Possible intermixing of neurons from the subthalamic nucleus and substantia nigra pars compacta in the guinea-pig

A population of cells in the anterior substantia nigra pars compacta (SNPc) of the guinea-pig have been reported previously that differ from classical dopaminergic neurons in terms of their active and passive membrane properties. To investigate this population further, anterior nigral neurons (n=17) were compared with neurons in the adjacent subthalamic nucleus (STN; n=26). The anterior nigral neurons were found to be indistinguishable from STN neurons in their action potential characteristics, firing rate, resting membrane potential and input resistance. A low-threshold calcium conductance and anomalous rectification could be demonstated in cells from both groups. Furthermore, the gross morphological characteristics of anterior nigral neurons and STN neurons were very similar, as assessed following the intracellular injection of biocytin. A further similarity was seen in the response of the two cell groups to cyanide (200 M) and apomorphine (500 M). Cyanide hyperpolarised the membrane potential of all STN neurons and the majority (77.8%) of anterior nigral neurons, in both cases producing a concomitant reduction in firing rate. These changes were accompanied by an increase in membrane conductance for potassium ions. Apomorphine depolarised the membrane potential of all STN neurons and anterior nigral neurons, in most cases increasing the input resistance (83.3% of STN neurons and 100% of anterior nigral neurons). In both groups of cells, when firing rate was affected, an increase was usually seen. Given the physiological, morphological and pharmacological similarities of STN and anterior nigral neurons, the most parsimonious interpretation is that the anterior nigral neurons belong to the STN. However, the anterior nigral neurons were found in slices that, when resectioned, contained tyrosine hydroxylase (TH)-immunoreactive cell bodies in every section, in a location corresponding to the SNPc. The implication is that in the guinea pig the SNPc and STN (usually considered to be anatomically distinct nuclei) intermix at this level for several hundred microns. This close association of the STN and the compacta was further demonstrated by the presence of TH-positive varicose and non-varicose neuronal processes within the STN.     

Stimulants and lesions of the substantia nigra and red nucleus

The interaction of altered activity levels by stimulants and brainstem lesions was examined. Lesions of the substantia nigra and red nucleus significantly increased activity over control levels in albino rats. The stimulant action of d-amphetamine and methylphenidate was additive with lesion effects. In addition, stimulants disrupted the normal light-dark relationship with activity while the lesions did not. It is suggested that there are two functionally separate systems regulating locomotor activity.     

Increases in TH immunoreactivity, neuromelanin and degeneration in the substantia nigra of middle aged mice

The dopaminergic (DArgic) neurons in the substantia nigra (SN) are particularly vulnerable to oxidative stress and during aging. The present study was undertaken in order to determine whether aging is associated with changes in the DA synthesizing enzyme tyrosine hydroxylase (TH) as early as middle age by comparing 7- and 50-week-old mice. Quantitative analysis, performed by measuring the density of TH-immunopositive neurons, revealed that in the older animals, the number of DArgic neurons was decreased by 10% while TH immunodensity was 24 +/- 3% higher compared to the younger animals. Based on Masson-Fontana staining for neuromelanin (NM), the number of NM-containing neurons in the SN and the volume of NM per NM-positive neurons in the older animals were 5- and 11.6 +/- 0.1-fold higher, respectively. The silver stain-positive fibers, indicative of degeneration, were higher in the SN and striatum of the older animals, with the optical density 3.3 +/- 0.1- and 5.4 +/- 0.2-fold of the younger animals. The present study demonstrates that aging is associated with changes in the DA synthesizing enzyme TH as early as middle age and that this is associated with dramatic increases in the number of NM-containing neurons, volume of NM per cell, and degeneration.     

Age-dependent changes in dopaminergic projections from the substantia nigra pars compacta to the neostriatum

Age-dependent changes in dopaminergic (DA) innervation of the neostriatum (Str) were studied in male F344/N rats. Projections from the substantia nigra pars compacta (SNc) to the neostriatum were quantified using electrophysiological methods at age points from 6 to 24 months. The percentage of DA neurons activated antidromically by electrical stimulation (P-index) of Str increased between 18 and 24 months. Additionally, the percentage of DA neurons showing multiple antidromic latencies from striatal stimulation (M-index), which suggests axonal branching of individual DA neurons, increased significantly between 6 and 12 months and 6 and 24 months. These results suggest that DA neurons exhibit increased axonal branching in the aged brain.     

