Synaptic connections between pars compacta and pars reticulata neurones: electrophysiological evidence for functional modules within the substantia nigra

Intracellular recordings were performed in vitro from both pars compacta and pars reticulata neurones of the substantia nigra, and their postsynaptic responses to electrical stimulation within the nucleus were analysed. Intracellular staining by biocytin was used to reveal the morphology and location of the recorded neurone and its position and distance to the stimulating electrode. Inhibitory postsynaptic potentials in pars compacta neurones were evoked exclusively from a specific region of stimulation within the pars reticulata; this field could be mapped out as a cone-shaped region surrounding the apical dendrite of the cell and perpendicular to the plane of the pars compacta. Furthermore, hemitransection, prior to the experiments which eliminated the most likely source of extrinsic inhibition (the GABAergic striatonigral pathway) affected neither the generation of inhibitory postsynaptic potentials nor its topographic pattern during pars reticulata stimulation. In contrast to the response of dopaminergic pars compacta neurones, pars reticulata neurones responded to stimulation over wide areas of substantia nigra, without any clear site-specific selectivity. It is concluded that within the substantia nigra, dopaminergic neurones are arranged in functional modules such that most inhibition is derived from a highly local circuit with the collaterals of adjacent pars reticulata cells. This module is intrinsic to the substantia nigra and may represent the basic functional unit of the nucleus.     

Unbiased morphometrical measurements show loss of pigmented nigral neurones with ageing

This study used the dissector method to evaluate pigmented nigral neuronal loss in the substantia nigra pars compacta with age. Dissector counts can be used to estimate the absolute and accurate total neurone numbers. In addition, the area and diameter of the neuronal cell body was estimated by using a computerized morphometric analysis in a single section of the substantia nigra pars compacta. Brain samples from 26 people with an age range from 17 to 90 years were studied. A significant decrease in the total number of pigmented neurones (r=-0.83, P<0.001) and their density (r=-0.83, P<0.001) with age was found in the substantia nigra pars compacta. The number of pigmented neurones counted from a single section also showed an age-dependent decline (r=-0.76, P<0.001). According to the regression equations, the total number of pigmented neurones estimated by dissector counts decreased by 9.8% per decade and the neuronal density decreased by 7.4% per decade. The area of the neuronal cell body decreased by 3.2% per decade. This latter change corresponds to an approximate 4.4% decrease per decade in neuronal volume. These findings show that both the number of pigmented neurones and their size in the substantia nigra pars compacta decreases with age. However, the reductions in the total number of pigmented neurones are more dramatic than the reduction in neurone size with ageing.     

Electrophysiological characterization of substantia nigra dopaminergic neurons in partially lesioned rats: effects of subthalamotomy and levodopa treatment

Progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta is the main histopathological characteristic of Parkinson's disease. We studied the electrophysiological characteristics of the spontaneous activity of substantia nigra pars compacta dopaminergic neurons in rats with a partial, unilateral, 6-hydroxydopamine lesion of the nigrostriatal pathway. In addition, the effects of subthalamotomy and prolonged levodopa treatment on the activity of dopaminergic neurons were investigated. As a result of the lesion ( approximately 50% neuronal loss), the number of spontaneously active neurons was significantly reduced. Basal firing rate, burst firing and responsiveness to intravenously administered apomorphine remained unchanged. In contrast, the variation coefficient, a measure of interspike interval regularity, was significantly increased. Ibotenic acid (10 microg) lesion of the ipsilateral subthalamic nucleus in lesioned rats did not modify the electrophysiological parameters. However, prolonged levodopa treatment (100 mg/kg/day + benserazide 25 mg/kg/day, 14 days) reversed the irregularity observed in cells from lesioned rats, while it induced an irregular firing pattern in cells from intact rats. Our results using an experimental model of moderate Parkinson's disease indicate that surviving substantia nigra pars compacta dopaminergic neurons fire irregularly. In this model, subthalamotomy does not modify the firing pattern while levodopa treatment efficiently restores normal firing of SNpc neurons and does not appear to be toxic to them.     

