The pro-oxidative drug WF-10 inhibits serial killing by primary human cytotoxic T-cells.

Cytotoxic T-cells (CTLs) play an important role in many immune-mediated inflammatory diseases. Targeting cytotoxicity of CTLs would allow to interfere with immune-mediated tissue destruction. Here we demonstrate that WF-10, a pro-oxidative compound, inhibits CTL-mediated cytotoxicity. WF-10 did not influence early steps of target-cell killing, but impaired the ability of CTLs to detach from the initial target cell and to move to a second target cell. This reduced serial killing was accompanied by stronger enrichment of the adhesion molecule LFA-1 in the cytolytic immune synapse. LFA-1 clustering requires activation of the actin-bundling protein L-plastin and was accordingly diminished in L-plastin knockdown cells. Interestingly, WF-10 likely acts through regulating L-plastin: (I) It induced L-plastin activation through phosphorylation leading to enhanced LFA-1-mediated cell adhesion, and, importantly, (II) WF-10 lost its influence on target-cell killing in L-plastin knockdown cells. Finally, we demonstrate that WF-10 can improve immunosuppression by conventional drugs. Thus, while cyclosporine A alone had no significant effect on cytotoxicity of CTLs, a combination of cyclosporine A and WF-10 blocked target-cell killing synergistically. Together, our findings suggest that WF-10 - either alone or in combination with conventional immunosuppressive drugs - may be efficient to control progression of diseases, in which CTLs are crucially involved.

Enhanced cyclooxygenase-2 expression in olfactory-limbic forebrain following kainate-induced seizures.

Cyclooxygenase-2 is expressed at low levels in a subset of neurons in CNS and is rapidly induced by a multiplicity of factors including seizure activity. A putative relationship exists between cyclooxygenase-2 induction and glutamatergic neurotransmission. Cyclooxygenase-1 is constitutively expressed in glial cells and has been specifically linked to microglia. In this study we evaluated cyclooxygenase-2 protein immunocytochemically and found markedly enhanced immunostaining primarily in olfactory-limbic regions at 2, 6 and 24 h following kainate-induced status epilepticus. Impressive enhanced cyclooxygenase-2 immunoreactivity was localized in anterior olfactory nucleus, tenia tecta, nucleus of the lateral olfactory tract, piriform cortex, lateral and basolateral amygdala, orbital frontal cortex, nucleus accumbens (shell) and associated areas of ventral striatum, entorhinal cortex, dentate gyrus granule cells and hilar neurons, hippocampal CA subfields and subiculum. Alternate sections were processed for dual immunocytochemical analysis utilizing c-Fos and cyclooxygenase-2 antiserum to examine the possibility that the neuronal induction of cyclooxygenase-2 was associated with seizure activity. Neurons that showed a timeline of cyclooxygenase-2 upregulation were found to possess c-Fos immunopositive nuclei. Additional results from all seizure groups showed cyclooxygenase-1 induction in microglia, which was confirmed by Western blot analysis of hippocampus. Western blot and real-time quantitative RT-PCR analysis showed significant upregulation of cyclooxygenase-2 expression, confirming its induction in neurons. These data indicate that cyclooxygenase-2 induction in a neuronal network can be a useful marker for pathways associated with seizure activity.

P2X7 receptor immunoreactive profile confined to resting and activated microglia in the epileptic brain.

