Immunohistochemical localization of N-methyl-D-aspartate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor subunits in the substantia nigra pars compacta of the rat

Ionotropic glutamate receptors in the substantia nigra pars compacta regulate the activity of dopamine neurons. We have used dual-label immunofluoresence and confocal laser microscopy to study the localization of subunits of two types of ionotropic receptors within the substantia nigra pars compacta of the rat. Immunostaining for N-methyl-D-aspartate receptor 1 and glutamate receptor 2/3 was prominent in the soma and proximal dendrites of all tyrosine hydroxylase-immunopositive cells, while only low amounts of N-methyl-D-aspartate receptor 2A and N-methyl-D-aspartate receptor 2B were present. Selective antibodies were used to determine the isoforms of N-methyl-D-aspartate receptor 1 present. Immunostaining for the N1, C1 and C2 variably spliced segments of N-methyl-D-aspartate receptor 1 were scarce in the substantia nigra pars compacta, while immunoreactivity for the alternative C2' terminus of N-methyl-D-aspartate receptor 1 was quite abundant. Staining for glutamate receptor 1 was heterogeneous; about half of the tyrosine hydroxylase immunopositive cells stained intensely, while the other half were immunonegative. The glutamate receptor 1-stained cells were concentrated in the ventral tier of the substantia nigra pars compacta. Glutamate receptor 4 was not found in tyrosine hydroxylase-immunopositive cells within the substantia nigra pars compacta. Together, these data demonstrate that dopaminergic neurons in the substantia nigra pars compacta express primarily glutamate receptor 1, glutamate receptor 2/3 and N-methyl-D-aspartate receptor 1 isoforms containing the alternative C2' terminus.     

Synaptic localization of ionotropic glutamate receptors in the rat substantia nigra

Glutamatergic neurotransmission in the substantia nigra pars compacta and pars reticulata is mediated through N-methyl-D-aspartate and alpha-amino-3-hydroxy-5-methyl-4-isoxaline propionic acid/kainate (AMPA) type receptors as well as other glutamate receptors and is critical for basal ganglia functioning. A major glutamatergic input to the substantia nigra originates in the subthalamic nucleus, and the long-lasting stimulation of the dopaminergic cells of the substantia nigra pars compacta by the subthalamic neurons has been implicated in the pathophysiology of Parkinson's disease. The objectives of the present study were to determine the subcellular and subsynaptic localization of subunits of the N-methyl-D-aspartate and AMPA receptors in the substantia nigra, and also to determine whether co-localization of N-methyl-D-aspartate and AMPA receptor subunits occur at individual synapses. To achieve this, pre-embedding and post-embedding immunocytochemistry was applied to sections of substantia nigra using antibodies that recognize the NR1 and NR2A/B subunits of the N-methyl-D-aspartate receptor, and GluR2/3 subunits of the AMPA receptor.In both regions of the substantia nigra, immunolabelling for each of the subunits was observed in numerous perikarya and proximal dendrites. At the subcellular level, silver-intensified immunogold particles localizing N-methyl-D-aspartate and AMPA receptor subunits were most commonly present within dendrites where they were associated with a variety of intracellular organelles and with the internal surface of the plasma membrane. Post-embedding immunogold labelling revealed immunoparticles labelling for NR1, NR2A/B and GluR2/3 to be enriched at asymmetric synaptic specializations, although a large proportion of asymmetric synapses were immunonegative. Double immunolabelling revealed, in addition to single-labelled synapses, the co-localization of subunits of the N-methyl-D-aspartate receptor and subunits of the AMPA receptor at individual asymmetric synapses. Similarly, double immunolabelling also revealed the co-localization of the NRl and NR2A/B subunits of the N-methyl-D-aspartate receptor at individual asymmetric synapses. Labelling for NR1 and GluR2/3 was, on average, relatively evenly distributed across the width of the synapse with a gradual reduction towards the periphery when analysed in single sections.In summary, the present results demonstrate that AMPA and N-methyl-D-aspartate receptors are selectively localized at a subpopulation of asymmetric synapses in the substantia nigra pars compacta and reticulata and that the two receptor types, at least partially co-localize at individual synapses. It is concluded that glutamatergic transmission in the substantia nigra pars compacta and pars reticulata occurs primarily at asymmetric synapses and, at least in part, is mediated by both N-methyl-D-aspartate and AMPA receptors.     