Effects of aging on the ventral and dorsal substantia nigra using diffusion tensor imaging

Dopaminergic neurons in the substantia nigra produce dopamine for the nigrostriatal pathway that facilitates motor function. Postmortem examinations demonstrate an age-related loss of cells in the substantia nigra, with most of the cell loss focused on the dorsal substantia nigra compared with the ventral substantia nigra. The current study used diffusion tensor imaging (DTI) to provide the first in vivo assessment of age-related degeneration in specific segments of the substantia nigra of humans. Measures extracted from DTI of 16 young adults (19-27 years) and 15 older adults (55-71 years) showed that in the dorsal substantia nigra, fractional anisotropy was reduced and radial diffusivity was increased with age. In the ventral substantia nigra and red nucleus, there were no differences across age for the DTI measures. DTI provides a noninvasive technique that accurately reflects the established pattern of age-related cell loss in the dorsal and ventral substantia nigra, further suggesting the robust potential for using DTI to characterize degeneration in the nigrostriatal pathway in both health and disease.     

Observations on the fine structure of the substantia nigra in the cat

Summary A light and electron microscopical investigation has been undertaken of the substantia nigra in the normal cat. The pars reticulata partly contains the arborization of dendrites whose cell bodies are located in the so-called pars compacta. There is a considerable overlap of the dendritic fields in the rostrocaudal direction, while the dendritic fields are very restricted in the mediolateral extension of the substantia nigra. The secondary and all subsequent branches of the dendrites of nigral cells are for considerable distances completely covered by boutons. Only few boutons contact the cell bodies. Three types of boutons are distinguished in the substantia nigra in the cat. Type I, about 90 % of the total, is of the terminal type, contains pleomorphic vesicles and establishes symmetrical synapses with nigral cell soma, dendritic trunks and spines. The type II bouton (about 10 % of the total number) is most commonly of the terminal type, contains spherical vesicles and establishes asymmetrical synapses with cell bodies and dendritic trunks of nigral cells. The type III bouton (about 2 % of the boutons) is always of the en passage type, contains pleomorphic vesicles and establishes symmetrical contacts with dendrites. All boutons in the cat's substantia nigra contain several large (700–1200 Å) dense core vesicles. Occasional axo-axonic contacts between type I and type III boutons are observed. Type I bouton is invariably presynaptic to the other.The findings are discussed in relation to some relevant problems.On leave of absence from the Anatomical Institute of the Medical Faculty, Charles' University in Prague, with an IBRO grant nr. E. 29.99-1.We gratefully acknowledge the valuable technical assistance of Mrs. J.L. Vaaland and the skilful help by Mrs. B.E. Branil in the preparation of the microphotographs.     

Activity of neurons in the cat substantia nigra pars reticulata during drinking

Summary Extracellular activity of single neurons in the pars reticulata of the Substantia Nigra (SNpr) was recorded in cats during drinking. Two groups of cells were distinguished: I. Somatosensory cells which responded by a short decrease in firing rate to the arrival of water against the upper lip. We suggest that these stimulus-related responses reflect a complex process linked to preparation of buccolingual movements. II. Action-related cells which were de-activated at the beginning or during the entire drinking period but without modulation in relation to the individual movements of jaws and tongue. We suggest that de-activation of these cells during drinking operates as a gating mechanism which allows implementation of complex motor sequences by cortical and/or subcortical structures.     

Influence of aging on the calbindin-D-28k immunoreactive positive dopaminergic neurons in the substantia nigra pars compacta of rats

We studied the relationship between aging and the vulnerability of substantia nigra pars compacta (SNc) calbindin-D-28k immunoreactive positive (CB⁺) dopaminergic (DA) neurons. Immunohistochemistry and cell counting were used to determine the number of CB⁺ DA neuron in aged rats (24 mon) compared to adult rats (5 mon). Furthermore, the expression of CB mRNA and protein levels in SN was studied by semi-quantitative RT-PCR and Western blotting. An 11% loss of CB⁺ DA neurons was detected in both the rostral (8.9%) and caudal (1.7%) segments but not in the intermedial segment of SNc in aged rats compared to adult rats (P < 0.05). No difference was detected in CB mRNA and protein levels between aged and adult rats (P > 0.05). These data suggest that expression levels of CB mRNA and protein may increase in the existing SNc DA neurons, which may compensate for the partial age dependent loss of CB⁺ DA neurons in the SNc.     

