Efferent projections of the subthalamic nucleus in the rat: light and electron microscopic analysis with the PHA-L method

Efferent projections of rat subthalamic nucleus were studied by use of the axonal transport of phaseolus vulgaris-leucoagglutinin (PHA-L), and the results were analyzed with light and electron microscopes. PHA-L injections in the subthalamic nucleus (STH) resulted in heavy labeling of fiber plexus with en passant boutons and terminals in the pallidal complex, i.e., the entopeduncular nucleus (EP), the globus pallidus (GP) and the ventral pallidum (VP), and the substantia nigra pars reticulata (SNR). Labeling in GP was characterized by two distinct bands of labeled terminals oriented dorsoventrally, whereas labeling in SNR was patchy. STH efferents to the pallidum and SNR displayed a mediolateral topographic organization. With regard to dorsoventral organization, projections to GP were inverted, but those to SNR were not. There were moderate projections to the neostriatum and sparse projections to the frontal cortex, substantia innominata, substantia nigra pars compacta (SNC), pedunculopontine tegmental nucleus, ventral part of the central gray matter including the dorsal raphe nucleus, and the mesencephalic and pontine reticular formation. PHA-L injections in the zona incerta and the lateral hypothalamic area resulted in fiber and terminal labelings in many structures, including the basal forebrain, EP, SNC, and other brainstem areas that overlap with some of the terminal sites of STH projections. Ultrastructural observations of PHA-L labeled processes in GP and SNR revealed that STH terminals in both structures contained small pleomorphic vesicles and formed asymmetrical contacts. These contacts were mainly on dendritic shafts, but some were on somata. It also was observed that the myelinated axons of STH neurons lost their myelin after reaching their target areas and the synaptic boutons arose from relatively thin unmyelinated axons.     

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.     

The pallido-subthalamic projection in rat: Anatomical and biochemical studies

Horseradish peroxidase injected into the rat globus pallidus was transported retrogradely to subthalamic nucleus neuronal cell bodies and anterogradely to axon terminals in the subthalamic nucleus. Electron microscopic observations revealed that the labeled axon terminals made symmetrical axosomatic and axo-dendritic synaptic contacts with labeled subthalamic nucleus perikarya and dendrites. Injection of kainic acid in the globus pallidus several days prior to the horseradish peroxidase injection abolished the anterograde but not the retrograde transport of the tracer. This suggested the anterograde labeling observed in the subthalamic nucleus originated from neuronal cell bodies in the globus pallidus.Kainic acid lesions identical to those employed in the above anatomical studies resulted in a loss of neuronal cell bodies throughout the globus pallidus and caused a drop in glutamic acid decar☐ylase and choline acetyltransferase levels in the globus pallidus. Levels of these two enzymes were not changed in the subthalamic nucleus after the globus pallidus kainic acid lesions, but both showed small, statistically significant decreases in the substantia nigra. It was concluded that there is a massive pathway from the globus pallidus to the subthalamic nucleus, which terminates on subthalamic nucleus neurons projecting back to the globus pallidus. Neither γ-aminobutyric acid nor acetylcholine is the major neurotransmitter in the massive pallido-subthalamic pathway.     

Efferent connections of the caudate nucleus, including cortical projections of the striatum and other basal ganglia: an autoradiographic and horseradish peroxidase investigation in the cat

