Evidence for Functional Differences between Entopeduncular Nucleus and Substantia Nigra: Effects of APV (DL-2-amino-5-phosphonovaleric acid) Microinfusion on Reaction Time Performance in the Rat

Overactivity of the excitatory amino acid outputs of the subthalamic nucleus (STN) has recently been found to be one of the cascade of subsequent disruptions caused by nigrostriatal dopaminergic degeneration in Parkinson's disease. The respective contribution of the excitatory glutamatergic output structures of the STN [i.e. the globus pallidus (GP), entopeduncular nucleus (EP) and substantia nigra pars reticulata (SNr)] to the control of movement is not known, however. To investigate further the function of glutamatergic transmission through NMDA receptor subtypes in these three structures, the effects of discrete local infusion of a competitive receptor antagonist, DL-2-amino-5-phosphonovaleric acid (APV), into the EP, GP and SNr were tested in rats performing a reaction time task. Bilateral infusion of APV into the different output structures of the STN differentially impaired the performance of rats trained to release a lever after the onset of a visual stimulus within a time limit to obtain a food reward. Infusion of APV (0.25 and 0.5 μg/0.5 μl) into the SNr was found to induce behavioural deficits characterized by a dramatic increase in the number of premature lever releases and decreased mean reaction time. In contrast, the infusion of APV at a dose of 0.25 μg into the GP or EP was found to induce a motor initiation deficit characterized by an increased number of delayed responses (lever release after the time limit) and increased mean reaction time. At a dose of 0.5 μg, a premature responding deficit was added to the previous motor impairment. Interestingly, when APV was infused simultaneously into the GP and SNr in the same animals, the behavioural effects tended to be similar to those observed after a single infusion into the SNr. Altogether, these results reveal that the different functional weight of the three main output pathways originating at the STN level is t.o. The behavioural deficits induced by NMDA receptor blockade in the SNr were similar to those observed previously after a neurotoxic lesion of the STN, suggesting that NMDA receptors in this structure play a major role as a functional output of the STN. Furthermore, regarding the differential effects produced by the same dose of APV in the SNr and the EP, these two structures, which are classically believed to be functionally linked should not be considered as the same functional entity in the organization of basal ganglia outflow.     

Single unit responses of substantia nigra pars reticula neurons to apomorphine: Effects of striatal lesions and anesthesia

Many of the behavioral consequences of dopamine system activation are thought to be mediated by substantia nigra pars reticulata output pathways. Extracellular, single unit recording studies were conducted to determine how i.v. administration of the dopamine agonist, apomorphine, affects the activity of these pars reticulata neurons. Results revealed that a 320 μg/kg dose of the drug, considered sufficient to stimulate striatal postsynaptic dopamine receptors, caused affinity variable changes in reticulata cell firing. Cells exhibited increases, decreases, or no changes in firing. Many cells also displayed marked minute to minute changes in firing. This non-uniform pattern of responses was not related to state of consciousness since similar responses were observed in both chloral hydrate-anesthetized as well as conscious, paralyzed rats. Both the increases and decreases could be reversed by subsequent administration of haloperidol. The variable responses to apomorphine were reduced but not totally prevented by striatal kainic acid lesions, suggesting that changes in striatonigral transmission may account for some but not all of the firing changes which were observed. A lower dose of apomorphine (20 μg/kg), thought to act primarily at dopamine cell autoreceptors, had little effect on reticula cell firing and did not modify the variable responses normally observed after the higher dose. These results contrast strikingly with the consistent excitatory responses to apomorphine which have previously been observed in the globus pallidus and suggest that complex or multiple indirect effects of the drug may contribute to the varied reticulata responses.     