MPTP对小鼠黑质与纹状体KIF基因表达的影响

目的: 揭示1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)对C57BL小鼠黑质与纹状体神经元驱动蛋白超家族(KIF)基因表达的影响。方法: 腹腔注射MPTP建立小鼠帕金森病动物模型,通过RT-PCR方法检测KIF1A、KIF2、KIF3A、KIF4与KIF5A基因的表达。结果: 在黑质中,MPTP造成KIF基因表达的普遍下降,只有KIF2基因表达无明显变化。在纹状体中则有所不同,KIF1A、KIF3A与KIF4基因表达上升,而KIF2与KIF5A表达的变化与在黑质中相似。结论: MPTP造成的神经黑质多巴胺能神经元丧失很可能与KIF基因表达的降低有关。     

Activity of substantia nigra neurons in the cat brain during a self-initiated behavioral act

Cats were trained to perform a self-initiated behavioral act in the form of an operant food-obtaining reflex with defined time requirements. Activity was recorded from 50 dopaminergic neurons (identified in terms of their low frequency of background activity and long action potentials) and 67 nondopaminergic neurons of the substantia nigra and adjacent region. Dopaminergic neurons were the more responsive. Prior to EMG activation, the activity of 33 (66%) of these cells changed, and 44 (88%) showed changes in activity on movement. Dopaminergic neurosn showed increased activity during the period of waiting for the conditioned stimulus, predicting the release of reinforcement or its absence. These cells were more frequently activated in response to a positive signal and reinforcement and were more frequently inhibited in the absence of reinforcement. The high reactivity of dopaminergic neurons during execution of a movement task could be explained by the involvement of a cognitive component, i.e., determining the point at which the movement should start. Department of Human and Animal Physiology and Biophysics, Simferopol' State University, 4 Yaltinskaya, 333036 Simferopol', Ukraine. Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova Vol. 83, No. 1–2, pp. 28–34, January–February, 1997. Original article     

A non-cholinergic function for acetylcholinesterase in the substantia nigra: Behavioural evidence

Summary Acetylcholinesterase is released from substantia nigra neurons, independently of cholinergic transmission. In an attempt to discover the functional significance of this phenomenon, the behavioural effects of injecting acetylcholinesterase into one substantia nigra of the rat were investigated. Following a single injection of the enzyme, intraperitoneal amphetamine evoked circling behaviour in a direction away from the side of injection. Purified acetylcholinesterase with a similar electrophoretic mobility to the endogenous secreted form, was far more potent in eliciting circling than much higher activities of commercial enzyme, consisting of several molecular species of acetylcholinesterase. Similar infusions of butyrylcholinesterase did not induce circling. Depending upon the amount of enzyme initially given, the behavioural effects of a single injection of acetylcholinesterase persisted for up to thirty days. During this period apomorphine, administered systemically, induced transient circling towards the acetylcholinesterase-treated side. It is concluded that secreted acetylcholinesterase has a functional significance within the substantia nigra, independent of cholinergic transmission. This released enzyme could exert long-term changes in the activity of the nigrostriatal system, involving modification of dopamine striatal receptors.This work was supported by the M.R.C., Project Grant G810/ 79/93 N.     

Studies on the role of the NMDA receptor in the substantia nigra pars reticulata and entopeduncular nucleus in the development of the high pressure neurological syndrome in rats

Summary The effect of the focal injection of N-methyl-D-aspartate (NMDA) and 2-amino-7-phosphonoheptanoate (APH) into the substantia nigra pars reticulata (SNR) and entopeduncular nucleus (EP) on behavioural signs of the high pressure neurological syndrome (HPNS) in rats was studied. Doses of 1, 5 and 10 nmoles of NMDA or APH were injected into the SNR or EP, 10–30 min prior to the exposure of animals to a high pressure. Injection of NMDA into either SNR or EP results in a lowering of the threshold pressure for tremor by about 30%. Injection of NMDA into the SNR has no significant effect on clonic seizures whereas its injection into the EP results in a decrease of threshold pressure for clonic seizures. NMDA also facilitates the occurrence of forelimb clonus when injected into the EP. Injection of the NMDA antagonist, APH, into the SNR or EP significantly increases the threshold pressure of tremor (32.8 and 48.2% respectively). Seizure threshold is also increased by the injection of APH into either area, but nigral injections (especially the higher doses) are more protective against seizures than the EP injections. Comparing the two sites blockade of NMDA receptors within the EP is more protective against tremor, whereas in the SNR NMDA blockade is more protective against seizures.     