Tritiated tracer was injected into the head of the caudate nucleus in cats. Following such injections, labeling is present within extensive regions of both the globus pallidus and entopeduncular nucleus, where it presents a mottled or meshlike appearance. These projections are topographically organized in that there is simple correspondence between the mediolateral, dorsoventral, and rostrocaudal origin of the caudate projection and its input to the globus pallidus and entopeduncular nucleus. Transported tracer is also present within the substantia nigra, where it is most abundant within the pars reticularis. However, distinct labeling also overlies cells of the pars compacta, and lesser amounts of labeling are present within the pars lateralis and within the retrorubral area. Following injections of horseradish peroxidase into the caudate nucleus, and subsequent tissue processing by the tetramethylbenzidine (TMB) method of Mesulam ('78), labeled anterograde fibers are present in abundance within the globus pallidus, entopeduncular nucleus, and all subdivisions of the substantia nigra, thus confirming the autoradiographic findings. Also, it is especially obvious in this HRP material that, contrary to previous degeneration studies, both the rostromedial and caudolateral parts of the pars lateralis of the substantia nigra contain numerous anterogradely labeled fibers. Retrogradely labeled neurons are also present within the substantia nigra of these same tissue sections, where they are most abundant within the pars compacta, but lesser numbers of labeled neurons are also present within the pars reticularis, pars lateralis, retrorubral area, and ventral tegmental area on the ipsilateral side, and all of these same subdivisions of the substantia nigra on the contralateral side. Also, within the subthalamic nucleus in these experiments, there are anterogradely labeled fibers, as well as retrogradely labeled neurons, which are interpreted to represent a reciprocal connection between the subthalamic nucleus and the striatum. In a separate series of experiments, horseradish peroxidase was injected into the motor cortex-specifically into the anterior sigmoidal gyrus. Following such injections, labeled neurons representing afferents to the motor cortex are found in all subcortical nuclei commonly known as the "basal ganglia," including the caudate nucleus, putamen, globus pallidus, entopeduncular nucleus, substantia innominata, nucleus of the diagonal band of Broca, medial septal nucleus, claustrum, and basolateral amygdaloid nucleus.     

Erratum

Tritiated tracer was injected into the head of the caudate nucleus in cats. Following such injections, labeling is present within extensive regions of both the globus pallidus and entopeduncular nucleus, where it presents a mottled or meshlike appearance. These projections are topographically organized in that there is simple correspondence between the mediolateral, dorsoventral, and rostrocaudal origin of the caudate projection and its input to the globus pallidus and entopeduncular nucleus. Transported tracer is also present within the substantia nigra, where it is most abundant within the pars reticularis. However, distinct labeling also overlies cells of the pars corapacta, and lesser amounts of labeling are present within the pars lateralis and within the retrorubral area. Following injections of horseradish peroxidase into the caudate nucleus, and subsequent tissue processing by the tetramethylbenzidine (TMB) method of Mesulam (1978), labeled anterograde fibers are present in abundance within the globus pallidus, entopeduncular nucleus, and all subdivisions of the substantia nigra, thus confirming the autoradiographic findings. Also, it is especially obvious in this HRP material that, contrary to previous degeneration studied, both the rostromedial and caudolateral parts of the pars reticularis of the substantia nigra contain numerous anterogradely labeled fibers. Retrogradely labeled neurons are also present within the substantia nigra of these same tissue sections, where they are most abundant within the pars compacta, but lesser numbers of labeled neurons are also present within the pars reticularis, pars lateralis, retrorubral area, and ventral tegmental area on the ipsilateral side, and all of these same subdivisions of the substantia nigra on the contralateral side. Also, within the subthalamic nucleus in these experiments, there are anterogradely labeled fibers, as well as retrogradely labeled neurons, which are interpreted to represent a reciprocal connection between the subthalamic nucleus and the striatum. In a separate series of experiments, horseradish peroxidase was injected into the motor cortex–specifically into the anterior sigmoidal gyrus. Following such injections, labeled neurons representing afferents to the motor cortex are found in all subcortical nuclei commonly known as the “basal ganglia,” including the caudate nucleus, putamen, globus pallidus, entopeduncular nucleus, substantia innominata, nucleus of the diagonal band of Broca, medial septal nucleus, claustrum, and basolateral amygdaloid nucleus.     