Reduction of anterograde degeneration in brain damaged rats by GM1-gangliosides

After receiving a nigrostriatal hemitransection in the left hemisphere and an electrolytic caudate nucleus (CN) lesion in the right hemisphere, rats were given intraperitoneal injections of saline or GM1-gangliosides. Significantly smaller areas of terminal degeneration were seen in the substantia nigra pars reticulata (SNr) ipsilateral to the caudate lesion of animals treated with GM1. No statistical differences were seen in the number of degenerating terminals in the CN and SNr on the side with the hemitransection. Exogenous GM1 may thus be effective in preventing anterograde degeneration following brain injury.     

Excitatory neurotransmission within substantia nigra pars reticulata regulates threshold for seizures produced by pilocarpine in rats: effects of intranigral 2-amino-7-phosphonoheptanoate and N-methyl-D-aspartate

Seizures produced by pilocarpine given i.p. to rats provide an animal model for studying the initiation, spread and generalisation of convulsive activity within the forebrain. Pilocarpine, 380 mg/kg, produces a sequence of behavioural and electroencephalographic alterations indicative of motor limbic seizures and status epilepticus, which is followed by widespread damage to the limbic forebrain resembling that occurring subsequent to prolonged intractable seizures. Microinjections of a selective antagonist at the N-methyl-D-aspartate receptor, (+/-)-2-amino-7-phosphonoheptanoate, into the substantia nigra pars reticulata, bilaterally, protects against the behavioural, electrographic and morphological features of seizures produced by pilocarpine, 380 mg/kg, with an ED50 of 0.0007 mumol (0.0004-0.0011). Microinjections of (+/-)-2-amino-7-phosphonoheptanoate, 0.005 or 0.01 mumol, into the substantia nigra pars compacta or into the dorsal part of mid-anterior striatum do not modify the electrographic and morphological sequelae of pilocarpine, 380 mg/kg. In rats pretreated with microinjections of N-methyl-D-aspartate into the substantia nigra pars reticulata, a non-convulsive dose of pilocarpine, 100 mg/kg, results in recurrent motor limbic seizures and status epilepticus. The ED50 of N-methyl-D-aspartate for the generation of seizures after pilocarpine, 100 mg/kg, is 0.0014 mumol (0.001-0.0019). Electrographic monitoring shows a pattern and sequence of evolution of convulsant activity within the hippocampus and cortex similar to that produced with pilocarpine, 380 mg/kg, alone. Morphological examination of brains from rats treated with N-methyl-D-aspartate in the substantia nigra pars reticulata and subsequently given pilocarpine, 100 mg/kg, which underwent status epilepticus, reveals widespread damage to the amygdala, thalamus, olfactory cortex, substantia nigra, neocortex, and hippocampus. Microinjections of N-methyl-D-aspartate, 0.002 mumol, into either the substantia nigra pars compacta or dorsal striatum, bilaterally, do not augment seizures produced by pilocarpine, 100 mg/kg. The results indicate that the threshold for pilocarpine-induced seizures in rats is modulated by excitatory amino acid neurotransmission within the substantia nigra pars reticulata.     

m-Chlorophenylpiperazine excites non-dopaminergic neurons in the rat substantia nigra and ventral tegmental area by activating serotonin-2C receptors

In vivo electrophysiological techniques were used to study the effect of m-chlorophenylpiperazine, a non-selective serotonin-2C receptor agonist, on the activity of non-dopaminergic neurons in the substantia nigra pars reticulata and the ventral tegmental area of anesthetized rats. Intravenous administration of m-chlorophenylpiperazine (5-320 microg/kg) caused a dose-dependent increase in the basal firing rate of a subpopulation of nigral neurons which do not respond to a footpinch stimulus [P(0) neurons], whereas it did not affect the activity of neurons which are responsive to the footpinch [P(+) neurons]. However, m-chlorophenylpiperazine (5-320 microg/kg) excited all non-dopaminergic neurons sampled in the ventral tegmental area. Moreover, microiontophoretic application of m-chlorophenylpiperazine (10-40 nA) caused an excitation of P(0) nigral and ventral tegmental area neurons. Pretreatment with the selective serotonin-2C receptor antagonist SB 242084 (200 microg/kg, i.v.) completely blocked the excitatory effect of i.v. m-chlorophenylpiperazine (5-320 microg/kg), both in the substantia nigra pars reticulata and in the ventral tegmental area. It is concluded that stimulation of serotonin-2C receptors by m-chlorophenylpiperazine activates non-dopaminergic (presumably GABA-containing) neurons in the substantia nigra pars reticulata and ventral tegmental area.     