Nitric oxide synthase inhibition and MPTP-induced toxicity in the common marmoset

Nitric oxide, produced following activation of N-methyl-D-aspartate (NMDA) receptors, may be involved in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity since NMDA receptor antagonists have been shown to prevent MPTP induced nigral cell loss in primates. Common marmosets were treated with either saline or MPTP or L-N^Gnitro arginine methyl ester (L-NAME) or MPTP and L-NAME. MPTP-treated common marmosets showed motor deficits including bradykinesia, rigidity, and tremor accompanied by a marked loss of tyrosine hydroxylase-immunoreactive neurones in the substantia nigra pars compacta and of [³H]-mazindol binding in the caudate-putamen. MPTP treatment also caused an increase in glial fibrillary acidic protein (GFAP) staining in the substantia nigra compared to controls. However, MPTP treatment did not alter the number of constitutive nitric oxide synthase-immunoreactive neurones in the caudate-putamen. Furthermore, neurones or glial cells immunoreactive for inducible nitric oxide synthase were not observed in the substantia nigra pars compacta following MPTP treatment. L-NAME treatment alone did not produce any behavioural changes in marmosets and did not alter the number of tyrosine hydroxylase-immunoreactive cells in the substantia nigra pars compacta, the number of constitutive nitric oxide synthase-immunoreactive neurones or [³H]-mazindol binding in the caudate-putamen compared to saline-treated control animals. Furthermore, L-NAME did not affect the motor deficits, loss of tyrosine hydroxylase-immunoreactive neurones in the substantia nigra pars compacta, loss of [³H]-mazindol binding in the caudate-putamen, or the increase in GFAP staining in the substantia nigra induced by MPTP treatment of common marmosets. The failure of L-NAME to protect against MPTP-induced toxicity in the marmoset suggests that nitric oxide does not play a major role in such toxicity and casts doubt over the involvement of the NMDA:nitric oxide system in neurodegeneration in MPTP-treated primates. Synapse 26:301–316, 1997. © 1997 Wiley-Liss, Inc.     

Neurotensin depolarizes substantia nigra dopamine neurones

Neurotensin and the C-terminal 8–13 amino acid fragment were found to excite substantia nigra compacta dopamine neurones in vitro in a dose-dependent manner. The N-terminal 1–8 amino acid fragment was not active at the concentration used. Desensitization was not observed. Intracellular recordings from the neurones responding to dopamine showed neurotensin-depolarized cells with an increase or no change in input resistance. These actions persisted in zero calcium, 8 mM magnesium artificial CSF suggesting a post-synaptic action.     

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.     

Dopaminergic and non-dopaminergic neurons in substantia nigra: Differential response to bromocriptine

Summary Bromocriptine reduces the spontaneous firing rate of neurons in the pars compacta of the substantia nigra but does not change the electrical activity of the neurons located in the pars reticulata. On the other hand, bromocriptine induces contralateral circling behaviour in rats with unilateral 6-hydroxydopamine nigral lesion. This increased motor activity follows an initial period of hypomotility. The decrease of the neuronal firing rate in the pars compacta of the substantia nigra coincides with the hypomotility observed in the lesioned rats.     

Evidence for the existence of a projection from the dorsal column nuclei to the substantia nigra in the cat.

Discrete injections of horseradish peroxidase in the substantia nigra of cats resulted in moderate to scant retrograde neuronal labeling of the contralateral dorsal column nuclei (nucleus gracilis and nucleus cuneatus). A moderate number of degenerating synaptic boutons emanating from the axons of stereotaxically lesioned dorsal column nuclei were identified by electron microscopy in the neuropil of the contralateral substantia nigra zona compacta and its dorsal division-nucleus parabrachialis pigmentosus. The present findings furnish evidence for a moderate, entirely crossed afferent projection of the dopaminergic neurons of the mesencephalic tegmentum, arising in the dorsal column nuclei.     

The functional role of the nucleus accumbens in the control of the substantia nigra: Electrophysiological investigations in intact and striatum-globus pallidus lesioned rats

The effects of electrical stimulation of the nucleus accumbens on the activity of identified substantia nigra neurons were studied in intact and lesioned rats. The latter had both the caudate-putamen complex and globus pallidus destroyed by electrolytic lesions. In intact rats a total of 42 of 107 neurons (39.2%) responded to stimulation of the nucleus accumbens. Of the 107 neurons 32 (29.8%) were inhibited and 10 (9.4%) were excited. Pure short inhibitions, long latency inhibitions and excitations followed by inhibition were found in both parts of the substantia nigra. Pure long lasting inhibitions were determined on pars compacta cells only. In lesioned animals, in which the coactivation of striatal and/or cortical fibers traversing the accumbens region was avoided, the percentage of responsive neurons decreased to 20% (23/115). The predominant responses recorded in this situation were pure inhibitions of pars compacta cells (14/46) and long latency inhibitions of pars reticulata neurons (7/69). No pure excitation or excitation-inhibition sequence was recorded. In the two sets of experiments 5 cells were activated antidromically from the nucleus accumbens. The results provide electrophysiological evidence for an inhibitory pathway from the nucleus accumbens to the substantia nigra. The low percentage of responsive neurons, the lack of excitatory responses, the paucity of reciprocal connections and the different inhibitory effects on the two populations of nigral neurons demonstrate that the functional role of the nucleus accumbens in controlling the substantia nigra differs from that exerted by the striatum.     