Anterograde and retrograde axonal transport of Phaseolus vulgaris leucoagglutinin (PHA-L) from the globus pallidus to the striatum of the rat

Iontophoretic administration of PHA-L into the globus pallidus of rats resulted in the labeling of neuronal perikarya in the striatum as well as axons and terminals in the striatum, entopeduncular nucleus, subthalamus and substantia nigra. The labeled striatal perikarya were densely stained in Golgi fashion with virtually complete filling of the dendrites and spines. It is concluded that the striatal cells were filled by the retrograde transport of PHA-L and represent either striatopallidal cells, or striatonigral cells whose axons were interrupted as they passed through the injection site. The anterogradely labeled axon terminals in the striatum were observed in close apposition to the dendrites of the retrogradely labeled neurons suggesting the existence of synaptic contacts between the two groups of cells. This study demonstrates that PHA-L can be transported retrogradely as well as anterogradely following iontophoretic injections.     

Topographical and Synaptic Organization of the GABA-Containing Pallidosubthalamic Projection in the Rat

The anterograde transport of Phaseolus vulgaris-leucoagglutinin (PHA-L) was combined with postembedding immunocytochemistry for gamma-aminobutyric acid (GABA) to study the topography, the synaptic organization and the neurotransmitter content of the pallidosubthalamic projection in the rat. After injections of PHA-L in different parts of the globus pallidus a rich plexus of anterogradely labelled fibres and terminals was found in the ipsilateral subthalamic nucleus. The immunoreactive elements were distributed according to a mediolateral and rostrocaudal topography. Injections of PHA-L restricted to the lateral two-thirds of the globus pallidus gave rise to a massive anterograde labelling confined to the lateral half of the subthalamic nucleus. On the other hand, injections of PHA-L strictly confined to the medial part of the globus pallidus resulted in anterograde labelling that occupied the ventromedial pole of the subthalamic nucleus. In some cases a few retrogradely labelled cells were found in the subthalamic nucleus after PHA-L injections in the globus pallidus. The perikarya and the primary dendrites of these labelled cells were sometimes surrounded by anterogradely labelled terminals suggesting a close reciprocal connection between the globus pallidus and the subthalamic nucleus. Electron microscopic analysis of the PHA-L-labelled terminals revealed that they contain many mitochondria, numerous small round or slightly pleomorphic vesicles and occasionally one or two large dense core vesicles. They form symmetrical synaptic contacts predominantly with the proximal dendrites (39%) and less frequently with the perikarya (31%) and the distal dendrites (30%) of the subthalamic cells. Quantitative measurements showed that the pallidosubthalamic varicosities have a diameter ranging from 0.7 to 4.5 microm and a mean cross-sectional area of 0.79 +/- 0.26 microm2 (Mean +/- SD). Postembedding immunocytochemistry for GABA revealed that the PHA-L-immunoreactive pallidosubthalamic axon terminals display GABA immunoreactivity. The results of our study demonstrate that the pallidosubthalamic projection is organized according to a mediolateral and rostrocaudal topography and that the proximal dendrites of the subthalamic cells are the major targets of the GABA-immunoreactive pallidosubthalamic terminals. This suggests that the globus pallidus exerts a powerful control over the subthalamic cells through an inhibitory GABAergic pathway.     

Alterations in local cerebral glucose utilization during electrical stimulation of the striatum and globus pallidus in rats

Alterations in local cerebral glucose utilization (LCGU) in conscious rats during electrical stimulation of the striatum and the globus pallidus were investigated using the [14C]deoxyglucose method. Stimulation of the globus pallidus produced a marked contraversive circling behavior, while stimulation of the striatum led only to contraversive head turning. Unilateral stimulation of the striatum increased LCGU bilaterally in the globus pallidus and substantia nigra pars compacta, but only ipsilaterally in the entopeduncular nucleus, substantia nigra pars reticulata and subthalamic nucleus. Similar stimulation of the globus pallidus increased LGCU in the globus pallidus, substantia nigra pars reticulata and compacta, entopeduncular nucleus, subthalamic nucleus, lateral habenular nucleus, parafascicular nucleus of the thalamus, deep layers of the superior colliculus and pedunculopontine nucleus, exclusively on the ipsilateral side. These results indicate that the electrical stimulation induces LCGU changes in the respective structures having both monosynaptic and transsynaptic neuronal inputs. Some changes may also be mediated by antidromic activation. They also suggest that activation of a synaptic process whether excitatory or inhibitory results in increases in LCGU. The bilateral modulatory effects of striatal stimulation may cancel out the circling behavior seen during pallidal stimulation, and cause only head turning.     

