Design, Fabrication, Simulation and Characterization of a Novel Dual-Sided Microelectrode Array for Deep Brain Recording and Stimulation.

In this paper, a novel dual-sided microelectrode array is specially designed and fabricated for a rat Parkinson's disease (PD) model to study the mechanisms of deep brain stimulation (DBS). The fabricated microelectrode array can stimulate the subthalamic nucleus and simultaneously record electrophysiological information from multiple nuclei of the basal ganglia system. The fabricated microelectrode array has a long shaft of 9 mm and each planar surface is equipped with three stimulating sites (diameter of 100 µm), seven electrophysiological recording sites (diameter of 20 µm) and four sites with diameter of 50 µm used for neurotransmitter measurements in future work. The performances of the fabricated microelectrode array were characterized by scanning electron microscopy (SEM), electrochemical impedance spectroscopy (EIS) and cyclic voltammetry. In addition, the stimulating effects of the fabricated microelectrode were evaluated by finite element modeling (FEM). Preliminary animal experiments demonstrated that the designed microelectrode arrays can record spontaneous discharge signals from the striatum, the subthalamic nucleus and the globus pallidus interna. The designed and fabricated microelectrode arrays provide a powerful research tool for studying the mechanisms of DBS in rat PD models.

Quantitative and ultrastructural study of serotonin innervation of the globus pallidus in squirrel monkeys.

The present immunohistochemical study was aimed at characterizing the serotonin (5-HT) innervation of the internal (GPi) and external (GPe) pallidal segments in the squirrel monkey (Saimiri sciureus) with an antibody against the 5-HT transporter (SERT). At the light microscopic level, unbiased counts of SERT+ axon varicosities showed that the density of innervation is similar in the GPi (0.57 ± 0.03 × 10(6)  varicosities/mm(3) of tissue) and the GPe (0.60 ± 0.04 × 10(6) ), with the anterior half of both segments being more densely innervated than the posterior half. Dorsoventral and mediolateral decreasing gradients of SERT varicosities occur in both pallidal segments, but are statistically significant only in the GPi. The neuronal density being significantly greater in the GPe (3.41 ± 0.23 × 10(3)  neurons/mm(3) ) than in the GPi (2.90 ± 0.11 × 103), the number of 5-HT axon varicosities per pallidal neuron was found to be superior in the GPi (201 ± 27) than in the GPe (156 ± 26). At the electron microscopic level, SERT+ axon varicosities are comparable in size and vesicular content in GPi and GPe, where they establish mainly asynaptic contacts with unlabeled profiles. Less than 25% of SERT+ varicosities display a synaptic specialization, which is of the symmetrical or asymmetrical type and occurs exclusively on pallidal dendrites. No SERT+ axo-axonic synapses are present, suggesting that 5-HT exerts its well-established modulatory action upon various pallidal afferents mainly through diffuse transmission, whereas its direct control of pallidal neurons results from both volumic and synaptic release of the transmitter.

[Changes of the structural organization of the nucleus raphe pallidus after the decrease of endogenous serotonin level in the prenatal period of development in rats].

The role of serotonin in the nucleus raphe pallidus (NRP) development and the dynamics of its serotonin-producing neurons were studied during various time points of the postnatal period in normal Wistar rats and in animals developing prenatally under the conditions of serotonin deficiency. It was shown that NRP contained 2 populations of serotoninergic neurons with different morphological characteristics. At the initial stages of postnatal development (Day 5) serotonin-producing neurons included only large neurons, while the synthetic activity of small neurons appeared later (by Day 10). With age, under normal conditions,the size of large neurons and their number were increased which is indicative of continuing process of differentiation and/or functional load augmentation. The size and number of small neurons were practically unchanged with age. Serotonin deficiency during prenatal development lead to the disturbance of NRP structural organization. In comparison with the control animals, the size and the number of serotonin-producing neurons of both populations was decreased, their size remained unchanged with the age. Part of the neurons underwent degeneration, resulting in the reduction of their numbers. The damage observed may change the serotoninergic innervation of the medullary nuclei, responsible for the cardiorespiratory the control, thus causing the disturbances of cardio-vascular and respiratory systems.

Regulation of archicortical arealization by the transcription factor Zbtb20.