Biochemical changes accompanying unilateral 6-hydroxydopamine lesions in the rat substantia nigra

The biochemical consequences of a unilateral 6-hydroxydopamine injection into the substantia nigra of the rat brain were investigated. Projections of dopaminergic neurons from the A8-A9-A10 regions to a number of forebrain areas were confirmed. No innervation to the hypothalamus, including the median eminence, or to the brain stem, could be found with the present techniques. No destruction of serotonergic or GABAergic fibers could be demonstrated in the lesioned substantia nigra. Increases in glutamic acid decarboxylase activity were found restricted to the caudate and zona compacta of the substantia nigra ipsilateral to the lesion, indicating the possibility of a physiological interaction between GABAergic and dopaminergic systems.The neuroanatomical localization of the nigral dopamine-sensitive adenylate cyclase was also studied. No change in enzyme activity was found after destruction of a great proportion of the dopaminergic cells, suggesting that this enzyme has an extradopaminergic localization in the substantia nigra.     

The influence of striatal stimulation and putative neurotransmitters on identified neurones in the rat substantia nigra

The response of two populations of neurones in the substantia nigra (nigro-striatal compacta neurones and reticulata neurones) to microelectrophoretically administered putative neurotransmitters and stimulation of the ipsilateral striatum has been investigated in anaesthetized rats. There were marked differences between compacta and reticulata neurones in respect to their action potential configurations, spontaneous firing rates and their responses to striatal stimulation. However, both compacta and reticulata neurones were excited and/or inhibited by striatal stimulation, although inhibition was usually the predominant response in both neuronal populations. Compacta neurones were strongly inhibited by noradrenaline (NA) and dopamine (DA) but were unaffected by acetylcholine (ACh) and 5-hydroxytryptamine (5-HT). Reticulata neurones were excited by ACh and showed mixed responses to 5-HT, DA and NA. Excitant amino acids overdepolarized compacta neurones preventing them from firing rapidly, but induced large increases in reticulata neurone firing rate; effects that were readily antagonized by D-alpha-aminoadipate. Compacta neurones were less sensitive than reticulata neurones to GABA and glycine. The action of these inhibitory amino acids were selectively and reversibly antagonized by bicuculline methochloride and strychnine, respectively. The striatal-evoked inhibition of both compacta and reticulata neurones was reversibly reduced by bicuculline methochloride and irreversibly reduced by tetanus toxin, but was unaffected by strychnine. These results demonstrate that nigrostriatal-compacta neurones and reticulata neurones are physiologically and pharmacologically distinct neuronal populations and both receive inhibitory GABAergic and excitatory striatal inputs.     

Nuclear factor kappa-B p50 and p65 subunits expression in dementia with Lewy bodies

Dementia with Lewy bodies (DLB) is the second most common cause of neurodegenerative dementia after Alzheimer's disease (AD). Parkinsonism in DLB is mainly caused by neuronal loss with Lewy bodies (LBs) in the substantia nigra, thereby inducing degeneration of the nigrostriatal dopaminergic pathway similar to that in Parkinson's disease (PD). To clarify the pathogenesis of DLB, it is important to investigate the mechanisms involved in the degenerative process of LB-bearing neurones. Several reports suggest a role for nuclear factor kappa-B (NFkappaB) in the manifestation of neurodegenerative conditions such as AD and PD. The aim of the present study was to investigate whether NFkappaB subunits are involved in the pathogenesis of neurodegeneration in DLB by measuring tyrosine hydroxylase (TH), NFkappaB p65 and p50 protein expression in frontal cortex and substantia nigra pars compacta of DLB and control human brains. An increase, although not statistically significant, in nigral TH expression in DLB cases was observed. There were no differences in the cortical and nigral expression levels of NFkappaB p65 subunit between control and DLB cases. Western blots of the frontal cortex showed no differences in the expression levels of NFkappaB p50 subunit. However, NFkappaB p50 levels were significantly decreased (P < 0.05) in the pars compacta of the substantia nigra in the DLB cases in comparison with controls. The decrease in the expression of the p50 subunit in the substantia nigra of DLB cases achieved in the present study may increase the vulnerability of the dopaminergic neurones to a possible neurotoxic effect of p65 subunit. Thus, normal levels of NFkappaB p65 might be toxic in neurones with a low expression of the NFkappaB p50 subunit.     