Immunohistochemical and biochemical studies on Lys8-Asn9-neurotensin8-13 (LANT6)-related peptides in the basal ganglia of pigeons, turtles, and hamsters

The distribution of the neurotensin-related hexapeptide LANT6 within the basal ganglia and its projection targets was studied in turtles, pigeons, and hamsters by using immunohistochemical techniques, radioimmunoassay (RIA), gel chromatography, and high performance liquid chromatography (HPLC). The results in turtles and pigeons were fundamentally similar. Within the basal ganglia, LANT6-like immunoreactivity (LLI) was observed in a population of large striatal neurons (comprising 1-5% of the total number of striatal neurons) and in essentially all of the medium-large pallidal neurons. In addition, LLI was observed in neurons of such other "striatal" and "pallidal" cell groups as the olfactory tubercle and ventral pallidum, respectively. Within the dopaminergic cell fields of the tegmentum, to which the pallidal cell groups project, LLI-containing fibers were abundant. Knife-cut studies confirmed that the majority of these LLI-containing fibers arise from telencephalic levels. Biochemical studies with RIA and HPLC showed large amounts of immunoreactive LANT6 (iLANT6) in the basal telencephalon (477 pmol/g) and tegmentum of pigeons (259 pmol/g), and this material was indistinguishable from the synthetic peptide. Lower levels of iLANT6 were demonstrated in the basal telencephalon (82 pmol/g) and tegmentum (156 pmol/g) of turtles, and the majority of this activity appeared to be associated with larger molecular forms of LANT6 or a peptide related to LANT6. In addition, one or more substances resembling Neuromedin N (NMN), a mammalian counterpart to LANT6, were detected in the turtle nervous system. The labeling patterns in hamsters were similar to those in pigeons and turtles, except that in hamsters fewer neurons were labeled and the labeling was generally lighter. The lighter level of labeling may reflect a difference between the LANT6-like material present in hamster nervous system and authentic LANT6. Biochemical studies revealed that a Neuromedin N-like substance, as well as high molecular weight forms of a LANT6-like substance, are present in hamster brain. In hamsters, neurons within globus pallidus, the entopeduncular nucleus, the ventral pallidum, and the polymorph layer of the olfactory tubercle were labeled for the presence of LANT6. Fiber labeling for LANT6 in the dopaminergic tegmental cell groups that receive pallidal input was, however, light. Thus, the present results establish that LANT6 in pigeons and LANT6-related peptides in turtles and hamsters are present within many pallidal neurons. In pigeons and turtles, these pallidal neurons give rise to a major LLI-containing projection to the dopaminergic cell groups of the tegmentum.(ABSTRACT TRUNCATED AT 400 WORDS)     

Efferent projections of the subthalamic nucleus in the squirrel monkey as studied by the PHA-L anterograde tracing method

The organization of the efferent connections of the subthalamic nucleus was studied in the squirrel monkey (Saimiri sciureus) by using the lectin Phaseolus vulgaris-leucoagglutinin (PHA-L) as an anterograde tracer. At the level of the basal forebrain, anterogradely labeled fibers and axon terminals were mostly found in the striatopallidal complex and the substantia innominata. In cases in which the PHA-L injection sites were placed in the central or the lateral third of the subthalamic nucleus, numerous anterogradely labeled fibers were seen to arise from the injection loci and innervate massively the globus pallidus. At pallidal levels the fibers formed bands lying parallel and adjacent to the medullary laminae. The number and the complexity of the topographical organization of these bands varied with the size and the location of the PHA-L injection site. When examined at a higher magnification, the bands of subthalamopallidal fibers appeared as rich plexuses of short axon collaterals with small bulbous enlargements that closely surrounded the cell bodies and primary dendrites of pallidal cells. In contrast, PHA-L injection involving the medial tip of the subthalamic nucleus did not produce bandlike fiber patterns in the globus pallidus. Instead, the labeled fibers formed a diffuse plexus occupying the ventral part of the rostral pole of the globus pallidus as well as the subcommissural pallidal region. The substantia innominata contained a moderate number of labeled fibers and axon terminals following injection of PHA-L in the medial tip of the subthalamic nucleus. A small to moderate number of anterogradely labeled fibers were seen in the putamen after all PHA-L injections. These subthalamostriatal fibers were long, linear, and branched infrequently. At midbrain level the substantia nigra contained a significant number of anterogradely labeled fibers and axon terminals following PHA-L injection in the subthalamic nucleus. The subthalamonigral fibers descended along the ventromedial part of the cerebral peduncle and swept laterally to reach their target. Most of these fibers formed small plexuses along the base of the pars reticulata, whereas a few others ascended along the cell columns of the pars compacta that impinged deeply within the pars reticulata. More caudally in the brainstem, a small number of fibers occurred in the area of the pedunculopontine nucleus and in the periaqueductal gray. These findings indicate that besides its well-known connection with the pallidum, the subthalamic nucleus gives rise to widespread projections to other components of the basal ganglia in primates.     

