Narcolepsy patients have antibodies that stain distinct cell populations in rat brain and influence sleep patterns.

Narcolepsy is a chronic sleep disorder, likely with an autoimmune component. During 2009 and 2010, a link between A(H1N1)pdm09 Pandemrix vaccination and onset of narcolepsy was suggested in Scandinavia. In this study, we searched for autoantibodies related to narcolepsy using a neuroanatomical array: rat brain sections were processed for immunohistochemistry/double labeling using patient sera/cerebrospinal fluid as primary antibodies. Sera from 89 narcoleptic patients, 52 patients with other sleep-related disorders (OSRDs), and 137 healthy controls were examined. Three distinct patterns of immunoreactivity were of particular interest: pattern A, hypothalamic melanin-concentrating hormone and proopiomelanocortin but not hypocretin/orexin neurons; pattern B, GABAergic cortical interneurons; and pattern C, mainly globus pallidus neurons. Altogether, 24 of 89 (27%) narcoleptics exhibited pattern A or B or C. None of the patterns were exclusive for narcolepsy but were also detected in the OSRD group at significantly lower numbers. Also, some healthy controls exhibited these patterns. The antigen of pattern A autoantibodies was identified as the common C-terminal epitope of neuropeptide glutamic acid-isoleucine/α-melanocyte-stimulating hormone (NEI/αMSH) peptides. Passive transfer experiments on rat showed significant effects of pattern A human IgGs on rapid eye movement and slow-wave sleep time parameters in the inactive phase and EEG θ-power in the active phase. We suggest that NEI/αMSH autoantibodies may interfere with the fine regulation of sleep, contributing to the complex pathogenesis of narcolepsy and OSRDs. Also, patterns B and C are potentially interesting, because recent data suggest a relevance of those brain regions/neuron populations in the regulation of sleep/arousal.

Neuroimmune aspects of the pathogenesis of Tourette's syndrome and experience in the use of immunoglobulins in children.

A total of 60 patients aged 6-16 years with tics and Tourette's syndrome were studied. Antibodies to caudate nucleus proteins were assayed by western blot hybridization. Ten patients with Tourette's syndrome were found to have antibodies to caudate nucleus protein. Seven patients with neuroleptic-resistant types of Tourette's syndrome received single transfusions of immunoglobulin preparations, which produced regression of vocal and motor hyperkinesias and improvement in behavior (remission for more than six months). The observation of antibodies to caudate nucleus proteins and the positive effects seen on administration of immunoglobulins to patients with Tourette's syndrome support previous data on the possibility of using immunoglobulin therapy in the treatment of tic-type hyperkinesias and provide evidence of the involvement of autoimmune mechanisms inducing damage to the dopaminergic system of the striatum.

[Neuroimmunological aspects of the pathogenesis of Tourette syndrome and use of immunoglobulins in the treatment of children].

Sixty children, aged from 6 to 16 years, with tics and Tourette syndrome have been studied. Western immunoblotting techniques has been used to evaluate the serum antibodies against the caudate nucleus. These antibodies have been detected in 10 cases. Seven patients with Tourette syndrome who were treated with neuroleptics for a long time without any therapeutical effect received the immunomodulatory therapy with intravenous immunoglobulin. The regression of motor and vocal tics as well as improvement of behavioral symptoms were observed (duration of remission was more than 6 months). These findings and successful immunomodulatory therapy of patients with Tourette syndrome confirm previous reports in the literature and support the idea of immunologically triggered disturbance of the striatal dopaminergic system.

Anti-basal ganglia antibodies in PANDAS.

An autoimmune-mediated mechanism involving molecular mimicry has been proposed for a variety of pediatric movement disorders that occur after a streptococcal infection. In this study, anti-basal ganglia antibodies (ABGA) were measured in 15 children with the diagnosis of pediatric autoimmune neuropsychiatric disorder associated with streptococcal infection (PANDAS) and compared with those in 15 controls. ELISA and Western immunoblotting (WB) methods were used to detect ABGA against supernatant (S1), pellet (P2), and synaptosomal preparations from adult postmortem caudate, putamen, and globus pallidus. ELISA optical density values did not differ between PANDAS patients and controls across all preparations. Immunoblotting identified multiple bands in all subjects with no differences in the number of bands or their total density. Discriminant analysis, used to assess mean binding patterns, showed that PANDAS patients differed from controls only for the caudate S1 fraction (Wilks' lambda = 0.0236, P < 0.0002), with PANDAS-primarily tic subjects providing the greatest discrimination. Among the epitopes contributing to differences between PANDAS and control in the caudate S1 fraction, mean binding to the epitope at 183 kDa was the most different between groups. In conclusion, ELISA measurements do not differentiate between PANDAS and controls, suggesting a lack of major antibody changes in this disorder. Further immunoblot analyses using a caudate supernatant fraction are required to completely exclude the possibility of minor antibody repertoire differences in PANDAS subjects, especially in those who primarily have tics.