The purpose of this study was to identify the CNS cellular constituent immunoreactive for specific P2X7 receptor antiserum in the kainate-induced seizure and non-seizure rat brain. Analysis of P2X7 immunocytochemistry (ICC) revealed small immunoreactive cells with processes showing distinct morphological changes as seizures progressed in time. These morphological changes were reminiscent of reactive glia during CNS injury. In order to determine the identity of this non-neuronal cellular constituent, we employed dual ICC techniques using sequential antibody incubations and reacted the sections with contrasting chromagens. Specific glial markers tested in the series included Iba1 (microglia), COX-1 (microglia), and GFAP (astroglia). Results of this study revealed distinct colocalization when sections immunostained for P2X7 were dual immunostained with antisera specific for microglia (Iba1, COX-1). In contrast, no colocalization was evident when sections were dual immunostained with P2X7 and GFAP, an astrocytic marker. In the latter experiment, dual ICC revealed two distinct cell populations with contrasting color demonstrating a population of distinct GFAP immunopositive cells and a population of distinct P2X7 immunopositive cells. We conclude that P2X7 antiserum used in this study is specific for and identifies microglia in rat and that there exists a timeline of progressive changes in microglia morphology that can be demonstrated following kainate-induced seizures. In addition, the morphological changes in microglia following seizure induction that can be identified with P2X7 antisera or with antisera specific for microglia suggest a neuroinflammatory milieu in areas of CNS seizure activity.

AMPA receptor alterations precede mossy fiber sprouting in young children with temporal lobe epilepsy.

Following neurological injury early in life numerous events, including excitotoxicity, neural degeneration, gliosis, neosynaptogenesis, and circuitry reorganization, may alone or in concert contribute to hyperexcitability and recurrent seizures in temporal lobe epilepsy. Our studies provide new evidence regarding the temporal sequence of key elements of hippocampal reorganization, mossy fiber sprouting and glutamate receptor subunit up-regulation, in a subset of young temporal lobe epileptic patients. Without evidence of mossy fiber sprouting, the youngest age group (3-10 years old) of mesial temporal lobe epileptic patients demonstrated enhanced glutamate receptor subunit profiles, suggesting that the dendritic change precedes axonal sprouting. However, sclerotic hippocampal specimens from epileptic patients ages 12-15 years old had the characteristic features of glutamate receptor up-regulation and mossy fiber sprouting first identified in the adult, indicating that reconstructed circuits appear early in the course of the disease. Non-sclerotic hippocampal specimens from lesion associated temporal lobe epileptic patients of all age groups showed minimal cell loss, sparse staining of glutamate receptor subunits in the dentate gyrus, and little or no mossy fiber sprouting. These compelling findings suggest a progressive sequence of events in the reorganization of the dentate gyrus of sclerotic hippocampal specimens. We suggest that cell loss and up-regulation of glutamate receptor subunits appear early in temporal lobe epilepsy and contribute to the synaptic plasticity that may facilitate the subsequent sprouting of mossy fiber collaterals which compound an already precipitous state of decline. The combination of pre-synaptic and post-synaptic changes serves as a potential substrate for hyperexcitability.

Adrenocorticotropic hormone immunoreactivity in the hippocampal formation of temporal lobe epilepsy patients.

PURPOSE: We wished to identify immunocytochemically the distribution of proopiomelanocortin-related peptides in the hippocampal formation of patients with epilepsy. METHODS: Surgical hippocampal specimens from temporal lobe epilepsy (TLE) patients and autopsy control tissue were examined immunocytochemically for ACTH, alpha-melanocyte-stimulating hormone (alpha-MSH) and beta-endorphin. RESULTS: There was a dense distribution of ACTH-immunoreactive neurons in the hippocampal formation of patients with mesial TLE syndrome (MTLE). These hippocampal specimens showed significant cell loss. ACTH-positive neurons were most prominent in the subiculum, with scattered ACTH-immunoreactive neuronal elements distributed in the cornu ammonis fields and hilus. Light ACTH immunoreactivity was detected in the tumor-related epileptic hippocampal specimens, which showed minimal cell loss. Although autopsy control tissue from the hypothalamus showed intense ACTH staining patterns in cells and fibers, there was little or no ACTH immunoreactivity in the autopsy hippocampal tissue. The expression of ACTH immunoreactive elements was correlated with patterns of cell loss. No alpha-MSH- or beta-endorphin-immunoreactive neurons were detected in any of the hippocampal specimens. CONCLUSIONS: ACTH has anticonvulsant properties, and its novel expression in the glutamatergic subicular neurons, which provide the main outflow of the hippocampal formation, may represent an attempt by the damaged hippocampal circuit to restore the balance of excitatory/inhibitory neurotransmission in TLE.