The striatum and the globus pallidus send convergent synaptic inputs onto single cells in the entopeduncular nucleus of the rat: a double anterograde labelling study combined with postembedding immunocytochemistry for GABA.

The entopeduncular nucleus is one of the major output stations of the basal ganglia. In order to better understand the role of this structure in information flow through the basal ganglia, experiments have been performed in the rat to examine the chemical nature, morphology, and synaptology of the projections from the globus pallidus and striatum to the entopeduncular nucleus. In order to examine the morphology and synaptology of pallidoentopeduncular terminals and striatoentopeduncular terminals, rats were subjected to a double anterograde labelling study. The globus pallidus was injected with Phaseolus vulgaris-leucoagglutinin (PHA-L), and on the same side of the brain, the striatum was injected with biocytin. The entopeduncular nuclei of these animals were then examined for anterogradely labelled pallidal and striatal terminals. Rich plexuses of PHA-L-labelled pallidal terminals and biocytin-labelled striatal terminals were identified throughout the entopeduncular nucleus. At the electron microscopic level, the pallidal boutons were classified as two types. The majority (Type 1), were large boutons that formed symmetrical synapses with the dendrites and perikarya of neurones in the entopeduncular nucleus. Type 2 PHA-L-labelled terminals were much rarer, slightly smaller, and formed asymmetrical synapses. It is suggested that the Type 2 boutons are not derived from the globus pallidus but from the subthalamic nucleus. The biocytin-labelled terminals from the striatum had the typical morphological features of striatal terminals and formed symmetrical synapses. The distribution of the postsynaptic targets of the pallidal terminals and the striatal terminals differed in that the pallidal terminals preferentially made synaptic contact with the more proximal regions of the neurones in the entopeduncular nucleus, whereas the striatal terminals were located more distally on the dendritic trees. Examination in the electron microscope of areas where there was an overlap of the two sets of anterogradely labelled boutons revealed that terminals from the globus pallidus and the striatum made convergent synaptic contact with the perikarya and dendrites of individual neurones in the entopeduncular nucleus. In order to examine the chemical nature of the input to the entopeduncular nucleus from the globus pallidus and the striatum, ultrathin sections were immunostained by the postembedding method to reveal endogenous GABA. Three classes of GABA-containing terminals were identified; two of them formed symmetrical synapses and one rare type formed asymmetrical synapses. The combination of the GABA immunocytochemistry and anterograde labelling revealed that both the striatal and pallidal afferents that make symmetrical synapses with neurones in the entopeduncular nucleus, including those involved in convergent inputs, are GABAergic.(ABSTRACT TRUNCATED AT 400 WORDS)     

Neurons of the substantia nigra reticulata receive a dense GABA-containing input from the globus pallidus in the rat

The lectin Phaseolus vulgaris leucoagglutinin (PHA-L) was used as an anterograde tracer to study the topographical distribution and synaptic organization of pallidonigral fibres in the rat. Injections of PHA-L in the lateral part of the globus pallidus led to anterograde labelling of a rich plexus of varicose fibres that arborized profusely in the central core of the rostral three quarters of the substantia nigra pars reticulata (SNr). However, few fibres were detected in SNr after PHA-L injection restricted to the most medial part of the globus pallidus. A small number of fibres was seen in the substantia nigra pars compacta after each injection. The most characteristic feature of the pallidonigral terminals was the formation of baskets around the perikarya and primary dendrites of SNr cells. Electron microscopic analysis revealed that the pallidonigral terminals contain pleomorphic vesicles and a large number of mitochondria and that they form symmetrical synaptic contacts. Furthermore, postembedding immunocytochemistry for gamma-aminobutyric acid (GABA) showed that they display GABA immunoreactivity. These findings demonstrate that, in the rat, the pallidonigral projection is a major source of GABA-containing terminals innervating pars reticulata cells and that the pattern of innervation is such that they may exert a powerful inhibitory control over these cells.     