A murine model for neuropsychiatric disorders associated with group A beta-hemolytic streptococcal infection.

A syndrome of motoric and neuropsychiatric symptoms comprising various elements, including chorea, hyperactivity, tics, emotional lability, and obsessive-compulsive symptoms, can occur in association with group A beta-hemolytic streptococcal (GABHS) infection. We tested the hypothesis that an immune response to GABHS can result in behavioral abnormalities. Female SJL/J mice were immunized and boosted with a GABHS homogenate in Freund's adjuvant, whereas controls received Freund's adjuvant alone. When sera from GABHS-immunized mice were tested for immunoreactivity to mouse brain, a subset was found to be immunoreactive to several brain regions, including deep cerebellar nuclei (DCN), globus pallidus, and thalamus. GABHS-immunized mice having serum immunoreactivity to DCN also had increased IgG deposits in DCN and exhibited increased rearing behavior in open-field and hole-board tests compared with controls and with GABHS-immunized mice lacking serum anti-DCN antibodies. Rearing and ambulatory behavior were correlated with IgG deposits in the DCN and with serum immunoreactivity to GABHS proteins in Western blot. In addition, serum from a GABHS mouse reacted with normal mouse cerebellum in nondenaturing Western blots and immunoprecipitated C4 complement protein and alpha-2-macroglobulin. These results are consistent with the hypothesis that immune response to GABHS can result in motoric and behavioral disturbances and suggest that anti-GABHS antibodies cross-reactive with brain components may play a role in their pathophysiology.

Pallido-nigral spheroids in nonhuman primates: accumulation of heat shock proteins in astroglial processes.

Alpha-B crystallin, ubiquitin and heat shock protein 27 (hsp27) belong to a class of proteins that are overexpressed in response to pathological conditions associated with increased cellular stress. In the present study, brain sections of old rhesus monkeys ( Macaca mulatta; n=10; mean age, 29.4 years) and baboons ( Papio anubis; n=8; mean age, 18.3 years) were examined for ubiquitin, alpha-B crystallin and hsp27-immunopositive structures. In both species, immunoreactive spheroid-like bodies were found in the globus pallidus and in the substantia nigra, pars reticulata. These structures frequently were associated with abnormally swollen cellular processes. To further clarify the origin of the pallido-nigral spheroids, single- and double-immunostaining was performed for hsp27, alpha-B crystallin and the astroglial marker glial fibrillary acidic protein (GFAP) as well as for neuronal markers against neurofilament and dendritic microtubule-associated protein 2. Confocal microscopic analysis demonstrated that spheroids were localized in swollen astroglial processes, whereas they were not seen in neuronal structures. Thus, pallido-nigral spheroids can be classified as astroglial accumulations of heat shock proteins. Further investigations of these structures may provide information pertinent to our understanding of astroglial heat shock protein inclusions developing in degenerative human brain diseases.

Spatiotemporal expression gradients of the carbohydrate antigen (CD15) (Lewis X) during development of the human basal ganglia.