Ventral pallidostriatal pathway in the monkey: evidence for modulation of basal ganglia circuits.

This study describes the organization of the ventral and dorsal pallidostriatal pathway in the monkey. Both retrograde and anterograde tracers were injected into various regions of the ventral and dorsal pallidum as well as into the striatum. The data indicate that the pallidostriatal pathway is an extensive pathway in the monkey. The projections are organized in a topographic manner preserving a general, but not strict medial-to-lateral and ventral-to-dorsal organization. The terminal arrangement of pallidostriatal fibers is widespread. Non-adjacent pallidal regions send fibers to the striatum which overlap considerably, suggesting convergence of terminals from different pallidal regions. The pallidostriatal pathway is found to have a reciprocal but also a large non-reciprocal component to the striatopallidal pathway. On the basis of these data it is concluded that segregation of different corticobasal ganglia-cortical pathways is maintained in the striatopallidal direction as described earlier (Haber et al. [1990] (J. Comp. Neurol. 293:282-298). However, the pallidostriatal projection to a large region of the striatum allows the modulation of several cortico-basal ganglia circuits.

Distribution of AMPA receptor subunits in the hippocampal formation of temporal lobe epilepsy patients.

The immunocytochemical distribution of the AMPA-selective receptor subunits GluR1 and GluR2/3 were mapped in the human hippocampal formation obtained from surgery for medically intractable temporal lobe epilepsy. GluR2/3 immunoreactivity was detected in all principal cell types of the hippocampal formation, including hilar neurons, granule cells of the dentate gyrus, and pyramidal cells of the cornu ammonis fields and subiculum. GluR2/3 immunostaining typically filled the cell bodies and processes of neurons. A comparison of GluR2/3 immunoreactivity in a sclerotic specimen versus a non-sclerotic specimen demonstrated a profound loss of staining, specifically in the areas where neuronal dropout was occurring, including CA1, CA3 and the hilus. An analysis of GluR1 immunoreactivity in non-sclerotic specimens revealed that it was predominantly localized to cellular processes throughout the cornu ammonis fields, with a sparse staining of the dentate gyrus outer molecular layer and little to no staining of the dentate gyrus inner molecular layer. Similar to the GluR2/3-immunostained patterns, GluR1 immunoreactivity was lost in the cornu ammonis fields of sclerotic hippocampal specimens, corresponding to patterns of neuronal dropout. Our most compelling finding was a unique extensive pattern of GluR1 and Glu2/3 immunoreactivity throughout the molecular layers of the dentate gyrus of severely compromised hippocampi. The altered staining of GluR1 and GluR2/3 complements some of the patterns of axonal sprouting already described for the dentate gyrus, with a conjecture that their anatomy and distribution pattern underlies to some degree the reorganization of the sclerotic hippocampus. A combination of enhanced glutamatergic transmission and changes in neuropeptides that modulate hippocampal circuitry could greatly affect the degree of excitability in the hippocampal formation. The alterations of GluR1 and GluR2/3 immunoreactivity in the dentate gyrus add another component to the concept of reorganization in the epileptic sclerotic hippocampus.

NF-kappa B transcription factor subunits in rat brain: colocalization of p65 and alpha-MSH.

The subunit proteins p50 and p65 of the transcription factor NF-kappa B inhibitory protein were immunocytochemically identified and mapped in rat brain. The p65 subunit was localized to the cytoplasm of neurons in the lateral hypothalamus and colocalized with alpha-MSH in neurons identified as the alpha-2 component of the alpha-MSH system. The p50 subunit protein was distributed throughout the neocortex, basal ganglia, thalamic, and hypothalamic nuclei, and certain nuclei of the pons and medulla. The I-kappa B protein, which is necessary for the cytoplasmic sequestration of the NF-kappa B transcription factor complex, was identified specifically in regions of limbic, hypothalamic, and autonomic nuclei.

The orbital and medial prefrontal circuit through the primate basal ganglia.