The molecular mechanisms of regionalization of the medial pallium (MP), the anlage of the hippocampus, and transitional (cingulate and retrosplenial) cortices are largely unknown. Previous analyses have outlined an important role of the transcription factor (TF) Zbtb20 for hippocampal CA1 field specification (Nielsen et al. (2007) Development 134:1133-1140; Nielsen et al. (2010) Cereb Cortex 20:1904-1914; Xie et al. (2010) Proc Natl Acad Sci USA 107:6510-6515). Here, we present novel data showing that Zbtb20 exhibits a ventral(high)-to-dorsal(low) gradient of expression in MP progenitors as well as an expression in postmitotic cells at the transitional cortex/neocortex border. Our detailed pattern analysis revealed that in Zbtb20 loss-of-function the molecular borders between neocortical, transitional, and hippocampal fields are progressively shifted ventrally, leading to an ectopic positioning of all dorsal fields into the neighboring ventrally located areas. Thus, in addition to its known importance for the specification of the hippocampal CA1 sector, the graded expression of TF Zbtb20 in ventricular zone of MP appears to translate early positional information for establishment of all developing MP fields. Our data also suggest that the signaling factor Wnt3a is a putative molecular partner of TF Zbtb20 in this patterning process.

Ultrastructural localization and function of dopamine D1-like receptors in the substantia nigra pars reticulata and the internal segment of the globus pallidus of parkinsonian monkeys.

The motor symptoms of Parkinson's disease (PD) are commonly attributed to striatal dopamine loss, but reduced dopamine innervation of basal ganglia output nuclei, the internal globus pallidus (GPi) and the substantia nigra pars reticulata (SNr) may also contribute to symptoms and signs of PD. Both structures express dopamine D1 and D5 receptors under normal conditions, and we have recently demonstrated that their local activation reduces neuronal discharge rates and enhances bursts and oscillatory activity in both nuclei of normal monkeys [M.A. Kliem et al. (2007)J. Neurophysiol., 89, 1489-1500]. Here, we determined the ultrastructural localization and function of D1-like receptors in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated parkinsonian monkeys. In both normal and MPTP-treated monkeys, most of the D1 and D5 receptor immunoreactivity was associated with unmyelinated axons, but we also found significant postsynaptic D5 receptor immunostaining in dendrites of GPi and SNr neurons. A significant proportion of axonal D1 immunostaining was bound to the plasma membrane in both normal and MPTP-treated monkeys. Local microinjections of the D1/D5 receptor agonist SKF82958 significantly reduced discharge rates in GPi and SNr neurons, while they increased burst firing and oscillatory activity in the 3-15-Hz band in SNr, but not in GPi, of parkinsonian monkeys. Together with our recent findings from normal monkeys, these data provide evidence that functional D1/D5 receptors are expressed in GPi and SNr in both normal and parkinsonian states, and that their activation by endogenous dopamine (under normal conditions) or dopamine receptor agonists (in parkinsonism) may regulate basal ganglia outflow.

Deep brain stimulation electrode used for radiofrequency lesion of the globus pallidus internus in dystonia.

BACKGROUND: There have recently been increasing case reports in the literature of deep brain stimulation (DBS) electrodes used for lesioning with satisfactory clinical success in the treatment of Parkinson disease and tremor. METHODS: After preliminary experiments of radiofrequency (RF) lesioning with a quadripolar DBS lead, a paediatric case of generalized primary dystonia was treated by RF lesioning of the globus pallidus internus (Gpi) with an electrode previously used for chronic stimulation. In order to study electrode damage related to the RF procedure, an electron microscopy study (SEM) at different magnifications (x40 and x300) was performed. RESULTS: Nine months after the unilateral pallidotomy, the patient had a good and stable control of dystonia. The MR study showed a T(1)-weighted hyperintensity signal corresponding to the electrode contacts used for lesions. The SEM scans of the DBS electrode used for RF lesioning did not show alterations of the ultrastructure. CONCLUSIONS: The RF lesioning technique by a DBS electrode allows small and staged lesions and could also be performed in a bilateral target. The versatility, efficacy, safety and low cost of the device make this approach suitable in selected cases.

Autonomous initiation and propagation of action potentials in neurons of the subthalamic nucleus.