Distribution of GABA-immunoreactive neurons in the basal ganglia of the squirrel monkey (Saimiri sciureus)

The distribution of GABA-immunoreactive neurons was visualized in the basal ganglia of the squirrel monkey (Saimiri sciureus), by using a highly specific antiserum raised against GABA-glutaraldehyde-lysyl-protein conjugate and revealed by the indirect peroxidase-antiperoxidase immunohistochemical method. In the dorsal striatum, GABA-immunoreactive nerve cell bodies were small to medium in size (sectional area ranging from 90 to 125 microns2), but some larger ones (500-600 microns2) were also found. These cells displayed no obvious clustering but were significantly more numerous in the caudate nucleus than in the putamen; their number was also markedly greater at caudal than at rostral striatal levels. A moderate number of evenly distributed positive axon terminals were visible in both the caudate nucleus and the putamen. In the ventral striatum, GABA-immunoreactive nerve cell bodies and axon terminals were seen in fair number within the nucleus accumbens and in the deep layers of the olfactory tubercle. Many positive terminals but no somata were found in the islands of Calleja. In the globus pallidus, virtually all nerve cell bodies were GABA-immunoreactive and the neuropil exhibited a multitude of positive terminals. In the substantia innominata, clusters of small, globular GABA-immunoreactive somata were scattered among aggregates of larger, nonimmunoreactive neurons belonging to the nucleus basalis, and the whole region showed a low to moderate number of evenly spread GABA-positive terminals. In the subthalamic nucleus, nerve cell bodies were generally surrounded by several GABA-positive terminals but were not themselves immunoreactive. The substantia nigra showed many GABA-immunoreactive somata, which predominated in the pars lateralis and diminished progressively in number along the lateromedial axis of the pars reticulata. These cells formed a rather pleomorphic group comprising round, fusiform, or polygonal elements of relatively large size (sectional area ranging from 200 to 800 microns2). In the pars compacta and ventral tegmental area, a few GABA-immunoreactive neurons of small size were dispersed among larger, unreactive neurons. In both pars lateralis and pars reticulata of the substantia nigra, the number of GABA-positive terminals was high and their distribution was rather uniform; a smaller number were visible in the pars compacta of the substantia nigra and in the ventral tegmental area. The present results demonstrate that GABA-containing neurons are widely and heterogeneously distributed in the various components of the squirrel monkey's basal ganglia.(ABSTRACT TRUNCATED AT 400 WORDS)     

Projections of the feline globus pallidus

The organization of globus pallidus (GP) projections was studied in cats using autoradiographic and horseradish peroxidase (HRP) techniques. Both methods confirmed the existence of a topographically organized projection to subthalamic nucleus (STN). Although all but the most caudal GP projects to STN, the heaviest projection is to the lateral two-thirds. In addition, HRP studies showed that the GP projection to the medial part of substantia nigra, pars reticulata receives projections from the rostral lateral GP, while lateral substantia nigra receives input from caudal GP. There is in addition a small projection from caudal GP to the caudal lateral mesencephalon. This most caudal projection of GP arises from the portion of GP which projects the least to the subthalamic nucleus. Mesencephalic and pontine cells labeled after injection of horseradish peroxidase into STN were in areas receiving projections from GP and entopeduncular nucleus, suggesting there may be reciprocal relationships between these areas. Labeled cells were located in the lateral part of subthalamic nucleus after injection into the lateral portion of substantia nigra pars reticulata, but cells were not labeled after medial injection. Pontine injections of HRP also revealed that cells in fields of Forel and zona incerta project to pons but very few subthalamic nucleus cells project there.     

Demonstration of a pallido-nigral projection innervating dopaminergic neurons.