Activation of metabotropic glutamate receptor 1 inhibits glutamatergic transmission in the substantia nigra pars reticulata

The substantia nigra pars reticulata is a primary output nucleus of the basal ganglia motor circuit and is controlled by a fine balance between excitatory and inhibitory inputs. The major excitatory input to GABAergic neurons in the substantia nigra arises from glutamatergic neurons in the subthalamic nucleus, whereas inhibitory inputs arise mainly from the striatum and the globus pallidus. Anatomical studies revealed that metabotropic glutamate receptors (mGluRs) are highly expressed throughout the basal ganglia. Interestingly, mRNA for group I mGluRs are abundant in neurons of the subthalamic nucleus and the substantia nigra pars reticulata. Thus, it is possible that group I mGluRs play a role in the modulation of glutamatergic synaptic transmission at excitatory subthalamonigral synapses. To test this hypothesis, we investigated the effects of group I mGluR activation on excitatory synaptic transmission in putative GABAergic neurons in the substantia nigra pars reticulata using the whole cell patch clamp recording approach in slices of rat midbrain. We report that activation of group I mGluRs by the selective agonist (R,S)-3,5-dihydroxyphenylglycine (100 microM) decreases synaptic transmission at excitatory synapses in the substantia nigra pars reticulata. This effect is selectively mediated by presynaptic activation of the group I mGluR subtype, mGluR1. Consistent with these data, electron microscopic immunocytochemical studies demonstrate the localization of mGluR1a at presynaptic sites in the rat substantia nigra pars reticulata.From this finding that group I mGluRs modulate the major excitatory inputs to GABAergic neurons in the substantia nigra pars reticulata we suggest that these receptors may play an important role in basal ganglia functions. Studying this effect, therefore, provides new insights into the modulatory role of glutamate in basal ganglia output nuclei in physiological and pathophysiological conditions.     

Nigral degeneration following striato-pallidal lesion in tissue type plasminogen activator deficient mice.

Tissue type plasminogen activator (tPA) has been suggested as a key factor in excitotoxic neuronal death in the hippocampus. Transneuronal degeneration of the substantia nigra pars reticulata (SNR) neurons after striato-pallidal lesions is attributable to excess excitatory glutamatergic inputs into the SNR following inhibitory GABAergic deafferentation and tPA may contribute to the mechanism of transneuronal degeneration of the SNR. To examine this possibility, we studied pathological changes in the SNR following striato-pallidal lesions produced by electrocoagulation in tPA-deficient mice. There was no difference in the degree of SNR degeneration, or in microglial activation and proliferation in the degenerating SNR of tPA-deficient and control mice. Our results indicate that tPA does not contribute to transneuronal degeneration in the SNR following striato-pallidal lesions in mice.     

Intrapallidal lipopolysaccharide injection increases iron and ferritin levels in glia of the rat substantia nigra and induces locomotor deficits

Increasing evidence suggests that abnormal iron handling may be involved in the pathogenesis of Parkinson's disease. The present study investigates the role of iron and the iron-storage protein ferritin in inflammation-induced degeneration of dopaminergic neurons of the substantia nigra pars compacta. Injection of lipopolysaccharide into the globus pallidus of young and middle-aged rats substantially decreased tyrosine hydroxylase immunostaining in substantia nigra pars compacta four weeks after injection. Loss of tyrosine hydroxylase expression was accompanied by increased iron and ferritin levels in glial cells of the substantia nigra pars reticulata. Despite greater increases in nigral iron levels, ferritin induction was less pronounced in older rats, suggesting the regulation of ferritin was compromised with age. Automated movement tracking analyses showed that young rats recovered from LPS-induced locomotor deficits within four weeks, yet older rats failed to improve on measures of speed and total distance moved. Intrapallidal lipopolysaccharide injection also increased expression of alpha-synuclein and ubiquitin in tyrosine hydroxylase-positive neurons of the substantia nigra pars compacta. These results suggest that pallidal inflammation significantly increases stress on dopamine-containing neurons in the substantia nigra pars compacta. Alterations in nigral iron levels and protein handing may increase the vulnerability of nigral neurons to degenerative processes.     

Effect of a functional impairment of corticostriatal transmission on cortically evoked expression of c-Fos and zif 268 in the rat basal ganglia.