The activity of the subthalamic nucleus (STN) is intimately related to movement and is generated, in part, by voltage-dependent Na(+) (Na(v)) channels that drive autonomous firing. In order to determine the principles underlying the initiation and propagation of action potentials in STN neurons, 2-photon laser scanning microscopy was used to guide tight-seal whole-cell somatic and loose-seal cell-attached axonal/dendritic patch-clamp recordings and compartment-selective ion channel manipulation in rat brain slices. Action potentials were first detected in a region that corresponded most closely to the unmyelinated axon initial segment, as defined by Golgi and ankyrin G labelling. Following initiation, action potentials propagated reliably into axonal and somatodendritic compartments with conduction velocities of approximately 5 m s(-1) and approximately 0.7 m s(-1), respectively. Action potentials generated by neurons with axons truncated within or beyond the axon initial segment were not significantly different. However, axon initial segment and somatic but not dendritic or more distal axonal application of low [Na(+)] ACSF or the selective Na(v) channel blocker tetrodotoxin consistently depolarized action potential threshold. Finally, somatodendritic but not axonal application of GABA evoked large, rapid inhibitory currents in concordance with electron microscopic analyses, which revealed that the somatodendritic compartment was the principal target of putative inhibitory inputs. Together the data are consistent with the conclusions that in STN neurons the axon initial segment and soma express an excess of Na(v) channels for the generation of autonomous activity, while synaptic activation of somatodendritic GABA(A) receptors regulates the axonal initiation of action potentials.

Differential localization of the GluR1 and GluR2 subunits of the AMPA-type glutamate receptor among striatal neuron types in rats.

Differences among the various striatal projection neuron and interneuron types in cortical input, function, and vulnerability to degenerative insults may be related to differences among them in AMPA-type glutamate receptor abundance and subunit configuration. We therefore used immunolabeling to assess the frequency and abundance of GluR1 and GluR2, the most common AMPA subunits in striatum, in the main striatal neuron types. All neurons projecting to the external pallidum (GPe), internal pallidum (GPi) or substantia nigra, as identified by retrograde labeling, possessed perikaryal GluR2, while GluR1 was more common in striato-GPe than striato-GPi perikarya. The frequency and intensity of immunostaining indicated the rank order of their perikaryal GluR1:GluR2 ratio to be striato-GPe>striatonigral>striato-GPi. Ultrastructural studies suggested a differential localization of GluR1 and GluR2 to striatal projection neuron dendritic spines as well, with GluR1 seemingly more common in striato-GPe spines and GluR2 more common in striato-GPi and/or striatonigral spines. Comparisons among projection neurons and interneurons revealed GluR1 to be most common and abundant in parvalbuminergic interneurons, and GluR2 most common and abundant in projection neurons, with the rank order for the GluR1:GluR2 ratio being parvalbuminergic interneurons>calretinergic interneurons>cholinergic interneurons>projection neurons>somatostatinergic interneurons. Striosomal projection neurons had a higher GluR1:GluR2 ratio than did matrix projection neurons. The abundance of both GluR1 and GluR2 in striatal parvalbuminergic interneurons and projection neurons is consistent with their prominent cortical input and susceptibility to excitotoxic insult, while differences in GluR1:GluR2 ratio among projection neurons are likely to yield differences in Ca(2+) permeability, desensitization, and single channel current, which may contribute to differences among them in plasticity, synaptic integration, and excitotoxic vulnerability. The apparent association of the GluR1 subunit with synaptic plasticity, in particular, suggests striato-GPe neuron spines as a particular site of corticostriatal synaptic plasticity, presumably associated with motor learning.

Chemical anatomy of pallidal afferents in primates.

Neurons of the globus pallidus receive massive inputs from the striatum and the subthalamic nucleus, but their activity, as well as those of their striatal and subthalamic inputs, are modulated by brainstem afferents. These include serotonin (5-HT) projections from the dorsal raphe nucleus, cholinergic (ACh) inputs from the pedunculopontine tegmental nucleus, and dopamine (DA) afferents from the substantia nigra pars compacta. This review summarizes our recent findings on the distribution, quantitative and ultrastructural aspects of pallidal 5-HT, ACh and DA innervations. These results have led to the elaboration of a new model of the pallidal neuron based on a precise knowledge of the hierarchy and chemical features of the various synaptic inputs. The dense 5-HT, ACh and DA innervations disclosed in the associative and limbic pallidal territories suggest that these brainstem inputs contribute principally to the planification of motor behaviors and the regulation of attention and mood. Although 5-HT, ACh and DA inputs were found to modulate pallidal neurons and their afferents mainly through asynaptic (volume) transmission, genuine synaptic contacts occur between these chemospecific axon varicosities and pallidal dendrites, revealing that these brainstem projections have a direct access to pallidal neurons, in addition to their indirect input through the striatum and subthalamic nucleus. Altogether, these findings reveal that the brainstem 5-HT, ACh and DA pallidal afferents act in concert with the more robust GABAergic inhibitory striatopallidal and glutamatergic excitatory subthalamopallidal inputs. We hypothesize that a fragile equilibrium between forebrain and brainstem pallidal afferents plays a key role in the functional organization of the primate basal ganglia, in both health and disease.