The organization of the descending ventral pallidal projections in the monkey

We studied the distribution and light- and electron-microscopic morphology of neurons in the hippocampal formation containing nitric oxide synthase (NOS), and thus likely to release nitric oxide, a freely diffusible neuromediator implicated in long-term potentiation. Only a small fraction of hippocampal neurons contained NOS or its marker, NADPH diaphorase. Most of the positive neurons were in the pyramidal layer of the subiculum, stratum radiatum of Ammon's horn, and subgranular zone of the dentate gyrus. Positive neurons were also conspicuous in the molecular layer of the dentate gyrus and in the pyramidal layer of CA3, sparse in the pyramidal layer of CA2 and CA1, and almost absent from presubiculum and parasubiculum. Numerous positive fibers were seen, especially in stratum radiatum and stratum lacunosum-moleculare of Ammon's horn. Double staining experiments demonstrated that nearly all NADPH diaphorase-positive neurons in the hippocampus also contained γ-aminobutyric acid. On the basis of their morphology, distribution, and inhibitory neurotransmitter content, most NOS-positive cells in the hippocampus are probably local circuit neurons. These data suggest that nitric oxide in CA1 may function as a paracrine agent, rather than a spatially precise messenger, in long-term potentiation. © 1993 Wiley-Liss, Inc.     

Convergent Synaptic Input From the Neostriatum and the Subthalamus Onto Identified Nigrothalamic Neurons in the Rat

The two major afferents of the substantia nigra pars reticulata are the subthalamic nucleus and the striatum. Stimulation of these afferents has opposing physiological effects on the output neurons of the substantia nigra pars reticulata. In order to better understand the role of these afferents in the flow of information through the basal ganglia and to better understand the ways in which they might interact, experiments have been performed to test the possibility that single-output neurons of the substantia nigra pars reticulata receive convergent synaptic input from the subthalamic nucleus and the neostriatum. To address this, rats received iontophoretic deposits of the anterograde tracer Phaseolus vulgaris leucoagglutinin in the subthalamic nucleus, injections of the anterograde tracer biocytin in the neostriatum and injections of the retrograde tracer horseradish peroxidase conjugated to wheat-germ agglutinin in the ventral medial nucleus of the thalamus. Following appropriate survival times the animals were perfusion-fixed and sections of the substantia nigra were processed to reveal the transported tracers and prepared for electron microscopy. Light microscopic examination revealed that the substantia nigra contained rich plexuses of anterogradely labelled subthalamic and striatal terminals, as well as many retrogradely labelled nigrothalamic neurons. The anterogradely labelled terminals were often seen apposed to the retrogradely labelled neurons. In the electron microscope the subthalamic terminals were seen to form asymmetrical synaptic contacts (subthalamic type 1) with the identified nigrothalamic neurons as well as unlabelled perikarya and both proximal and distal dendrites. In confirmation of previous findings, the striatal terminals made symmetrical synaptic contact with the nigrothalamic neurons as well as unlabelled neurons. In areas of overlap between the two classes of terminals, identified nigrothalamic neurons and unlabelled nigral neurons were found to receive convergent synaptic input from the subthalamic nucleus and the neostriatum. In addition to the anterogradely labelled subthalamic terminals that formed asymmetrical synaptic specializations, a second, much rarer class was also observed (subthalamic type 2). These terminals were much larger and formed symmetrical synapses; several lines of evidence suggest that they originated not in the subthalamic nucleus but in the globus pallidus. These terminals were found to make synaptic contacts with identified nigrothalamic neurons and non-labelled neurons and to form convergent synaptic contacts with subthalamic type 1 terminals and striatal terminals. It is concluded that the topographical and synaptic organization of the so-called direct (striatum to substantia nigra pars reticulata) and indirect pathways (i.e. pathways involving the subthalamic nucleus andlor the globus pallidus) of information flow through the basal ganglia underlies the inhibition and excitation of the output neurons of the substantia nigra pars reticulata that occur following stimulation of the striatum.     