The activity-dependent induction of immediate-early genes is commonly used to map activated neuronal networks. In a previous analysis of the cortico-basal ganglia circuits, we have shown that a cortical stimulation produces Fos protein expression in the striatum and the subthalamic nucleus, with a pattern which conforms to the anatomical organization of cortical projections [Sgambato V. et al. (1996) Neuroscience 81, 93-112]. In the present study, we examined the effects of a unilateral blockade of the corticostriatal transmission on c-fos and zif 268 messenger RNA expression evoked in the substantia nigra pars reticulata and the subthalamic nucleus following stimulation of the ipsilateral motor cortex. The blockade of the corticostriatal pathway was performed either by an excitotoxic striatal lesion or by an application of the AMPA/kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione within the striatum. After application of the glutamate receptor antagonist, which prevented the cortical stimulation activating the GABAergic striatonigral pathway, the induction of both c-fos and zif 268 messenger RNAs was facilitated in the ipsilateral substantia nigra pars reticulata. In the subthalamic nucleus ipsilateral to the application of 6-cyano7-nitroquinoxaline-2,3-dione, the cellular discharges evoked by stimulation of the cortex were considerably shortened as a result of the blockade of the disinhibitory striato-pallido-subthalamic circuit. However, a strong expression of immediate-early genes was still induced by the cortical stimulation. By contrast, after unilateral kainate lesion of the striatum, the cortical stimulation was no longer able to induce c-fos and zif 268 messenger RNA expression in the ipsilateral subthalamic nucleus and in the substantia nigra pars reticulata bilaterally. The lack of immediate-early gene induction strongly contrasted with the neuronal discharges evoked in these nuclei by the cortical stimulation. Comparison between the cortically evoked neuronal activities and the pattern of immediate-early gene expression suggests that the induction of immediate-early genes in the basal ganglia mainly reflects the level of synaptic activity rather than the frequency of discharge of the postsynaptic neurons. Moreover, the results stress that modifications of immediate-early gene expression observed in the basal ganglia after an acute or a chronic interruption of the corticostriatal transmission are not superimposable.     

Neonatal hypoxic-ischemic or excitotoxic striatal injury results in a decreased adult number of substantia nigra neurons.

It has been shown that morphologic and biochemical presynaptic markers of dopaminergic terminals are preserved in a unilateral experimental model of neonatal hypoxic-ischemic injury to the striatum. As the substantia nigra is spared direct injury in this model, we anticipated that the number of tyrosine hydroxylase-positive dopaminergic neurons projecting to the striatum would also be normal. We have found, however, that following unilateral neonatal striatal injury the number of ipsilateral tyrosine hydroxylase-positive neurons is decreased, as is the mean area of the substantia nigra pars compacta. The decrease in neurons is correlated with the decrease in striatal size (r = 0.7, P = 0.01). Neuron loss is most pronounced in the substantia nigra pars reticulata, where it is 50%. Calbindin-positive neurons in the dorsal tier of the substantia nigra pars compacta appear to be preserved. We also examined effects on the nigra following a neonatal excitotoxic striatal lesion made with quinolinic acid. We observed a decrease in the number of substantia nigra tyrosine hydroxylase-positive neurons in the absence of direct nigral injury, and the decrease was closely correlated with reductions in striatal area (r = 0.91, p < 0.01). While there are a number of possible explanations for these observations, one major possibility is that there has been a reduction in tyrosine hydroxylase-positive neurons due to a diminution in developmental target-derived trophic support from the striatum. If striatum-derived trophic support plays a role in the developmental regulation of substantia nigra neuron number, then abnormalities in this supportive relationship may play a role in the loss of these neurons in some animal models of developmental nigral degeneration, and some forms of human parkinsonism.     

Changes in dopamine D1 and D2 receptor binding in the substantia nigra following intrastriatal injection of a retrograde neurotoxin (volkensin).

The neurotoxic properties of the proposed retrograde neurotoxin volkensin were investigated. Unilateral intrastriatal injections of volkensin (n = 8) caused a 60-79% decrease in substantia nigra pars compacta (SNc) cell number on the ipsilateral side as compared to the contralateral side. This decrease was associated with a 35-56% decrease in [3H]sulpiride binding to dopamine D2 receptors in the SNc. In the substantia nigra pars reticulata (SNr) there was a 17-24% decrease in [3H]SCH 23390 binding to dopamine D1 receptors on the ipsilateral as compared to the contralateral side. The cell loss and decrease in D2 binding is attributed to the retrograde neurotoxic properties of volkensin. The decrease in D1 binding is believed to reflect loss of presynaptic receptors from terminals of striato-nigral neurons, and thus the anterograde neurotoxicity of volkensin.     