Ultrastructural localization of nitrotyrosine within the caudate-putamen nucleus and the globus pallidus of normal rat brain.

Nitration of protein tyrosine residues by nitric oxide (NO)-derived reactive species results in the production of stable nitrotyrosine (NT) moieties that are immunochemically detectable in many regions of normal brain and enriched in those areas containing constitutive nitric oxide synthase (cNOS). These include the caudate-putamen nucleus (CPN) and the globus pallidus, which receives major inhibitory input from the CPN. To determine the functional sites for NT production in these critical motor nuclei, we examined the electron microscopic immunocytochemical localization of NT and cNOS in rat brain. In the CPN, NT was localized to the somata and dendrites of cNOS-containing interneurons and spiny neurons, some of which received input from cNOS-labeled terminals. The NT immunoreactivity was most prevalent on outer mitochondrial membranes and nearby segments of the plasma membranes in dendrites and within asymmetric synapses on dendritic spines. In the CPN and globus pallidus, there was also a prominent labeling of NT in astrocytic processes, small axons, and tubulovesicles and/or synaptic vesicles in axon terminals. These terminals formed mainly asymmetric synapses in the CPN and inhibitory-type synapses in the globus pallidus where they often apposed cNOS-containing terminals that also formed asymmetric, excitatory-type synapses. Our results suggest that NT is generated by mechanisms requiring the dual actions of excitatory transmitters and NO derived either from interneurons in the CPN or from excitatory afferents in the globus pallidus. The findings also implicate NT in the physiological actions of NO within the striatal circuitry and, particularly, in striatopallidal neurons severely affected in Huntington's disease.

Synaptic and extrasynaptic GABA-A and GABA-B receptors in the globus pallidus: an electron microscopic immunogold analysis in monkeys.

GABA-A and GABA-B receptors mediate differential effects in the CNS. To better understand the role of these receptors in regulating pallidal functions, we compared their subcellular and subsynaptic localization in the external and internal segments of the globus pallidus (GPe and GPi) in monkeys, using pre- and post-embedding immunocytochemistry with antibodies against GABA-A (alpha1, beta2/3 subunits) and GABA-BR1 receptor subtype. Our results demonstrate that GABA-A and GABA-B receptors display a differential pattern of subcellular and subsynaptic localization in both segments of the globus pallidus. The majority of GABA-BR1 immunolabeling is intracellular, whereas immunoreactivity for GABA-A receptor subunits is mostly bound to the plasma membrane. A significant proportion of both GABA-BR1 and GABA-A receptor immunolabeling is extrasynaptic, but GABA-A receptor subunits also aggregate in the main body of putative GABAergic symmetric synapses established by striatal- and pallidal-like terminals. GABA-BR1 immunoreactivity is expressed presynaptically in putative glutamatergic terminals, while GABA-A alpha1 and beta2/3 receptor subunits are exclusively post-synaptic and often coexist at individual symmetric synapses in both GPe and GPi. In conclusion, our findings corroborate the concept that ionotropic and metabotropic GABA receptors are located to subserve different effects in pallidal neurons. Although the aggregation of GABA-A receptors at symmetric synapses is consistent with their role in fast inhibitory synaptic transmission, the extrasynaptic distribution of both GABA-A and GABA-B receptors provides a substrate for complex modulatory functions that rely predominantly on the spillover of GABA.

Electron microscopy of tissue adherent to explanted electrodes in dystonia and Parkinson's disease.