The developmental expression pattern of the carbohydrate epitope CD15 (Lewis X, Le X) (alpha1-->3-fucosyl-N-acetyl-lactosamine) has been immunocytochemically evaluated in paraffin sections within the human basal ganglia from 10 weeks gestation to three years after birth. At 11 weeks of gestation, CD15 (Le X) positive radial glial cells were located in the anterior and dorsal parts of the lateral ganglionic eminence. Their processes ran from the subventricular zone radially in a highly ordered fashion to the dorsolateral margin of the caudate nucleus and further to the lateral rim of the putamen. At 12 weeks of gestation, strands of CD15 (Le X) material continued to the pial surface, forming a continuous CD15 (Le X) positive borderline separating the accumbens nucleus and olfactory tubercle from the piriform cortex. At 13 weeks of gestation the dorsal putamen was completely CD15 (Le X) immunoreactive along its perimeter and CD15 (Le X) patches, consisting of fine granular material, appeared at the dorsolateral margin of the putamen at this age; while the first CD15 (Le X) patches in the caudate nucleus were observed four weeks later. The matrix compartment of the caudate and dorsal putamen became gradually stained by granular CD15 (Le X) positive material into which CD15 (Le X) immunoreactive somata were embedded. The striking contrast in staining between patch and matrix compartments disappeared shortly after birth. The ventral striatum did not become immunoreactive until the last few weeks before birth. After the formation of CD15 (Le X) positive patches in the striatum (from 12 weeks of gestation), delicate CD15 (Le X) fibres, often accumulated in bundles and related to the striatal patches, became apparent coursing towards the external pallidal lamina and the globus pallidus. Immunoreactivity in the globus pallidus itself was transient, emerging from 16 weeks of gestation, reaching a peak at 21 weeks of gestation and disappearing by birth. Both processes, i.e. the occurrence of CD15 (Le X) striatopallidal fibres and the emerging immunoreactivity in their pallidal target, may be interrelated, so that ingrowing CD15 (Le X) positive axons from the striatum provoke CD15 (Le X) expression in the external and internal pallidum. The variable patterns and intensities of CD15 (Le X) expression are possibly related to periods of maturation of the striatum and the establishment of functional interactions within the basal ganglia. Differential staining of patch and matrix in the developing neostriatum suggests that a distinct phase of cellular adhesion or dishesion mediated by the CD15 (Le X) epitope occurs during establishment of the patch and matrix regions.

Differential distribution of protein kinase C (PKC alpha beta and PKC gamma) isoenzyme immunoreactivity in the chick brain.

Protein kinase C (PKC) is involved in neural plasticity. The phosphorylation of the myristoylated alanine-rich protein kinase C substrate (MARCKS) in the left intermediate and medial hyperstriatum ventrale (IMHV) of the chick brain has been shown previously to correlate significantly with the strength of learning in filial imprinting. The distribution of PKC alpha, beta I, beta II and PKC gamma in the brain of 1-day-old dark-reared chicks was determined immunocytochemically, using the monoclonal antibodies MC5 and 36G9, raised against purified PKC alpha beta and PKC gamma, respectively. PKC gamma-stained cells were distributed widely in the telencephalon, including all hyperstriatal structures (including the IMHV), the hippocampus, neostriatum, ectostriatum and archistriatum. There were fewer stained cells in the septum and the least cellular staining was in the paleostriatum primitivum. Fluorescent double-labelling with neuron-specific enolase (NSE) and with the glial calcium-binding protein S100 suggested that PKC gamma immunoreactivity was present in neurones but not in glia. The distribution of PKC alpha beta-stained cells was more limited, with staining in the archistriatum, hippocampus and septum but not in the hyperstriatum. However, there was PKC alpha beta-staining of some fibres in the IMHV (but little elsewhere in the hyperstriatum ventrale), in the neostriatum, paleostriatal complex and the lobus parolfactorius. Double-labelling with NSE and S100 revealed PKC alpha beta/S100-positive glial cells present in the paleostriatal region only. There was some PKC alpha beta-staining of putative neurones in the hippocampus, septum and archistriatum. The differential distribution of PKC isoenzymes suggests that in the IMHV some axonal inputs contain PKC alpha beta whereas some postsynaptic cells contain the gamma form of PKC.

A subset of striatopallidal neurons are Fos-immunopositive following acute monoamine depletion in the rat.

Experiments were conducted to characterise the Fos-immunopositive neurons that are observed in the dorsal rim of the striatum following monoamine depletion by the systemic administration of reserpine. Using a retrograde tract-tracer, some of these neurons could be shown to project to the globus pallidus but none were seen to project to the entopeduncular nucleus. In addition, these neurons were located in a region of both poor calbindin immunoreactivity and cholinesterase activity. It can be concluded that Fos levels are increased only in a subset of striatopallidal neurons following monoamine depletion. This subset of neurons is located in the dorsal region of the striatum where it has previously been shown that neurons can preferentially be induced to undergo apoptosis upon monoamine depletion.

Evidence for a preferential loss of enkephalin immunoreactivity in the external globus pallidus in low grade Huntington's disease using high resolution image analysis.