Localization of nigrostriatal, nigrothalamic and nigrotectal neurons in ventricular coordinates in macaques

The topography of the substantia nigra and its subdivisions was first analysed in macaques by using a topographic technique based on ventricular landmarks. This study shows the stability of the contours of the substantia nigra and its subdivisions in various species of macaques. The anteroposterior sequence of four subdivisions was standardized by defining eight verticotransverse levels, regularly interspaced and systemically used for each experimental case. Neurons of the substantia nigra sending axons to the striatum, thalamus and superior colliculus were identified by the technique of retrograde transport of horseradish peroxidase. The nigrostriatal labeled neurons were essentially located in the ipsilateral pars compacta but also scattered dorsally in the pars mixta and ventrally in the pars reticulata. In addition, the existence of a crossed nigrostriatal pathway was demonstrated in monkeys. Nigrothalamic labeled neurons were found in the whole pars reticulata at rostral levels and only in the medial part at more caudal levels. Comparatively, nigrotectal labeled neurons were also found in the whole pars reticulata at rostral levels, but caudally, they were confined to the lateral part of the pars reticulata and the pars lateralis. It thus appears that these three nigral components may overlap at some levels of the substantia nigra. This is discussed in relation to the existence of branched axons already documented. However, the present results underline the strong tendency of the nigrotectal neurons to be segregated from the nigrothalamic ones and to be laterally located in monkeys. In addition, two nigrotectal components have been identified on the basis of their topography and their somata size: one with large somata located in the pars lateralis, probably specific to primates, and the other with smaller somata located in the pars reticulata. These two components may indicate the existence of two different functional systems.     

GM1 gangliosides stimulate neuronal reorganization and reduce rotational asymmetry after hemitransections of the nigro-striatal pathway

Summary The effects of monosialoganglioside (GM1) injections on neuronal reorganization and behavioral recovery were studied in rats with unilateral transections of the nigro-striatal pathway. In Experiment 1, animals were treated daily with injections of saline or GM1 for not more than 14 days. At 2 days after surgery, GM1-treated animals exhibited less amphetamine-induced rotational asymmetry than did saline treated counterparts. This difference was still apparent at day 12, but vanished at post-operative day 39. Apomorphine-induced rotational asymmetry was equal in both groups at day 15, but by day 42, asymmetries increased in saline controls while remaining unchanged in GM1-treated animals. Rats were killed at either post-operative days 3, 15, or 45 after having received injections of horseradish peroxidase (HRP) into the denervated caudate nucleus. The number of neurons labelled by retrograde HRP-transport were counted in the ipsilateral substantia nigra pars compacta (iSNc), ipsilateral ventral tegmental area (iVTA), frontal cortex, and in the contralateral substantia nigra pars compacta (cSNc). Anterograde transport was also examined in the ipsilateral substantia nigra pars reticulata (iSNr). A significant loss of retrograde labelling in iSNc and iVTA was observed for both groups at post-operative day 3. At day 15, however, GM1-treated animals showed more labelling in these structures as well as in the cSNc. At 45 days after surgery comparable labelling was seen in both lesion groups. The total area of anterograde HRP-labelling in the iSNr significantly increased over time, with no differences between treatment groups. In Experiment 2, rats given the same hemitransections as in Experiment 1, were treated with daily injections of saline or GM1 for 14 days, and then received unilateral injections of 6-hydroxydopamine into the iSNc and iVTA. Nine days later, brain tissue was stained for examination of anterograde degeneration. Significantly more degenerating axons and terminals were found in the caudate nucleus of GM1-treated rats than in salinetreated controls. We propose that the early reduction of behavioral deficits may be related to a ganglioside-induced reduction of secondary degeneration or edema. The effect of gangliosides on later behavioral recovery is to accelerate neuronal reorganization. This reorganization probably involves terminal proliferation of ascending, intact striatal afferents spared by the hemitransection.     

Two pathways for the activation of small-conductance potassium channels in neurons of substantia nigra pars reticulata