Deep brain stimulation (DBS) is used to treat a variety of severe medically intractable movement disorders, including Parkinson's disease, tremor and dystonia. There have been few studies examining the effect of chronic DBS on the brains of Parkinson's disease patients. Most of these post mortem studies concluded that chronic DBS caused mild gliosis around the lead track and did not damage brain tissue. There have been no similar histopathological studies on brains from dystonic patients who have undergone DBS. In this study, our objective was to discover whether tissue would be attached to DBS electrodes removed from patients for routine clinical reasons. We hoped that by examining explanted DBS electrodes using scanning (SEM) and/or transmission (TEM) electron microscopy we might visualize any attached tissue and thus understand the electrode-human brain tissue interaction more accurately. Initially, SEM was performed on one control DBS electrode that had not been implanted. Then 21 (one subthalamic nucleus and 20 globus pallidus internus) explanted DBS electrodes were prepared, after fixation in 3% glutaraldehyde, for SEM (n = 9) or TEM (n = 10), or both (n = 2), according to departmental protocol. The electrodes were sourced from two patients with Parkinson's disease, one with myoclonic dystonia, two with cervical dystonia and five with primary generalized dystonia, and had been in situ for 11 and 31 months (Parkinson's disease), 16 months (myoclonic dystonia), 14 and 24 months (cervical dystonia) and 3-24 months (primary generalized dystonia). Our results showed that a foreign body multinucleate giant cell-type reaction was present in all TEM samples and in SEM samples, prewashed to remove surface blood and fibrin, regardless of the diagnosis. Some of the giant cells were >100 microm in diameter and might have originated from either fusion of parenchymal microglia, resident perivascular macrophage precursors and/or monocytes/macrophages invading from the blood stream. The presence of mononuclear macrophages containing lysosomes and sometimes having conspicuous filopodia was detected by TEM. Both types of cell contained highly electron-dense inclusions, which probably represent phagocytosed material. Similar material, the exact nature of which is unknown, was also seen in the vicinity of these cells. This reaction was present irrespective of the duration of implantation and may be a response to the polyurethane component of the electrodes' surface coat. These findings may be relevant to our understanding of the time course of the clinical response to DBS in Parkinson's disease and various forms of dystonia, as well as contributing to the design characteristics of future DBS electrodes.

Cerebral sponginess and GM3 gangliosidosis; ultrastructure and probable pathogenesis.

Extensive multifocal vacuolation of the cerebral hemispheres, brain stem, cerebellum, optic nerves and spinal cord were demonstrated in a 3 1/2 month-old infant. This co-existed with marked increases in cerebral and hepatic ganglioside GM3 (hematoside), absence of its higher homologues (GM1 and GM2) and absence of tissue N-acetylgalactosaminyl transferase. Ultrastructurally, there are major abnormalities in myelin and astroglia. The absence of identifiable "storage" material is believed to correlate with an enzymatic defect involved in ganglioside anabolism. A familial occurrence of this disorder is strongly suggested by the clinical history.

Electron microscopic analysis of the synaptic organization of the globus pallidus in the cat.

The synaptic organization of the feline globus pallidus (GP) was studied electron microscopically. The axon terminals were classified into five types on the basis of the size and shape of synaptic vesicles and the type of postsynaptic differentiations. Type I and II axon terminals were characterized by large, pleomorphic vesicles and by a symmetric and an asymmetric synaptic contact, respectively. Type III and IV axon terminals were characterized by small, pleomorphic vesicles and by a symmetric and an asymmetric synaptic contact, respectively. Type V axon terminals were characterized by elongated and large round vesicles and by a symmetric synaptic contact. The origins of these terminals were determined by a combined degeneration and HRP tracing technique. Following injections of HRP into the caudate nucleus or electrolytic lesions in this nucleus, type I terminals were anterogradely labeled with HRP or degenerated, respectively. Although type III, IV, and V terminals were labeled with HRP after HRP injections into the subthalamic nuclear region, only type IV and V terminals degenerated after lesions in that area. Type II terminals did not show any alterations following such treatment. These results suggest that type I terminals originate from the caudate nucleus, that type IV and V terminals come from the subthalamic nucleus or caudal to it, and that type III terminals are the terminals of intrinsic axon collaterals of GP neurons which send axons to the subthalamic nucleus. Occasionally convergence of different kinds of axon terminals on the same GP neuron was also observed. These terminals originated from the caudate nucleus and the subthalamic nucleus or caudal to it.(ABSTRACT TRUNCATED AT 250 WORDS)

The radial fibers in the globus pallidus.