Afferents to the substantia nigra from the neostriatum and globus pallidus were studied in the rat by means of the autoradiographic tracing technique. 3H-leucine was injected stereotaxically into either the globus pallidus or neostriatum. Twenty-four hours later the axoplasmic transport of labelled proteins to the substantia nigra was studied by light and electron microscopic autoradiography. In animals used for electron microscopy, degeneration of dopaminergic cells in the substantia nigra was induced by intraventricular injection of 6-hydroxydopamine 72 hours before sacrifice. After neostriatal injections, light microscopic analysis revealed heavy labelling of the globus pallidus and entopeduncular nucleus, but only background labelling of the subthalamic nucleus. There was preferential labelling of the zone reticulata of the substantia nigra, with significantly less labelling of the zone compacta. After pallidal injections, light microscopic analysis showed very light labelling of those parts of the caudate-putamen in the vicinity of the injection site. There was intense labelling of the subthalamic nucleus and heavy labelling of the entopeduncular nucleus. The zona compacta of the substantia nigra was also heavily labelled. There was considerably less labelling of the zona reticulata. The electron microscopic analyses showed that after neostriatal injections, autoradiographic grains in the substantia nigra were located preferentially over boutons which terminated on normal dendritic processes. After pallidal injections, however, grains were preferentially located over boutons synapsing with degenerating dendritic processes. The degeneration produced in these dopaminergic processes by 6-hydroxydopamine was invariably of the dark type. Except for the different association with degenerating vs. non-degenerating dendrites, the subcellular distribution of autoradiographic grains in the substantia nigra was the same after injection into either the globus pallidue or caudate-putamen. Approximately 80 percent of the grains were over axons or boutons which invariably made symmetrical synaptic contacts. These observations demonstrate the existence of a pallido-nigral projection which terminates preferentially on dopaminergic cells in the pars compacta of the substantia nigra. They also confirm previous studies indicating that the strionigral projection terminates mainly in the pars reticulata. These terminations appear to be principally to non-dopaminergic cells.     

The Effects of Activation or Inhibition of the Subthalamic Nucleus on the Metabolic and Electrophysiological Activities Within the Pallidal Complex and Substantia Nigra in the Rat

The changes in local cerebral glucose utilization (LCGU) and neuronal unit activities in the subthalamic nucleus and its major target structures (the pars reticulata of the substantia nigra, the globus pallidus and the entopeduncular nucleus) following microinjection of a GABAergic antagonist (bicuculline methiodide, 0.08 nmol) or agonist (muscimol, 0.2 nmol) into the subthalamic nucleus were determined. The metabolic effect was assessed by measuring LCGU by quantitative [14C]-2-deoxyglucose autoradiography in ketamine-anaesthetized rats. Bicuculline methiodide induced increased LCGU in the ipsilateral globus pallidus, the entopeduncular nucleus and the substantia nigra pars reticulata. In contrast, muscimol decreased LCGU in these structures. The neuronal activities in the subthalamic nucleus and related structures increased following injection of bicuculline and decreased after injection of muscimol. The changes in LCGU within the structures directly related to the subthalamic nucleus were correlated with the changes in the unit activity either in the subthalamic nucleus and/or its projection structures. However, the amplitude of the relative changes in neuronal unit activity were greater than the changes in LCGU. Nevertheless, the results emphasize the functional role of the subthalamic nucleus as an activating structure within the basal ganglia.     

Efferent connections of the subthalamic region in the rat. I. The subthalamic nucleus of luys

The efferent connections of the subthalamic nucleus of Luys (STN) in the rat were investigated with the aid of the anterograde autoradiographic and the retrograde horseradish peroxidase (HRP) tracer techniques.A small microelectrophoretic injection of tritiated proline and leucine centered in the STN (case RST-4) was found to label fibers directed mainly at 3 ipsilateral structures: the substantia nigra (chiefly the ventral portions of this pars reticulata), the entopeduncular nucleus and the globus pallidus (including the ventral pallidum). In addition to this major labeling pattern, much sparser labeling was seen in striatal, thalamic, hypothalamic, pretectal, tectal and reticular territories. In another series of experiments, microelectrophoretic HRP injections confined to the substantia nigra or the globus pallidus consistently resulted in retrograde labeling of neurons in the ipsilateral STN. On the other hand, HRP injections of the vontromedial portion of the midbrain tegmentum (including the red nucleus), the superior colliculus, the pretectal area or a midbrain region at the lateral border of the central gray substance resulted in retrograde labeling of cells in the zona incerta, but no labeled cells appeared in these cases in the ventrally adjacent STN. These HRP results, together with autoradiographic data obtained in control cases, suggest that the minor projections to territories other than the substantia nigra and the pallidal complex originate in the zona incerta or the cerebral cortex rather than in STN.     