Transient increase of brain derived neurotrophic factor mRNA expression in substantia nigra reticulata after partial lesion of the nigrostriatal dopaminergic pathway

By using non-isostopic in situ hybridization we have demonstrated a transient increase of BDNF mRNA in the lateral subregion of the substantia nigra pars reticulata 1 week after intrastriatal application of 6-OH-DA. These changes correlate with a partial reduction of dopamine (DA) content in the striatum but with a normal tyrosine hydroxylase immunoreactivity in substantia nigra pars compacta. Our data suggest that non-DA, BDNF expressing cells in substantia nigra pars reticulata may play a role in neuronal protection after partial lesions of the DA nigrostriatal pathway.     

Dopaminergic sprouting in the rat striatum after partial lesion of the substantia nigra

The capacity of the dopaminergic nerve system to reinnervate the denervated adult striatum was analyzed in a model of partial 6-hydroxydopamine-induced unilateral lesion of rat substantia nigra pars compacta. Sprouting of dopaminergic fibers entering the ventrolateral part of the striatum from a narrow zone of the external capsule was detected on the lesioned side 4 and 7 months, but not 10 days, after lesioning. Ultrastructural examination of the zone of sprouting revealed hypertrophic dopaminergic fibers and growth-cone-like structures, confirming the existence of an ongoing process of spontaneous regrowth of dopaminergic fibers. The identification of the factors involved in the regrowth of dopaminergic fibers may help to orientate molecular research into new treatments for Parkinson's disease.     

Monosynaptic input from the nucleus accumbens-ventral striatum region to retrogradely labelled nigrostriatal neurones

After placement of lesions (either electrolytic or by injection of kainic acid) in an area including the nucleus accumbens and part of the ventral striatum in the rat, the ipsilateral substantia nigra was studied in the electron microscope. Degenerating axons and nerve terminals were found mainly in the zona reticulata and in the ventral layer of the zona compacta. Degenerating synaptic boutons were found in contact with cell bodies (symmetric synapses) and dendrites (mainly symmetric, but a few asymmetric).The postsynaptic target of some of the afferent fibres from the accumbens-ventral striatum was established by demonstrating degenerating synaptic boutons of the above types in contact with nigrostriatal neurones which had been identified by the retrograde transport of horseradish peroxidase (HRP) from the main body of the striatum. Some of the HRP-labelled cells were also impregnated by the Golgi stain and degenerating boutons were found in contact with their distal dendrites. We also observed two types of HRP-containing boutons (presumably labelled anterogradely) in the substantia nigra after injection of HRP into the main body of the striatum: type 1 boutons contained large spherical vesicles, and formed symmetrical synapses mainly on dendritic shafts in the zona reticulata and in one case the dendrite was from a nigrostriatal neurone; type 2 boutons had pleomorphic and flattened vesicles and formed symmetrical synapses with perikarya and proximal dendrites, especially in the zona compacta. The latter type of HRP-labelled bouton was frequently found in synaptic contact with the cell bodies of nigrostriatal neurones and the same neurones sometimes also received degenerating boutons originating from neurones in the nucleus accumbens-ventral striatum.It is concluded that part of the striato-nigro-striatal circuit includes a monosynaptic link between neurones in the ventral striatum-accumbens and some nigrostriatal neurones. The possible convergence of input from different regions of the striatum on to single nigrostriatal neurones is also suggested.     

Sensitivity of transient outward rectification to ion channel blocking agents in guinea-pig substantia nigra pars compacta neurones in vitro.