In our Golgi collection of adult monkey brains the striatal efferents, i.e., the radial fibers in the globus pallidus and the "comb" bundle fibers in the internal capsule and in the cerebral peduncle, are well impregnated in the horizontally sectioned brain and in a sagittal sectioned brain. Since collaterals emerging from radial fibers are seen only in the horizontal series and not in the saggittal series, the interpretation is that they proceed anteriorly and posteriorly only, following the curvature of the pallidal segments, and do not run superiorly or inferiorly as they emerge. Although radial fibers emitting collaterals in the lateral segment and in the medial segment of the globus pallidus have been observed, it has not been possible to observe the same radial fiber emitting collaterals in both pallidal segments and the prospects of ever doing so are not good. The radial fibers converging in the globus pallidus pursue many radii and there is little coincidence between the plane of section and the planes in which they travel. At most only severed radial fiber segments 100-150 microns in length can be found in the horizontal sections needed to observe the collaterals. Moreover, sagittal sections trodorsally, as they pass through the internal medullary lamina to enter the medial segment of the globus pallidus. The radial fibers in the medial segment of the globus pallidus are continuous with the "comb" bundle fibers and appear to be thinner than the radial fibers in the lateral segment of the globus pallidus. It is not proved; nonetheless, the view expressed here is that the radial fibers are thinner in the medial segment of the globus pallidus because they may be the same fibers that gave off collaterals in the lateral segment of the globus pallidus. This is discussed in the light of the electrophysiological disclosure of Yoshida et al. ('71, '72) that caudatopallidal fibers are collaterals off caudatonigral fibers. The afferent plexuses of fine, "bouton en passage" fibers, which completely ensheath the long radiating dendrites in the globus pallidus (Fox et al., '66) are well impregnated in the horizontal series. Obviously, they are formed by a number of ultimate branches converging from the collateral brances of a number of different radial fibers. The divergence, too, in this system must be considerable; however, its true extent can only be surmised from the several radial fibers and radial fiber collaterals seen in the incompletely impregnanted Golgi section. Continued.

Ultrastructural analyses of afferent terminals in the subthalamic nucleus of the cat with a combined degeneration and horseradish peroxidase tracing method.

The synaptic organization of the feline subthalamic nucleus (STN) was studied electron microscopically. Following horseradish peroxidase (HRP) injections into the globus pallidus (GP) and electrolytic lesions of the nucleus tegmenti pedunculopontinus pars compacta (TCP) in the same cat, both degenerating and HRP-labeled terminals were found in the STN with abundant retrogradely HRP-labeled neurons. Degenerating terminals of TPC origin were medium-sized and characterized by asymmetric synaptic contacts. They synapsed widely on the STN neuronal surface, including the somata, dendrites of varying dimensions, dendritic spines and vesicle-containing processes. They formed 25.1%, 65.1%, 4.7%, and 4.7%, respectively, of all TPC efferent terminals. Some of the postsynaptic components were labeled with HRP. Occasionally both degenerating terminals and HRP-labeled terminals were in synaptic contact with the same HRP-labeled neuron: therefore, afferents of TPC and GP converge on the same STN projection neuron. In order to discover the origin of these HRP-labeled terminals, a mixed solution containing HRP and kainic acid was injected into the GP. Numerous degenerating terminals were observed to synapse with HRP-labeled STN neurons, but no HRP-labeled terminal was observed. These degenerating terminals were similar in appearance to the above-mentioned HRP-labeled terminals. They were characterized by their relatively large size, predominantly symmetric synapses, and preferential distribution on the somata and large or medium-sized dendrites. They formed 39.6%, 20.1%, and 31.1%, respectively, of all GP efferent terminals. Therefore, it became clear that both the HRP-labeled terminals of the first experiment and the degenerating terminals of the second experiment originated from the GP. Following surgical ablations of the primary sensorimotor cortex (Cx), some axon terminals in the STN showed degeneration. These degenerating terminals were small and formed asymmetric synapses mainly with dendritic spines, small dendrites and vesicle-containing processes. They formed 48.0%, 28.0%, and 12.0%, respectively, of all Cx efferent terminals. These electron microscopic investigations reveal the convergence of TPC and GP afferents and that STN projection neurons relay the TPC and pallidal inputs directly to the GP.