Third group of neostriatofugal neurons: neurokinin B-producing neurons that send axons predominantly to the substantia innominata

Neostriatal neurons that produce neurokinin B were investigated immunocytochemically in the rat brain with an antibody against the C-terminal portion of the precursor prepropeptide of neurokinin B, preprotachykinin B (PPTB). PPTB-immunoreactive neurons were scattered throughout the neostriatum and constituted 5.1% of neostriatal neurons. They were immunopositive for projection neuron markers, such as precursor peptides of substance P, enkephalins, and dynorphins, but negative for intrinsic neuron markers, suggesting that PPTB was expressed in neostriatal projection neurons. However, PPTB-immunoreactive neurons were immunonegative for dopamine- and cyclic AMP-regulated phosphoprotein, which is known to be produced by striatopallidal and striatonigral neurons. Furthermore, almost no PPTB-immunoreactive axon terminals were observed in the substantia nigra or globus pallidus. The authors then made large kainic acid lesions in the neostriatum to reveal the target areas of PPTB-producing neurons and observed a decrease in PPTB-immunoreactive fibers in the sublenticular portion of the substantia innominata and, to much lesser extent, in the bed nucleus of the stria terminalis and central nucleus of the amygdala. After injection of wheat germ agglutinin into the substantia innominata, PPTB immunoreactivity was detected in many retrogradely labeled neostriatal neurons. In contrast, no PPTB immunoreactivity was observed in striatonigral or striatopallidal neurons after injection of retrograde tracers into the substantia nigra or globus pallidus. Thus, neurokinin B-producing neostriatal neurons were considered to send projection fibers predominantly to the substantia innominata. Furthermore, PPTB-immunoreactive axonal swellings were closely apposed to neurokinin B receptor-immunoreactive dendrites in the substantia innominata. Overall, the present results indicate that the rat brain possesses a chemically and hodologically unique neostriatofugal pathway in addition to the direct and indirect pathways.     

The pallidointralaminar and pallidonigral projections in primate as studied by retrograde double-labeling method

The cellular origin and degree of collateralization of the pallidointralaminar and pallidonigral projections in squirrel monkey (Saimiri sciureus) were studied using Evans blue (EB) and a mixture of DAPI-Primuline (DP) as fluorescent retrograde tracers. In a first series of experiments EB was injected into the VA/VL thalamic nuclei whereas DP was delivered into the CM/Pf complex. After these injections numerous EB-labeled cells were scattered throughout the central 'motor' zone of the internal segment of the globus pallidus (GPi), compared to a smaller number of DP-positive neurons forming two small but distinct clusters in central GPi. The majority of the neurons within these two clusters were double-labeled. In addition, EB-labeled cells were disclosed in the lateral two-thirds of substantia nigra pars reticulata (SNr), whereas DP-positive neurons occurred in a wide variety of structures including the nucleus reticularis thalami, the SNr, the periaqueductal gray, the superior colliculus, the midbrain raphe nuclei, the pedunculopontine nucleus (TPP) area (bilaterally), and the locus coeruleus. In a second series of experiments, EB was injected into the CM/Pf complex whereas DP was delivered into the TPP area of the midbrain tegmentum. After these injections two small clusters of EB-labeled cells and a larger number of more uniformly distributed DP-positive cells occurred in the core of GPi. The cell clusters were similar in size and location to those observed after VA/VL-CM/Pf injection, but contained only a minority of double-labeled neurons. The distribution of non-pallidal cells projecting to CM/Pf complex was similar to that reported above, whereas retrogradely labeled cells resulting from TPP injection were disclosed in the paraventricular hypothalamic nucleus, the central amygdaloid nucleus, the preoptico-hypothalamic complex, the lateral habenula, the ventral tegmental area, and the SNr where some double-labeled cells were present. In a third series of experiments DP alone was injected into the entire substantia nigra (SN), involving both pars compacta and pars reticulata. The SN injection produced retrograde cell labeling in numerous structures such as the striatum, the rostral intralaminar nuclei, the subthalamic nucleus, the TPP area (bilaterally), the dorsal raphe nucleus and the locus coeruleus. At pallidal levels, a moderate number of DP-labeled cells occurred within the dorsal half of the external segment of the globus pallidus (GPe), whereas the GPi was virtually devoid of labeled neurons. The GPi appeared nevertheless surrounded ventromedially by numerous large-sized DP-positive cells belonging to the substantia innominata.(ABSTRACT TRUNCATED AT 400 WORDS)     