The ventral striatum is considered an interface between limbic and motor systems. We followed the orbital and medial prefrontal circuit through the monkey basal ganglia by analyzing the projection from this cortical area to the ventral striatum and the representation of orbitofrontal cortex via the striatum, in the globus pallidus and substantia nigra. Following injections of Lucifer yellow and horse radish peroxidase into the medial ventral striatum, there is a very densely labeled distribution of cells in areas 13a and 13b, primarily in layers V and VI, and in medial prefrontal areas 32 and 25. Injections into the shell of the nucleus accumbens labeled primarily areas 25 and 32. The reaction product in the globus pallidus and the substantia nigra supports previous studies demonstrating that efferent projections from the ventral striatum are represented topographically in the ventral pallidum and nontopographically in the substantia nigra, pars compacta. Tritiated amino acid or PHA-L tracer injections into orbitofrontal cortex produce dense patches of terminal labeling along the medial edge of the caudate nucleus and the dorsal part of the nucleus accumbens. These results demonstrate that the orbital prefrontal cortex projects primarily to the medial edge of the ventral striatum and to the core of the nucleus accumbens. The arrangement of terminals in the globus pallidus and substantia nigra show two different patterns. Thus, the orbital and medial prefrontal cortex is represented in a confined region of the globus pallidus but throughout an extensive area of the dorsal substantia nigra. Terminals are extensive throughout the region of the dopaminergic neurons, suggesting that this input may influence a wide area of both the striatum and frontal cortex.

Primate striatonigral projections: a comparison of the sensorimotor-related striatum and the ventral striatum.

The striatum receives topographic cortical inputs with the limbic lobe terminating in the ventral striatum and sensorimotor cortical regions terminating in the dorsolateral striatum. The organization of striatonigral projections originating from these different striatal territories was examined in primate by using several anterograde tracers. The ventral striatum innervates a large area of the substantia nigra, including the medial pars reticulata and much of the pars compacta. Moreover, projections from separate areas of the ventral striatum overlap considerably in the substantia nigra. No mediolateral or rostrocaudal topographic order is apparent, and the area of the substantia nigra associated with the ventral striatum is extensive. In contrast, the sensorimotor-related striatum innervates a limited region of the ventrolateral substantia nigra. Similar to ventral striatonigral projections, projections originating from different areas of the sensorimotor-related striatum send converging inputs to the substantia nigra. Sensorimotor-related striatonigral projections avoid the region of the dopaminergic neurons in the dorsal pars compacta. Striatonigral projections from the sensorimotor-related and ventral striatum do not overlap in the substantia nigra. Examination of the outputs of discrete striatal loci indicates that the organization of striatonigral projections is more related to corticostriatal inputs than to a simple rostrocaudal, dorsoventral, or mediolateral topography of the striatum. Striatal projections that originate from different striatal territories are distinct and nonoverlapping, thus supporting the concept of segregated striatonigral circuits. However, areas of the striatum that receive common cortical inputs send converging inputs to the substantia nigra. This suggests that the substantia nigra is also an important link for integrating information between functionally related (sub)circuits.

The organization of midbrain projections to the ventral striatum in the primate.

Because the dopaminergic neurons of the midbrain form a continuum, boundaries between the ventral tegmental area, substantia nigra pars compacta, and retrorubral area are difficult to distinguish in the primate. Therefore, dopaminergic neurons have been subdivided into more readily discernible dorsal and ventral tiers. The projections from these dorsal and ventral tier neurons of the ventral mesencephalon to the ventral striatum were labeled by injections of horseradish peroxidase conjugated to wheatgerm agglutinin and Lucifer Yellow conjugated to dextran amines into different regions of the nucleus accumbens, the ventral caudate nucleus, and the rostral, ventral putamen in the primate. Neurons projecting to the ventral striatum are not topographically organized in the ventral mesencephalon. Retrogradely labeled neurons are found in the medial densocellular zone of the ventral tier following injections into all regions of the ventral striatum except the ventromedial shell region of the nucleus accumbens. These medial nigral neurons have diverging projections throughout the mediolateral extent of the ventral striatum. In addition, neurons of the dorsal tier project to all ventral striatal regions examined. Notably, neurons projecting to the shell region of the nucleus accumbens are limited to the dorsal tier, throughout the rostrocaudal extent of the substantia nigra. Both dorsal and ventral tier neurons innervate the ventral striatum. Not only do neurons of the ventral tegmental area project to the ventral striatum, but also many of the pars compacta. The projections to the shell region of the nucleus accumbens are more restricted, suggesting that the dopaminergic regulation of this accumbens subterritory is distinct from the rest of the ventral striatum.