Neurons in substantia nigra pars reticulata express the messenger RNA for SK2 but not for SK3 subunits that form small-conductance, Ca2+-dependent K+ channels in dopamine neurons. To determine pathways for the activation of small-conductance, Ca2+-dependent K+ channels in substantia nigra pars reticulata neurons of rats and mice, we studied effects of the selective blocker of small-conductance, Ca2+-dependent K+ channels, apamin (0.01 or 0.3 microM). Apamin diminished the afterhyperpolarization following each action potential and induced burst discharges in substantia nigra pars reticulata neurons. Apamin had a robust effect already at a low (10 nM) concentration consistent with the expression of the SK2 subunit. Afterhyperpolarizations were also reduced by the Ca2+ channel blockers Ni2+ (100 microM) and omega-conotoxin GVIA (1 microM). Depletion of intracellular Ca2+ stores did not change the afterhyperpolarization. However, we observed outward current pulses that occurred independently from action potentials and were abrogated by apamin. Apart from a faster time course, they shared all properties with spontaneous hyperpolarizations or outward currents that ryanodine receptor-mediated Ca2+ release from intracellular stores induces in juvenile dopamine neurons. Sensitization of ryanodine receptors by caffeine silenced substantia nigra pars reticulata neurons. This effect was abolished by the depletion of intracellular Ca2+ stores. We conclude that SK2 channels in substantia nigra pars reticulata neurons are activated by Ca2+ influx through at least two types of Ca2+ channels in the membrane and by ryanodine receptor-mediated Ca2+ release from intracellular stores. Ryanodine receptors do not amplify small-conductance, Ca2+-dependent K+ channel activation by the Ca2+ influx during a single spike. Yet, ryanodine receptor-mediated Ca2+ release and, thereby, an activation of small-conductance, Ca2+-dependent K+ channels by intracellular Ca2+ are available for excitability modulation in these output neurons of the basal ganglia system.     

Different neuronal location of [3H]SCH 23390 binding sites in pars reticulata and pars compacta of the substantia nigra in the rat

The precise neuronal localization of D1 receptors in the substantia nigra has been studied autoradiographically in the rat by measuring the alterations of [3H]SCH 23390 binding site densities in this brain area after 6-hydroxydopamine (6-OHDA) induced destruction of nigrostriatal dopaminergic neurons and after ibotenate-induced lesion of striatal afferents. 6-OHDA-induced nigral lesion provoked a total loss of [3H]SCH 23390 binding sites in the pars compacta and pars lateralis (but not in the pars reticulata) of the substantia nigra. In contrast, ibotenate-induced striatal lesion caused a large diminution of the [3H]ligand binding site density in the pars reticulata but not in the pars compacta and pars lateralis of the substantia nigra. These results suggest that D1 receptors in the pars compacta or pars lateralis of the substantia nigra are located on the dopaminergic perikarya whereas those D1 receptors present in the pars reticulata of the substantia nigra lie on the terminals of nigral afferents of striatal origin.     

Dopaminergic dendrites in the pars reticulata of the rat substantia nigra and their striatal input. Combined immunocytochemical localization of tyrosine hydroxylase and anterograde degeneration

An antiserum prepared in the rabbit against bovine adrenal gland tyrosine hydroxylase has been used to identify by the immunoperoxidase method dopaminergic neurons in the rat substantia nigra. The purpose of this identification was (i) to assess the storing compartments and the release sites in the dopamine-containing processes of the pars reticulata; (ii) to determine if these processes receive a direct input from the neostriatum.Immunoreactive neurons were present in the three divisions of the substantia nigra (pars compacta, pars lateralis and pars reticulata), but they were much more numerous in pars compacta. The caudal half and the most rostral end of pars reticulata contained single and small clusters of reactive neurons, which were absent from the remaining regions. Processes emerging from the positive neurons, exhibiting also immunoperoxidase reactivity, spread throughout the whole pars reticulata. The ultrastructural study was limited to the region of the pars reticulata free of reactive perikarya, in order to analyze the processes that originate from neurons located in the pars compacta. Five hundred and eighty well-preserved immunoreactive processes were analyzed. Almost all of them (578) displayed cytological features allowing their identification as dendrites. Two of them corresponded to thin unmyelinated, non-synaptic segments of axons, probably in their way to their terminal fields outside the substantia nigra. The large majority of the reactive dendrites (82%) were postsynaptic to one or several axon terminals and did not establish direct appositions with other dendritic elements. Only 4.35% of the labeled dendrites were directly apposed to other reactive or unreactive dendrites. Two of the labeled dendrites (0.35%) contained synaptic-like vesicles. In one of them, the vesicles were clustered against a restricted area of the plasma-membrane, forming an active zone.In two animals, kainic acid was used to destroy neurons located within the central region of the main body of the neostriatum. Their projections were traced to the ipsilateral substantia nigra, in which dopaminergic neurons were visualized by the immunoperoxidase method. The axons originating from the injured neurons in the striatum established direct synaptic contacts with the immunoreactive dendrites in pars reticulata.These findings indicate that (i) there is no dopaminergic recurrent collateral axonal plexus in pars reticulata; (ii) the dopamine-storing compartment in the dendritic processes is not vesicular; the cisterns of the smooth endoplasmic reticulum might be such a compartment; (iii) The differentiation of presynaptic dendrites which establish typical junctional synaptic complexes does not occur in the dopaminergic dendrites present in pars reticulata; (iv) The proportion of presynaptic release sites observed in dopaminergic dendrites (1 active zone out of the 578 analyzed dendrites) is too low to account for the dendritic release revealed by biochemical analysis (Nieoullou, Chéramy &; Glowinski, 1977a). Therefore, the modality of transmitter release from dopaminergic dendrites must be different from that supposed in the vesicular theory; (v) combined anterograde degeneration and immunocytochemistry has allowed us to demonstrate a direct striatal input to the dopamine-containing dendrites present within the pars reticulata.     