A direct neuronal projection from the entopeduncular nucleus to the globus pallidus. A PHA-L anterograde tracing study in the rat

The efferent projections of the rat entopeduncular nucleus were examined by use of the anterogradely transported lectin Phaseolus vulgaris-leucoagglutinin (PHA-L). Injections of PHA-L into different parts of the entopeduncular nucleus resulted in a moderate number of labeled nerve fibers in the ipsilateral globus pallidus. The fibers displayed a heterogeneous morphology: some were of small caliber with few delicate varicosities, others were of medium caliber with several more bulbous nerve terminals. Restricted injections in the dorsal and ventral parts of the entopeduncular nucleus, respectively, showed that the dorsal part of the entopeduncular nucleus projects to the dorsal and rostral parts of the dorsal pallidum and the ventral part to the ventral and caudal parts.     

The distribution and origin of glutamate decarboxylase and choline acetyltransferase in ventral pallidum and other basal forebrain regions.

The distribution of choline acetyltransferase (ChAT) and glutamate decarboxylase (GAD), and the histochemical reaction for acetylcholinesterase have been studied in the basal forebrain and globus pallidus of unoperated rats and in rats with an electrolytic lesion of the nucleus accumbens. ChAT was highly concentrated in the substriatal region, the neostriatum and the lateral part of the rostral substantia innominata. The strongest intensity of staining for acetylcholinesterase was found in the substriatal grey and the neostriatum. Very high GAD activity was found in the substantia innominata, being even slightly higher than that in the pars reticulata of the substantia nigra. The lateral preoptic area, the bed nucleus of the stria terminalis and the globus pallidus also showed high activity of GAD. After lesions of the nucleus accumbens the activity of GAD decreased significantly in the substantia innominata and in a restricted part of the rostroventral globus pallidus, but not in the other regions studied. ChAT activity and acetylcholinesterase staining were unaffected in all regions. The results indicate that a dense GABAergic projection originates in the nucleus accumbens and terminates in the rostral substantia innominata and rostroventral part of the globus pallidus. The study gives neurochemical support to the suggestion that nucleus accumbens may be regarded as a ventral part of the neostriatum and that the rostral substantia innominata may be regarded as a ventral part of the globus pallidus.     

Excitatory amino acid receptor regulation after subthalamic nucleus lesions in the rat

The subthalamic nucleus plays a pivotal role in the regulation of basal ganglia output. Recent electrophysiologic, lesion and immunocytochemical studies suggest that the subthalamic nucleus uses an excitatory amino acid as a neurotransmitter. After complete ablation of the subthalamic nucleus, we have examined the NMDA, AMPA, kainate and metabotropic subtypes of excitatory amino acid receptors in two major subthalamic projection areas (globus pallidus and substantia nigra pars reticulata) with quantitative autoradiography. Two weeks after ablation, binding sites for [³H]AMPA and [³H]kainate increased in substantia nigra pars reticulata ipsilateral to the lesion. In globus pallidus on the lesioned side, [³H]glutamate binding to the NMDA recognition site decreased. The results suggest that glutamate receptors regulate after interruption of subthalamic nucleus output.