Synaptic innervation of neurones in the internal pallidal segment by the subthalamic nucleus and the external pallidum in monkeys.

In order to better understand the way by which the subthalamic nucleus interacts with the globus pallidus to control the output of the basal ganglia, we carried out a series of experiments to investigate the pattern of synaptic innervation of the pallidal neurones by the subthalamic terminals in the squirrel monkey. To address this problem we used the anterograde transport of biocytin. Following injections of biocytin in the subthalamic nucleus, rich plexuses of labelled fibres and varicosities formed bands that lay along the medullary lamina in both segments of the ipsilateral pallidum. At the electron microscopic level, two populations of biocytin-containing terminals were identified in the internal pallidum (GPi). A first group of small to medium-sized terminals (type 1; mean cross-sectional area +/- S.D. = 0.41 +/- 0.04 microns 2) contained round vesicles and formed asymmetric synapses with dendritic shafts (95%) of mixed sizes (maximum diameter ranging from 0.3 to 4.0 microns) and spine-like structures (5%). The second group of terminals (type 2) contained pleiomorphic vesicles, had a larger cross-sectional area (mean +/- S.D. = 0.9 +/- 0.4 micron 2) and formed symmetric synapses predominantly with perikarya (41%) and large dendrites (57%). In some cases, the two types of terminals converged at the level of single GPi neurones. Postembedding immunogold method revealed that the type 2 terminals displayed gamma-aminobutyric acid (GABA) immunoreactivity, whereas the type 1 terminals did not. In the external pallidum (GPe), injections in the subthalamic nucleus labelled both type 1 or type 2 terminals. However, the labelled type 2 boutons were much less abundant in GPe than in GPi. The presence of biocytin-labelled perikarya in GPe and the fact that the type 2 terminals displayed GABA immunoreactivity led us to suspect that these terminals were derived from axons of GPe neurones. In agreement with this hypothesis, injections of Phaseolus vulgaris-leucoagglutinin (PHA-L) in GPe labelled terminals in GPi that displayed the morphological features and a pattern of synaptic organization similar to the type 2 terminals. In conclusion, the results of our study demonstrate that the subthalamopallidal terminals form asymmetric synapses that are distributed along the dendritic tree of GPe and GPi neurones. In contrast, the GPe projection to GPi gives rise to large GABA-containing terminals that form symmetric synapses predominantly with the proximal region of pallidal neurones.(ABSTRACT TRUNCATED AT 400 WORDS)

The neurons in the primate subthalamic nucleus: a Golgi and electron microscopic study.

In Golgi preparations of the adult monkey (Macaca mulatta) local interneurons and two varieties of principal neurons, radiating and elongated fusiform, are found in the subthalamic nucleus. The cell bodies of the radiating neurons have a few delicate, somatic spines some of which are occasionally bilobed and trilobed. Five to eight dendritic trunks give rise to branching, tapering dendrites, which may extend for over 400 microns. These dendrites are much thinner than the dendrites in the globus pallidus and the substantia nigra. Some neurons have many and some neurons have few dendritic spines. When numerous the dendritic spines are concentrated on the dendritic trunks and proximal dendrites. The relatively few elongated fusiform neurons are found not only in the capsule but also in the center of the nucleus. Most dendrites emerge from the opposite poles of their smooth surfaced cell bodies. They have a few dendritic spines. Some of these dendrites extend for more than 750 microsn. In 1-micron thick plastic sections lipofuscin granules are present in some but not all principal neuron cell bodies of the monkey (Macaca mulatta); but these granules are present in all principal neuron cell bodies of the pig-tail monkey (Macaca nemestrina) and of the squirrel monkey (Saimiri sciureus). The local interneurons have small cell bodies and a few relatively long undulating dendrites. The dendrites have bulbous dendritic appendages of varying complexity and beaded axon-like processes. The dendritic appendages and axon-like processes are more numerous distally and on the distal ends of the dendrites they form complex entanglements. Axons coming from the cell body have not been observed. The cell bodies of the local interneurons are identified in cresyl violet stained sections of the monkey (Macaca mulatta), in 1-micron thick plastic sections and electron micrographs of the squirrel monkey (Saimiri sciureus). They have relatively large nuclei surrounded by a thin rim of cytoplasm rich in polyribosomes.