Nigral GABAergic inhibition upon mesencephalic dopaminergic cell groups in rats

Synaptic inhibition from the substantia nigra pars reticulata (SNr) to the mesencephalic dopaminergic neurons, which was mediated by gamma (gamma)-amino-butyric acid (GABA), was investigated in a midbrain slice preparation of Wistar rats. Whole-cell patch-clamp recordings were used to record synaptic potentials/currents from the dopaminergic neurons (n = 93) located in the retrorubral field (n = 22), the substantia nigra pars compacta (n = 47) and the ventral tegmental area (n = 24). In the presence of ionotropic glutamate receptor antagonists electrical stimulation of the SNr induced inhibitory postsynaptic potentials (IPSPs) and/or currents (IPSCs) in 83 neurons. The IPSPs/IPSCs were comprised early and late components. The early IPSPs/IPSCs were mediated by chloride currents through GABA(A) receptors. The late IPSPs/IPSCs were mediated by potassium currents through GABA(B) receptors. Both GABA(A)- and GABA(B)-IPSPs were amplified by repetitive stimuli with frequencies between 25 and 200 Hz. This frequency range covers the firing frequencies of SNr neurons in vivo. It was observed that an application of a GABA(B) receptor antagonist increased the amplitude of the GABA(A)-IPSPs. The amplification was followed by a rebound depolarization that induced transient firing of dopaminergic neurons. These properties of the IPSPs were common in all of the three dopaminergic nuclei. These results suggest that postsynaptic GABA(A)- and GABA(B)-inhibition contribute to transient and persistent alternations of the excitability of dopaminergic neurons, respectively. These postsynaptic mechanisms may be, in turn, regulated by presynaptic GABA(B)-inhibition. Nigral GABAergic input may provide the temporospatial regulation of the background excitability of mesencephalic dopaminergic systems.     

Actions of Glycine on Non-dopaminergic Neurons of the Rat Substantia Nigra

The effects of glycine on non-dopaminergic cells in rat substantia nigra pars compacta and pars reticulata maintained in vitro were investigated using intracellular recording techniques. Glycine, superfused at a concentration between 30 μM and 1 mM, reversibly blocked the spontaneous firing of these neurons. The inhibition of firing discharge was associated with a hyperpolarization of the membrane (potassium acetate-filled electrodes) and an increase in conductance. Under voltage-clamp experiments (holding potential between -57 and -65 mV), glycine produced an outward response which reversed polarity at about -74 mV. However, when the recording electrodes were filled with KCI, the glycinergic response was mainly depolarizing/inward and reversed at about -43 mV. Thus, it appeared to be due to an increase in chloride permeability. Furthermore, the effects of glycine were reversibly antagonized by strychnine (between 300 nM and 1 μM). Our findings demonstrate that glycine is a potent inhibitory agent on non-dopaminergic cells of the substantia pars compacta and pars reticulata that acts by activating strychnine-sensitive receptors.     