NeuN is not a reliable marker of dopamine neurons in rat substantia nigra

Quantification of neuronal cell number is a key endpoint in the characterization of neurodegenerative disease models and neuroprotective regimens. Immunohistochemistry for phenotypic markers, followed by unbiased stereology is often used to quantify the relevant neuronal population. To control for loss of phenotypic markers in the absence of cell death, or to determine if other types of neurons are lost, a general neuronal marker is often desired. Vertebrate neuron-specific nuclear protein (NeuN) is reportedly expressed in most mammalian neurons. In Parkinson's disease models, NeuN has been widely used to determine if there is actual nigral dopamine neuron loss or simply loss of tyrosine hydroxylase expression, a prominent phenotypic marker. To date, the qualitative value of NeuN expression as such a marker in the substantia nigra has not been assessed. Midbrain tissue sections from control rats were stained for NeuN and tyrosine hydroxylase and assessed by light or confocal microscopy. Here we report that NeuN expression level in the rat substantia nigra was highly variable, with many faintly stained cells that would not be meet stereological scoring criteria. Additionally, dopamine neurons with little or no NeuN expression were readily identified. Subcellular compartmentalization of NeuN expression was also variable, with many cells dorsal and ventral to the nigra exhibiting expression in both the nucleus and cytoplasm. NeuN expression also appeared to be much higher in non-dopamine neurons within the ventral midbrain. This characterization of nigral NeuN expression suggests that it is not useful as a quantitative general neuronal marker in the substantia nigra.     

Collateral projections of neurons of the rat globus pallidus to the striatum and substantia nigra

Summary Double retrograde fluorescent tracing techniques were used to evaluate the possibility that ascending and descending projections from the globus pallidus arise from divergent axon collaterals. Appropriately placed injections of different tracers (True Blue, Nuclear Yellow) into the substantia nigra and the striatum resulted in the double labelling of neurons in the globus pallidus. Conversely, simultaneous injection of two different sites within the striatum did not produce significant double labelling of globus pallidus neurons. These results indicate that at least a portion of the neurons of the globus pallidus project to both the striatum and substantia nigra, and that individual pallidal neurons do not have widespread projections to the striatum.     

Monoaminergic synapses,including dendro-dendritic synapses in the rat substantia nigra

Summary Intraventricular administration of 1 or 2 mg of the osmiophilic false transmitter 5-hydroxydopamine (5-OHDA) was used to label monoamine storage and release sites in the rat substantia nigra. Vesicles containing unusually dense cores indicative of the presence of the marker were seen forming from the Golgi apparatus in the cell bodies of medium-sized neurons of the substantia nigra, pars compacta, and from smooth endoplasmic reticulum in the dendrites of those neurons and in small unmyelinated axons of unknown origin. In serial sections, both axons and dendrites containing synaptic vesicles marked with 5-OHDA were seen to form synapses en passage in pars compacta, and some presynaptic dendrites containing vesicles filled by the marker were also observed to form contacts with dendrites in pars reticulata. The only identified postsynaptic elements engaging in monoaminergic synapses in the substantia nigra were dendrites of medium-sized pars compacta neurons.     

Electrophysiological analysis of dopamine cells from the substantia nigra pars compacta of circling rats

Summary Extracellular single unit recordings were obtained from dopamine cells in the substantia nigra pars compacta during forced locomotion on a circular turntable treadmill. Stainless steel wire electrodes, 18 m diameter, insulated with Parylene C were used. During the entire recording session the rat was in the treadmill apparatus. The device was stopped while a cell was being sought. A cell was identified as dopaminergic by a frequency of 3 to 10 Hz and a biphasic or triphasic action potential of greater than 2 ms in duration. An attempt was made to record from cells under the following conditions: animal at rest, animal turning in one direction, at rest again, turning in the opposite direction and finally, at rest. If the cell was still firing after these recordings, haloperidol was injected i.p. to see that the presumed dopamine cell increased its firing rate. A cell was held for all the observations in 4 animals. In an additional 10 rats, recordings were made before, during and after movement in one direction. Three animals were recorded only before and during movement. In 6 of the total of 17 animals haloperidol was administered. Results showed that firing patterns of cells in awake animals were similar to those reported from dopamine cells of anesthetized rats. During either contralateral or ipsilateral turning the firing frequency and burst activity significantly increased. These results indicate that the activity of dopamine cells in substantia nigra is increased bilaterally during circling.     