The organization of midbrain projections to the striatum in the primate: sensorimotor-related striatum versus ventral striatum.

In order to examine the organization of nigrostriatal projections in the primate, the retrograde tracers Lucifer Yellow conjugated to dextran amines and horseradish peroxidase conjugated to wheatgerm agglutinin were injected into different regions of the dorsolateral and ventral striatum. Based on the topography of cortical inputs to the striatum, the dorsolateral striatum is associated with the motor system, and the ventral striatum is related to the limbic system. Our results indicate that although midbrain neurons projecting to the ventral and dorsolateral striatum are mostly separate, there are neurons projecting to these different striatal territories that overlap in the medial substantia nigra. The dopaminergic neurons of the ventral mesencephalon can be subdivided into dorsal and ventral tiers that include the cells of the ventral tegmental area, the substantia nigra pars compacta, and the retrorubral area. Neurons projecting to the ventral striatum are found in both the dorsal and ventral tiers. A large number of neurons occupying the medial densocellular zone of the ventral tier are labeled following injections into different regions of the ventral striatum. Neurons projecting to the sensorimotor-related striatum are derived almost exclusively from the ventral tier. Many of these neurons are located very ventrally in the substantia nigra, where clusters of neurons invade the pars reticulata. In addition, labeled neurons are found throughout the mediolateral extent of the densocellular zone of the pars compacta. Notably, neurons are labeled in the medial densocellular zone following injections into the dorsolateral and ventral striatum. Mesencephalic neurons projecting to different striatal territories are distinct in that dorsal tier neurons mainly innervate the ventral striatum, whereas the ventral columns of neurons in the ventral tier innervate the sensorimotor-related striatum. Thus, the dopaminergic regulation of the sensorimotor-related striatum and the ventral striatum may be different. However, a subgroup of dopaminergic neurons in the medial densocellular zone projects to both striatal territories. Such divergent projections may allow the substantia nigra to serve as a link, connecting different striatal territories, via their connections with the substantia nigra.

The organization of the descending ventral pallidal projections in the monkey.

This study describes the organization and topography of the descending efferent projections from the monkey ventral pallidum. The main efferent projections from the globus pallidus are to the subthalamus, to the thalamus, and to the substantia nigra. Although these projections have been well established for the dorsal pallidum, the projections of the ventral pallidum have not been explored in primates. The results of this study add an important link in how information from the limbic lobe is channeled through the basal ganglia in monkeys. Anterograde tracers, Phaseolus vulgaris-leucoagglutinin, and tritiated amino acids were injected into various regions of the ventral pallidum. The descending efferent projection from the ventral pallidum in monkeys terminates primarily in the subthalamic nucleus and adjacent lateral hypothalamus, in the substantia nigra, and in the lateral habenular nucleus. Although terminals are also found in the thalamus, these are relatively sparse. The projections to the subthalamic nucleus and the lateral hypothalamus are topographically arranged, while those to the substantia nigra are not. These results suggest that pathways from distinct pallidal regions that receive specific striatal input terminate in distinct regions of the subthalamic/hypothalamic regions, thus maintaining a topographic arrangement. Projections to the substantia nigra, however, overlap extensively, suggesting convergence of terminals from different ventral pallidal regions. The relatively small projection to the thalamus raises the question that without a prominent thalamic projection, is this system parallel to that described for the dorsal globus pallidus?