Early postnatal development of the monkey globus pallidus: a Golgi and electron microscopic study.

The globus pallidus of 20 monkeys ranging in age from newborn to 4 months was examined in Golgi-impregnated material and ultrastructurally. There was no discernible difference between the lateral and medial segments of the structure. At the light microscope level, all neuronal types described in the adult are found at birth. The most common, the large fusifom cell, shows initial signs of immaturity such as blunt protrusions and dendritic dilations at bifurcation points, as well as growth cones, filopodia, and filiform processes. These features become more rare with age, and by 4 months, the neurons appear fully mature save for the terminal dendritic arborizations which are still underdeveloped. From the earliest ages examined, the large globular cells and the interneurons are more mature than the previous type. The afferent radial fibers of striatal origin are observed from birth, but they are grouped in bundles only after 8 weeks. The density of their climbing branches increases over time, reaching a mature appearance by 16 weeks. Afferents entering from the ventral surface do not yet show clusters of varicosities at 2 weeks. At the latter age, plexuses of fine beaded fibers are already seen covering large extensions of the nucleus. The fine structure correlates well with the Golgi material. The basic features of the neuropil are present at birth, albeit with immature characteristics such as the incomplete covering of the dendrites with axonal boutons and the low level of myelination of the radial fibers. Growth cones and profiles with signs of degeneration are observed during the first month. In the early ages examined, most dendrites show large varicosities and protrusions, some of which are spinelike and can be postsynaptic to multiple terminals. The other dendritic type, with only an occasional axodendritic synapse, is also seen from birth and increases in size as a function of time. The type I axonal boutons, of probable striatal origin, are quite immature at birth, and their characteristic interdigitations are seen only after the first week. The type II, III, IV, and V boutons appear mature at all ages examined but crest synapses formed by the type III terminals are observed in the later stages of the study. Finally, postsynaptic vesicle-containing profiles are present at 4 weeks, but triadic synaptic arrangements are formed only by 16 weeks.(ABSTRACT TRUNCATED AT 400 WORDS)

Topographic intermingling of striatonigral and striatopallidal neurons in the rhesus monkey.

The topography and interrelationship of striatofugal neurons have been examined using a double retrograde tracing paradigm to label striatopallidal and striatonigral neurons in the same neostriatum. The rostral globus pallidus and the rostral substantia nigra in the same hemisphere were injected simultaneously with fluorescent tracers in three monkeys. In addition, the caudal globus pallidus and the caudal substantia nigra were injected separately in a fourth and fifth monkey with a fluorescent dye and wheat germ agglutinin-horseradish peroxidase (WGA-HRP), respectively. Digitized plots of fluorescent dye-labeled neurons revealed that large numbers of striatonigral projection neurons lie within both neostriatal nuclei, i.e., the caudate and putamen. Similarly, neurons innervating the globus pallidus were found in both caudate and putamen. The distribution of retrogradely labeled neurons observed was consistent with the topography of striatofugal projections that has been described previously, i.e., the rostrocaudal and mediolateral axes of the neostriatum are preserved in the striatopallidal and striatonigral projections (e.g., Szabo, '62, '67, '70, '72) and the dorsoventral axis is inverted in the projection of the neostriatum onto the nigra but not in the striatopallidal projection (Nauta and Domesick, '79; Gerfen, '85). Analysis of cases in which striatonigral and striatopallidal neurons were present in large numbers within the same region of the neostriatum disclosed that the two populations are intermingled such that small clusters of striatopallidal neurons are surrounded by striatonigral neurons and vice versa. The clustered arrangement of striatofugal neurons observed in the fluorescent cases was unambiguous in a case in which HRP was injected into the caudal substantia nigra. In this case, both anterogradely labeled terminals and retrogradely labeled neurons exhibited a striking, compartmental-like distribution in the posterior putamen. Our observations indicate that the matrix compartment of the neostriatum is comprised of a patchwork of interposed clusters of nigral and pallidal efferent neurons. We hypothesize that these clusters of efferent neurons may direct interdigitated cortical inputs into distinct nigro- and pallido-thalamic pathways. In view of the parallel nature of processing throughout the basal ganglia, it appears that convergence of these segregated nigral and pallidal loops must occur at the cortical level where prefrontal and premotor targets of the basal ganglia are interconnected via corticocortical projections (Selemon and Goldman-Rakic, '88).