: DBA/2 male mice (short lived) and C57BL/6 male mice (long lived). A representative sample of each age group was injected (IP) daily with a single dose of either DHET (1 mg/kg) or vehicle (0.9% saline) solution for one month. Total spontaneous motor activity was measured using a File apparatus to assess the functional ability of the selected brain areas. Histochemical parameters measured included the relative RNA and protein contents from a homogeneous population of neurons within each nuclei. The RNA and protein contents were assessed with a scanning microdensitometer using azure B and Coomassie staining protocols, respectively. The results of this study provide evidence that DHET does have significant effects on neuronal functioning in the motor compartments studied at the behavioral as well as the histochemical level for DBA/2 male mice. The C57BL/6 strain showed parallel, but less significant, changes in the histochemical parameters and no statistical differences in motor activity. In addition, DHET treatment produced no sign of neurotoxicity within any of the brain nuclei in either strain.     

Supersensitivity of substantia nigra pars reticulata neurons to GABAergic drugs after striatal lesions

Rats were given unilateral, intrastriatal injections of kainic acid in order to destroy striatal and pallidal GABAergic projections to the substantia nigra. Two to 3 weeks after the lesions were made, a population of neurons in the substantia nigra pars reticulata was found to be significantly more sensitive to the inhibitory actions of iontophoretically appled GABA, although their responsiveness to iontophoresed glycine was not significantly altered. The increased sensitivity was reflected by a 48% decrease in the IT₅₀ value for GABA. In addition, pars reticulata cells became more sensitive to the inhibitory actions of i.v. muscimol, a GABA agonist. While the change in sensitivity was not statistically significant at 2–3 weeks, cells were markedly more sensitive to i.v. muscimol by 5–6 weeks after the lesions were made. This increased sensitivity was indicated by a 2.5 fold shift to the left in the cumulative dose-response curve and a significant decrease in the ED₅₀ value for muscimol. These results (1) demonstrate that a population of substantia nigra pars reticulata neurons becomes “functionally” supersensitive to GABAergic agents after destruction of the striatonigral GABA pathway, and (2) support the idea that these cells lie postsynaptic to striatonigral GABAergic fibers. The implications of these findings with respect to the etiology and treatment of tardive dyskinesia are discussed.     

Regulation of c-Ret,GFRalpha1, and GFRalpha2 in the substantia nigra pars compacta in a rat model of Parkinson's disease

Glial cell line-derived neurotrophic factor (GDNF) family members have been proposed as candidates for the treatment of Parkinson's disease because they protect nigral dopaminergic neurons against various types of insult. However, the efficiency of these factors depends on the availability of their receptors after damage. We evaluated the changes in the expression of c-Ret, GFRalpha1, and GFRalpha2 in the substantia nigra pars compacta in a rat model of Parkinson's disease by in situ hybridization. Intrastriatal injection of 6-hydroxydopamine (6-OHDA) transiently increased c-Ret and GFRalpha1 mRNA levels in the substantia nigra pars compacta at 1 day postlesion. At later time points, 3 and 6 days, the expression of c-Ret and GFRalpha1 was downregulated. GFRalpha2 expression was differentially regulated, as it decreased only 6 days after 6-OHDA injection. Triple-labeling studies, using in situ hybridization for the GDNF family receptors and immunohistochemistry for neuronal or glial cell markers, showed that changes in the expression of c-Ret, GFRalpha1, and GFRalpha2 in the substantia nigra pars compacta were localized to neurons. In conclusion, our results show that nigral neurons differentially regulate the expression of GDNF family receptors as a transient and compensatory response to 6-OHDA lesion.     

The 12th J. A. F. Stevenson memorial lecture. Aging, Alzheimer's disease, and the cholinergic system

Aging does not affect tissues in a uniform fashion. Within the brain, substantial neuronal dropout occurs with age in the cholinergic medial basal forebrain complex, the noradrenergic locus coeruleus, and the dopaminergic substantia nigra pars compacta. These areas are also struck by diseases that are sharply age dependent. Alzheimer's disease causes neuronal destruction in the cholinergic cells of the medial basal forebrain and noradrenergic cells of the locus coeruleus. Parkinson's disease causes neuronal destruction mainly in the substantia nigra but with some destruction in the locus coeruleus. Parkinsonism-dementia affects all three areas. Alzheimer's disease is responsible for 50-60% of all cases of dementia. Severe dementia rises in frequency from less than 1% of the population at age 65-70 to over 15% by age 85. The cause of the disease is unknown. No method of prevention is known and present treatments are ineffective, although modest improvement has been reported for various therapeutic regimens designed to stimulate the cholinergic system. The neuronal systems identified as being affected in Alzheimer's disease and in the dementia of Parkinsonism correspond with those shown many years ago to be associated with the reticular activating system. This correspondence permits a new hypothesis of cognition and memory to be put forward, as well as a reinterpretation of data from animal research on the reticular activating system performed over a quarter of a century ago. The locus coeruleus is proposed as the noradrenergic element sensitizing the cortex to conscious recognition of real time events. The medial basal forebrain complex is proposed as the system registering the conscious event for storage and as the readout device when it is subsequently redisplayed in the cortex as memory. Storage could either be in the temporal lobe, in several areas of cortex with feedback to the medial basal forebrain, or in the cholinergic cells themselves.