Previous studies have shown that in advanced cases of Huntington's disease, enkephalin-immunoreactive striatal projections to the external globus pallidus may be more affected than substance P-containing striatal projections to the inner segment of the pallidum [Reiner A. et al. (1988) Proc. natn. Acad. Sci. U.S.A. 85, 5733-5737]. Other immunohistochemical [Ferrante R. J. et al. (1990) Soc. Neurosci. Abstr. 16, 1120] and neurochemical observations [Storey E. and Beal M.F. (1993) Brain 116, 1201-1222] suggest no difference in the loss of these peptide-containing pathways in Huntington's disease. In view of the potential significance of this issue for understanding the neuropathological process in Huntington's disease, we examined the globus pallidus in control and Huntington's disease brains, using a quantitative approach which involved high resolution image analysis of 7 microns frozen sections to determine the overall density of peptide-immunoreactive terminals. Results showed that in the controls there was no significant difference between the density of enkephalin- and substance P-immunoreactive terminals in the external and internal globus pallidus, respectively. In all Huntington's disease brains, including grade 1 cases, enkephalin-immunoreactive terminals in the external globus pallidus were significantly reduced compared to substance P-positive boutons in the internal segment of the adjacent section. In comparison to controls, enkephalin immunoreactivity in all Huntington's disease cases was significantly lower; substance P-immunoreactive terminals in the internal globus pallidus were significantly lower than controls in some of the grade 2 cases and in the grade 3 cases.(ABSTRACT TRUNCATED AT 250 WORDS)

Two new monoclonal antibodies provide immunohistochemical evidence for the unique biochemical similarity of the mouse globus pallidus, entopeduncular nucleus and substantia nigra pars reticulata.

Similarities in cellular morphology, afferentation, efferentation, and neurotransmitter content between the internal and external parts of the pallidum and the substantia nigra pars reticulata have long been noted. Here we present evidence that the globus pallidus, entopedunucular nucleus and substantia nigra pars reticulata are more closely related to each other antigenically than to any other anatomical subdivision in the murine central nervous system. In a monoclonal antibody library composed of 20 distinctive lines selected from 300 hybridomas screened immunohistochemically on mouse brain sagittal sections we found two antibodies whose staining patterns distinguish the pallidum and substantia nigra pars reticulata from all other brain gray matter regions but stain these two divisions similarly. One monoclonal antibody, F1-134, stains all brain gray matter regions moderately but gives intense staining of the globus pallidus, entopeduncular nucleus, and substantia nigra pars reticulata only. Another monoclonal antibody, F1-20, stains different brain gray matter regions to varying degrees but shows a complete and exclusive exclusion of staining from the globus pallidus, entopeduncular nucleus and substantia nigra pars reticulata. These results support the tripartite pallidum hypothesis. This study also provides an example of how the monoclonal antibody library strategy can be applied to general problems of brain organization.

Ventral striatopallidal parts of the basal ganglia in the rat: I. Neurochemical compartmentation as reflected by the distributions of neurotensin and substance P immunoreactivity.

The distribution of neurotensin immunoreactivity in the basal ganglia of the adult rat was evaluated by studying alternate serial vibratome sections that were exposed to antiserum against neurotensin, substance P, or cholecystokinin. It was observed that a heterogeneous distribution of neurotensin-immunoreactive fibers and terminals contributes to the neurochemical compartmentation of the ventral pallidum and ventral striatum, and that significant numbers of neurotensin-immunoreactive neurons occupy striatal districts of the olfactory tubercle, nucleus accumbens, and ventromedial caudate-putamen. An intense band of pallidal neurotensin immunoreactivity characterizes the medial part of the ventral pallidum adjacent to the nucleus accumbens, whose medial boundary is conveniently defined in sections incubated with cholecystokinin antiserum. Electron microscopic studies showed that the pallidal plexus of neurotensin-immunoreactive elements consists primarily of boutons, which contact large dendrites in arrangements that in all respects appear to be of the classical striatopallidal variety. A gradual decrease in immunolabel was observed approaching the lateral parts of the ventral pallidum, which display sparse neurotensin immunoreactivity. The results thus indicate the existence of a significant neurotensinergic striatopallidal pathway confined primarily, if not exclusively, to the medial part of the ventral striatopallidal system. The contribution of neurotensin-immunoreactive fibers and terminals to the compartmentation of ventral striatum is expressed most vividly in their exclusion from clusters of tightly packed medium-sized neurons, many of which are intensely substance P immunoreactive. Such clusters appear identical with those previously described as rich in opiate receptors and poor in acetylcholinesterase activity. In the ventral striatal region where the nucleus accumbens and ventromedial caudate-putamen merge, neurotensin-immunoreactive neurons are organized in clusters. Further rostral in the nucleus accumbens, they are more evenly distributed. Few were found in the dorsolateral quadrant of the neostriatum.