Subregional changes in discharge rate, pattern, and drug sensitivity of putative GABAergic nigral neurons in the kindling model of epilepsy

The substantia nigra pars reticulata (SNr) is thought to act as a seizure-gating mechanism in kindling and other epilepsy models. We investigated whether the kindling process induces site-specific (anterior-posterior) and seizure-outlasting alterations in the activity of putative GABAergic SNr neurons and in their response to pharmacological manipulation. Female Wistar rats were kindled via the basolateral amygdala by daily stimulation. In vivo extracellular single unit recordings of SNr neurons were performed in kindled rats 1 day after a generalized seizure in order to examine activity changes that outlast the kindled seizures. Sham-kindled and naive rats served as controls. We found a significant and seizure-outlasting increase of discharge rates within the posterior but not within the anterior SNr of kindled rats when compared to controls. Furthermore, kindling resulted in seizure-outlasting burst-like firing pattern of SNr neurons. The antiepileptic drug valproic acid (VPA; 100 mg/kg i.v.) significantly reduced SNr discharge rates in all animal groups. Interestingly, neurons located in the anterior SNr of kindled rats were significantly less depressed by VPA compared to the reduction obtained in naive controls. The present data disclose kindling induced functional plasticity within basal ganglia regions. The findings are relevant for a better understanding of the mechanisms underlying the seizure-gating function of the SNr and might provide new targets for rational therapeutic manipulations, which aim to establish a remote control of epileptic seizures.     

Dopamine D1/5 receptor stimulation induces c-fos expression in the subthalamic nucleus: possible involvement of local D5 receptors

The activity of neurons in the subthalamic nucleus controls various aspects of movement. The present study examined the action of dopamine receptor agonists on c-fos gene expression in the subthalamic nucleus in normal rats. We found that systemic administration of the dopamine D1/5 receptor agonist, SKF 82958 (1 mg/kg), induces c-fos expression in the subthalamic nucleus. In contrast, systemic administration of the dopamine D2/3 receptor agonist, quinelorane (2 mg/kg) had no effect. When combined, SKF 82958 and quinelorane induced c-fos expression in subthalamic neurons that was similar to that found following administration of SKF 82958 alone. We also examined c-fos expression in the substantia nigra pars reticulata, the major projection area for subthalamic neurons, and found that SKF 82958, but not quinelorane, caused an induction of c-fos expression in this area. In order to clarify the mechanisms underlying the SKF 82958-mediated induction of c-fos expression in the subthalamic nucleus and substantia nigra pars reticulata, in situ hybridization for the dopamine D1, D2, D3 and D5 receptor mRNAs was performed. The only significant observation was that D5 receptor mRNA is expressed in subthalamic neurons.The present data show that dopamine, via D1/D5 receptors, upregulates c-fos expression in subthalamic neurons, and that the high expression of D5 receptors in this area might be involved. Taken together, these data suggest that dopamine D1/5 receptors are more important for the action of dopamine in the so-called indirect pathway of the basal ganglia circuitry than what is recognized in current models of basal ganglia organization.     

Antiparkinsonian and behavioral effects of inactivation of the substantia nigra pars reticulata in hemiparkinsonian primates

Altered activity in one of the output nuclei of the basal ganglia, the internal segment of the globus pallidus, is known to play an important role in the generation of parkinsonism. These inactivation studies tested the hypothesis that altered activity in the second major output nucleus of the basal ganglia, the substantia nigra pars reticulata (SNr), also contributes to parkinsonian motor signs. To this end, three rhesus monkeys were rendered hemiparkinsonian by intracarotid injections of MPTP. The animals then received intra-SNr injections of the GABA(A) receptor agonist muscimol to inactivate small portions of the SNr. Before and after these injections, parkinsonian motor signs were evaluated with a battery of behavioral observation methods. Injections into the centrolateral SNr reduced contralateral limb akinesia and bradykinesia in two animals. By contrast, medial injections induced generalized activation, contralateral turning, and saccadic eye movements in all animals. Injections in the most lateral and posterior portions of the nucleus had no effects. Two of the animals also received ibotenic acid lesions of the SNr, followed by a series of similar observations. These injections induced improvements in limb akinesia, postural improvements, and turning. The experiments suggest that the anterolateral "motor" territory of the SNr is involved in the development of appendicular parkinsonian motor signs.