The sensitivity of a transient outward rectifier in guinea-pig substantia nigra pars compacta (SNC) neurones in vitro was tested to a variety of ion channel blocking agents. This rectification was not blocked by TEA (10 mM), 4-aminopyridine (4-AP) (5 mM), apamin (0.2-1 microM) or Cd2+ ions (200 microM). Cs+ ions (5 mM) blocked the inward 'anomalous' rectifier but had no effect on the transient outward rectification. However, replacing Ca2+ ions in the bath perfusate by Ba2+ blocked the transient outward but not the inward rectifier. It is suggested that an atypical barium-sensitive conductance is responsible for the transient outward rectification of these neurones.     

Microtopography of methionine-enkephalin, dopamine and noradrenaline in the ventral mesencephalon of human control and Parkinsonian brains

The topographical distributions of Met-enkephalin, dopamine and noradrenaline were determined in serial frontal sections of human substantia nigra (pars compacta and pars reticulata) and ventral tegmental area. Met-enkephalin was identified by Biogel and thin layer chromatography and assayed by a specific radioimmunoassay. In the substantia nigra (pars compacta and pars reticulata), the levels of Met-enkephalin increased progressively from the rostal to the caudal part of the structure. This pattern closely resembled that of dopamine levels, particularly in the pars compacta. Noradrenaline levels in the substantia nigra and those of Met-enkephalin, dopamine, and noradrenaline in the ventral tegmental area, exhibited only limited fluctuations from the anterior to the posterior part of each structure.Highly significant decreases in Met-enkephalin, dopamine and noradrenaline levels were observed in the substantia nigra and ventral tegmental area of Parkinsonian brains. This observation, together with the close topographical association of dopamine and Met-enkephalin in the substantia nigra, further supports the likely existence of important functional relationships between dopaminergic and enkephalinergic neurons in the human brain.     

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.     

The influence of striatum on the substantia nigra: a study using the spreading depression technique

Effects of changes in striatal neurone activity on substantia nigra (SN) unit activity were studied in rats. Striatal changes were produced by the spreading depression (STSD) induced by perfusing the head of striatum with 0.06 M KCl via a push-pull cannula. In the medial part (caudate) striatal neurones are silent, with the arrival of STSD they exhibit a brief excitation coinciding with the onset of a slow wave of depolarisation in the extracellular steady potential. In the lateral part (putamen) striatal neurones are active, they undergo an arrest of their spontaneous activity for about 1 minute following the brief excitation. Antidromic activation of SN reticulata (SNr) neurones was attempted from the superior colliculus (SC). The majority of the 54 SNr neurones (96%) exhibited changes in firing rate during STSD. Ten neurones underwent brief single or repetitive changes of the same duration as the brief striatal excitation. Eight presented an initial brief change of activity followed by a longer duration inverse change in firing rate. Eighteen exhibited multiple brief changes followed by or superimposed on a long lasting decrease of firing rate and 50% of them were nigrotectal neurones. Sixteen neurones presented long duration biphasic changes in firing rate. Two SNr neurones and the 12 substantia nigra compacta (SNc) neurones studied were unaffected. Some neurones received both inhibitory and facilitatory striatal controls. In total thirty-nine neurones were inhibited and 47 facilitated, the origin of these effects from caudate and putamen are discussed. The excitatory action may be due to either a direct excitatory pathway or to a relayed effect of the inhibitory GABAergic pathway.     

Amygdalonigral pathway: an anterograde study in the rat with Phaseolus vulgaris leucoagglutinin (PHA-L)

The projection from the central nucleus of the amygdala to the substantia nigra was labeled by injections of the anterograde tracer Phaseolus vulgaris leucoagglutinin into different subregions of the nucleus. A sparse projection of labeled bouton-like swellings was observed in the rostral, medial substantia nigra pars compacta and ventral tegmental area from all subregions of the central nucleus of the amygdala that were injected. A dense projection of labeled axons and bouton-like swellings was observed in the lateral part of the substantia nigra pars compacta and pars lateralis when the injection site included the dorsal and rostral central nucleus. Heavy labeling was also seen in the lateral retrorubral field in these cases. In no instances were labeled terminals observed in the substantia nigra pars reticulata. The same pattern of labeling in the lateral substantia nigra and retrorubral field was seen after injections rostral to the central nucleus or dorsal and medial to it in the sublenticular region. The results suggest that the amygdalonigral pathway contributes to the innervation of extensive areas of the substantia nigra pars compacta. The major component of the pathway, however, projects only to a subregion of the substantia nigra. The origin of this pathway is confined to a discrete region of the dorsal central nucleus of the amygdala but extends rostrally into an area that is part of the "extended amygdala."