Depletion of glial cell line-derived neurotrophic factor in substantia nigra neurons of Parkinson's disease brain

The distribution of nerve growth factor (NGF), ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), brain derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4) in substantia nigra pars compacta (SNc) of Parkinson's disease (PD) brains was investigated by immunofluorescence. Cases studied included four 69–77 year old neurologically normal male controls and four 72–79 year old male PD patients. Integrated optical densities (IODs) of immunofluorescence over individual neuromelanin-containing neurons and in areas of neuropil and the number of neurons on H & E stained adjacent sections were quantitated with the use of the BioQuant Image Analyzer. Data were statistically analyzed by ANOVA, including the unpaired two-tailed Student t-test and the Mann–Whitney test. The results showed 55.8% (P<0.0001) dropout of SNc neurons in PD brains compared to age-matched controls. Despite considerable neuronal dropout, immunofluorescent NTFs in the PD brains showed differential reductions that were consistent within the group as compared to age-matched controls: reductions were GDNF, 19.4%/neuron (P<0.0001), 20.2%/neuropil (P<0.0001); CNTF, 11.1%/neuron (P<0.0001), 9.4%/neuropil (P<0.0001); BDNF, 8.6%/neuron (P<0.0001), 2.5%/neuropil. NGF, NT-3 and NT-4 showed no significant differences within surviving neurons or neuropil. Since the depletion of GDNF both within surviving neurons and neuropil was twice as great as that of CNTF and BDNF and since the other NTFs showed no changes, GDNF, of the tested NTFs, is probably the most susceptible and the earliest to decrease in the surviving neurons of SNc. These observations suggest a role for decreased availability of GDNF in the process of SNc neurodegeneration in PD.     

Electrophysiological evidence for the dendritic localization of a calcium conductance in guinea-pig substantia nigra neurones in vitro

Summary Within the substantia nigra, anatomical, neurochemical and pharmacological findings strongly suggest that transmitter and protein are secreted from the dendrites of nigrostriatal neurones. This phenomenon may underlie a non classical modulatory cellular mechanism. Two conductances are generated in nigrostriatal neurones independent of somatic action potentials, that might mediate this modulation. However, these conductances have never been directly nor precisely located specifically within the dendrites. The aim of this study was to record the membrane properties of substantia nigra zona compacta neurones in response to selective sectioning of the population of long apical dendrites i.e. the removal of the zona reticulata. Intracellular recordings from substantia nigra zona compacta neurones were made from mesencephalic slices of the guinea-pig brain maintained in vitro. In cells without the apical dendrites, the membrane potential, input resistance and mean firing frequency was not significantly different from the control neurones. However, removal of the substantia nigra zona reticulata virtually abolished one conductance in particular. This conductance, seen in control neurones, is a long lasting slow depolarization which is resistant to tetrodotoxin blockade of sodium channels: rather, it is mediated by the entry of calcium ions and is optimally deinactivated at a hyperpolarised membrane potential. Hence, this study strongly suggests that this conductance is generated exclusively in the apical dendrites. It has been postulated that this long lasting calcium conductance is central to the modulation of nigrostriatal neuronal excitability. Thus, the apical dendrites could play a specific and active role in the functioning of nigrostriatal neurones.     

Opposite locomotor asymmetries elicited from the medial and lateral substantia nigra: role of the superior colliculus

The present study investigated the role of the superior colliculus (SC) in the expression of opposing locomotor asymmetries elicited from the medial and lateral substantia nigra pars compacta (SNC). In experiment one it was found that amphetamine stimulated ipsiversive circling produced by unilateral SC lesions was additive with the ipsiversive circling produced by alpha-flupenthixol microinjections into the lateral SNC but was not additive with the contraversive circling produced by such injections into the medial SNC. Experiment two showed that the amphetamine stimulated ipsiversive circling produced by unilateral SC lesions was additive with the contraversive circling produced by lateral SNC lesions but was not additive with the ipsiversive circling produced by medial SNC lesions. Both experiments were taken to suggest that the striato-nigral-colliculus system is an output path for medial SNC derived circling but is not an output path for the opposing circling behavior derived from the lateral SNC.     

Eye position and memory saccade related responses in substantia nigra pars reticulata

The substantia nigra pars reticulata (SNr), a major output nucleus of the basal ganglia, has been implicated anatomically, pharmacologically and physiologically in the generation of saccadic eye movements. However, the unique contribution of the SNr to saccade generation remains elusive. We studied the activity of SNr neurons while rhesus monkeys made saccades from different initial orbital positions, to determine what effects, if any, eye position had on SNr neuronal activity. We found that there was no effect of eye position on SNr neuronal responses. We also examined the responses of SNr neurons during memory-guided saccades to determine whether SNr discharges were affected by whether the target of the upcoming saccade was visible. We found that there was no change in response properties during memory saccade trials as compared to otherwise identical visually guided trials. SNr neurons appear to carry no information about either eye position or whether a movement is guided by a visible or remembered target. These results suggest that nigral signals are encoded in the same coordinate frame as those in the SC and FEF, but that unlike neuronal responses in these areas, SNr activity is not influenced by whether the saccade target remains visible until the movement is executed.