MK-801 is neuroprotective in gerbils when administered during the post-ischaemic period

The neuroprotective effects of the non-competitive N-methyl-D-aspartate receptor antagonist (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) have been evaluated in the gerbil hippocampus when the drug was administered i.p. at various times during and after a 5 min period of transient forebrain ischaemia, induced by bilateral common carotid artery occlusion. A single dose of 1, 3 or 10 mg/kg of MK-801 gave significant protection of hippocampal CA1 and CA2 pyramidal neurons when administered during the occlusion and up to 24 h following the period of ischaemia. A dose of 0.3 mg/kg was effective when administered during the occlusion period but gave no protection at 30 min or 2 h post-ischaemia. Experiments in which MK-801 was administered in repeated doses indicated that significant protection was achieved with 1 mg/kg of MK-801 repeated post-ischaemically and with 1 mg/kg MK-801 supplemented with repeated doses of 0.3 mg/kg of MK-801. However 0.3 mg/kg of MK-801 followed by repeated doses of 0.03 mg/kg administered post-ischaemically was not neuroprotective. These results indicate that MK-801 can protect hippocampal neurons from ischaemia-induced neuronal degeneration when it is administered up to 24 h after the insult. These data provide further evidence that therapeutic intervention in the post-ischaemic period can successfully prevent neurodegenerative events, and that the delayed degeneration of hippocampal neurons following an ischaemic insult occurs by an N-methyl-D-aspartate receptor-mediated process.     

Dopamine agonist-mediated rotation in rats with unilateral nigrostriatal lesions is not dependent on net inhibitions of rate in basal ganglia output nuclei.

Current models of basal ganglia function predict that dopamine agonist-induced motor activation is mediated by decreases in basal ganglia output. This study examines the relationship between dopamine agonist effects on firing rate in basal ganglia output nuclei and rotational behavior in rats with nigrostriatal lesions. Extracellular single-unit activity ipsilateral to the lesion was recorded in awake, locally-anesthetized rats. Separate rats were used for behavioral experiments. Low i.v. doses of D1 agonists (SKF 38393, SKF 81297, SKF 82958) were effective in producing rotation, yet did not change average firing rate in the substantia nigra pars reticulata or entopeduncular nucleus. At these doses, firing rate effects differed from neuron to neuron, and included increases, decreases, and no change. Higher i.v. doses of D1 agonists were effective in causing both rotation and a net decrease in rate of substantia nigra pars reticulata neurons. A low s.c. dose of the D1/D2 agonist apomorphine (0.05 mg/kg) produced both rotation and a robust average decrease in firing rate in the substantia nigra pars reticulata, yet the onset of the net firing rate decrease (at 13-16 min) was greatly delayed compared to the onset of rotation (at 3 min). Immunostaining for the immediate-early gene Fos indicated that a low i.v. dose of SKF 38393 (that produced rotation but not a net decrease in firing rate in basal ganglia output nuclei) induced Fos-like immunoreactivity in the striatum and subthalamic nucleus, suggesting an activation of both inhibitory and excitatory afferents to the substantia nigra and entopeduncular nucleus. In addition, D1 agonist-induced Fos expression in the striatum and subthalamic nucleus was equivalent in freely-moving and awake, locally-anesthetized rats. The results show that decreases in firing rate in basal ganglia output nuclei are not necessary for dopamine agonist-induced motor activation. Motor-activating actions of dopamine agonists may be mediated by firing rate decreases in a small subpopulation of output nucleus neurons, or may be mediated by other features of firing activity besides rate in these nuclei such as oscillatory firing pattern or interneuronal firing synchrony. Also, the results suggest that dopamine receptors in both the striatum and at extrastriatal sites (especially the subthalamic nucleus) are likely to be involved in dopamine agonist influences on firing rates in the substantia nigra pars reticulata and entopeduncular nucleus.     

Striatal, pallidal, and pars reticulata evoked inhibition of nigrostriatal dopaminergic neurons is mediated by GABA(A) receptors in vivo

Dopaminergic neurons express both GABA(A) and GABA(B) receptors and GABAergic inputs play a significant role in the afferent modulation of these neurons. Electrical stimulation of GABAergic pathways originating in neostriatum, globus pallidus or substantia nigra pars reticulata produces inhibition of dopaminergic neurons in vivo. Despite a number of prior studies, the identity of the GABAergic receptor subtype(s) mediating the inhibition evoked by electrical stimulation of neostriatum, globus pallidus, or the axon collaterals of the projection neurons from substantia nigra pars reticulata in vivo remain uncertain. Single-unit extracellular recordings were obtained from substantia nigra dopaminergic neurons in urethane anesthetized rats. The effects of local pressure application of the selective GABA(A) antagonists, bicuculline and picrotoxin, and the GABA(B) antagonists, saclofen and CGP-55845A, on the inhibition of dopaminergic neurons elicited by single-pulse electrical stimulation of striatum, globus pallidus, and the thalamic axon terminals of the substantia nigra pars reticulata projection neurons were recorded in vivo. Striatal, pallidal, and thalamic induced inhibition of dopaminergic neurons was always attenuated or completely abolished by local application of the GABA(A) antagonists. In contrast, the GABA(B) antagonists, saclofen or CGP-55845A, did not block or attenuate the stimulus-induced inhibition and at times even increased the magnitude and/or duration of the evoked inhibition. Train stimulation of globus pallidus and striatum also produced an inhibition of firing in dopaminergic neurons of longer duration. However this inhibition was largely insensitive to either GABA(A) or GABA(B) antagonists although the GABA(A) antagonists consistently blocked the early portion of the inhibitory period indicating the presence of a GABA(A) component. These data demonstrate that dopaminergic neurons of the substantia nigra pars compacta are inhibited by electrical stimulation of striatum, globus pallidus, and the projection neurons of substantia nigra pars reticulata in vivo. This inhibition appears to be mediated via the GABA(A) receptor subtype, and all three GABAergic afferents studied appear to possess inhibitory presynaptic GABA(B) autoreceptors that are active under physiological conditions in vivo.     

Striato-nigral dynorphin and substance P pathways in the rat

The effect of striatal ibotenic acid lesions on dynorphin-, substance P- and enkephalin-like immunoreactivities in the substantia nigra has been studied with immunohistochemistry as well as biochemistry. A comparison was made with the effects produced by intranigral ibotenic acid lesion and by 6-hydroxy-dopamine injection into the medial forebrain bundle. In addition, the effect of the striatal lesions on nigral glutamic acid decarboxylase (GAD)-positive structures was analysed with immunohistochemistry. The effect of the lesions was analysed functionally in the Ungerstedt rotational model, in order to obtain a preliminary evaluation of the extent of the lesions. The striatal lesions produced a parallel depletion of dynorphin and substance P levels in the substantia nigra, pars reticulata, ipsilateral to the treated side, which was dependent upon the extent and location of the lesion. Ibotenic acid lesions into the tail and the corpus of the striatum produced stronger nigral-peptide depletion than lesions in the head and the corpus of the striatum. Comparison of placement of lesions and localization of depleted area in the substantia nigra revealed a topographical relationship. Furthermore, the nigral depletion patterns of dynorphin and substance P were similar. The immunohistochemical analysis revealed that also GAD-positive fibers in the pars reticulata to a large extent disappeared after striatal lesions, in parallel to the dynorphin- and substance P-positive fibers. However, the depletion was less pronounced for GAD than for the peptides, probably related to presence of local GABA neurons in the zona reticulata of the substantia nigra. These results indicate that with the types of lesion used in this study it is not possible to provide evidence for a differential localization within the striatum of dynorphin-, substance P- and GABA-positive cell bodies projecting to the substantia nigra. The radioimmunoassay showed that (Leu)- but not (Met)-enkephalin was affected to the same extent as the dynorphin peptides, supporting the view that (Leu)-enkephalin in the pars reticulata of the substantia nigra is derived from proenkephalin B and not from proenkephalin A. In the immunohistochemical analysis (Met)-enkephalin-like immunoreactivity could only be detected in the pars compacta of the substantia nigra and did not seem to be affected by any of the lesions. The striatal lesions produced a behavioural asymmetry, which could be disclosed by stimulating the rats with apomorphine, which produced ipsilateral rotation.(ABSTRACT TRUNCATED AT 400 WORDS)