Cell shape regulates subcellular organelle location to control early Ca2+ signal dynamics in vascular smooth muscle cells.

The shape of the cell is connected to its function; however, we do not fully understand underlying mechanisms by which global shape regulates a cell's functional capabilities. Using theory, experiments and simulation, we investigated how physiologically relevant cell shape changes affect subcellular organization, and consequently intracellular signaling, to control information flow needed for phenotypic function. Vascular smooth muscle cells going from a proliferative and motile circular shape to a contractile fusiform shape show changes in the location of the sarcoplasmic reticulum, inter-organelle distances, and differential distribution of receptors in the plasma membrane. These factors together lead to the modulation of signals transduced by the M3 muscarinic receptor/Gq/PLCß pathway at the plasma membrane, amplifying Ca2+ dynamics in the cytoplasm, and the nucleus resulting in phenotypic changes, as determined by increased activity of myosin light chain kinase in the cytoplasm and enhanced nuclear localization of the transcription factor NFAT. Taken together, our observations show a systems level phenomenon whereby global cell shape affects subcellular organization to modulate signaling that enables phenotypic changes.

c-Myc-dependent transcriptional regulation of cell cycle and nucleosomal histones during oligodendrocyte differentiation.

Oligodendrocyte progenitor cells (OPCs) have the ability to divide or to growth arrest and differentiate into myelinating oligodendrocytes in the developing brain. Due to their high number and the persistence of their proliferative capacity in the adult brain, OPCs are being studied as potential targets for myelin repair and also as a potential source of brain tumors. This study addresses the molecular mechanisms regulating the transcriptional changes occurring at the critical transition between proliferation and cell cycle exit in cultured OPCs. Using bioinformatic analysis of existing datasets, we identified c-Myc as a key transcriptional regulator of this transition and confirmed direct binding of this transcription factor to identified target genes using chromatin immunoprecipitation. The expression of c-Myc was elevated in proliferating OPCs, where it also bound to the promoter of genes involved in cell cycle regulation (i.e. Cdc2) or chromosome organization (i.e. H2afz). Silencing of c-Myc was associated with decreased histone acetylation at target gene promoters and consequent decrease of gene transcripts. c-Myc silencing also induced a global increase of repressive histone methylation and premature peripheral nuclear chromatin compaction while promoting the progression towards differentiation. We conclude that c-Myc is an important modulator of the transition between proliferation and differentiation of OPCs, although its decrease is not sufficient to induce progression into a myelinating phenotype.

Continuously delivered ovarian steroids do not alter dendritic spine density or morphology in macaque dorsolateral prefrontal cortical neurons.

Aged ovariectomized (OVX) female monkeys, a model for menopause in humans, show a decline in spine density in the dorsolateral prefrontal cortex (dlPFC) and diminished performance in cognitive tasks requiring this brain region. Previous studies in our laboratory have shown that long-term cyclic treatment with 17ß-estradiol (E) produces an increase in spine density and in the proportion of thinner spines in layer III pyramidal neurons in the dlPFC of both young and aged OVX rhesus monkeys. Here we used 3D reconstruction of Lucifer yellow-loaded neurons to investigate whether clinically relevant schedules of hormone therapy would produce similar changes in prefrontal cortical neuronal morphology as long-term cyclic E treatment in young female monkeys. We found that continuously delivered E, with or without a cyclic progesterone treatment, did not alter spine density or morphology in the dlPFC of young adult OVX rhesus monkeys. We also found that the increased density of thinner spines evident in the dlPFC 24h after E administration in the context of long-term cyclic E therapy is no longer detectable 20days after E treatment. When compared with the results of our previously published investigations, our results suggest that cyclic fluctuations in serum E levels may cause corresponding fluctuations in the density of thin spines in the dlPFC. By contrast, continuous administration of E does not support sustained increases in thin spine density. Physiological fluctuations in E concentration may be necessary to maintain the morphological sensitivity of the dlPFC to E.

Interactive effects of age and estrogen on cortical neurons: implications for cognitive aging.

In the past few decades it has become clear that estrogen signaling plays a much larger role in modulating the cognitive centers of the brain than previously thought possible. We have developed a nonhuman primate (NHP) model to investigate the relationships between estradiol (E) and cognitive aging. Our studies of cyclical E treatment in ovariectomized (OVX) young and aged rhesus monkeys have revealed compelling cognitive and synaptic effects of E in the context of aging. Delayed response (DR), a task that is particularly dependent on integrity of dorsolateral prefrontal cortex (dlPFC) area 46 revealed the following: (1) that young OVX rhesus monkeys perform equally well whether treated with E or vehicle (V), and (2) that aged OVX animals given E perform as well as young adults with or without E, whereas OVX V-treated aged animals display significant DR impairment. We have analyzed the structure of layer III pyramidal cells in area 46 in these same monkeys. We found both age and treatment effects on these neurons that are consistent with behavioral data. Briefly, reconstructions of pyramidal neurons in area 46 from these monkeys showed that cyclical E increased the density of small, thin spines in both young and aged monkeys. However, this effect of E was against a background of age-related loss of small, thin spines, leaving aged V-treated monkeys with a particularly low density of these highly plastic spines, and vulnerable to cognitive decline. Our current interpretation is that E not only plays a critically important role in maintaining spine number, but also enables synaptic plasticity through a cyclical increase in small highly plastic spines that may be stabilized in the context of learning. Interestingly, recent studies demonstrate that chronic E is less effective at inducing spinogenesis than cyclical E. We have begun to link certain molecular attributes of excitatory synapses in area 46 to E effects and cognitive performance in these monkeys. Given the importance of synaptic estrogen receptor α (ER-α) in rat hippocampus, we focused our initial studies on synaptic ER-α in area 46. Three key findings have emerged from these studies: (1) synaptic ER-α is present in axospinous synapses in area 46; (2) it is stable across treatment and age groups (which is not the case in rat hippocampus); and (3) the abundance and distribution of synaptic ER-α is a key correlate of individual variation in cognitive performance in certain age and treatment groups. These findings have important implications for the design of hormone treatment strategies for both surgically and naturally menopausal women. This article is part of a Special Issue entitled: Neuroactive Steroids: Focus on Human Brain.

Estrogen and aging affect synaptic distribution of phosphorylated LIM kinase (pLIMK) in CA1 region of female rat hippocampus.

17beta-Estradiol (E) increases axospinous synapse density in the hippocampal CA1 region of young female rats, but not in aged rats. This may be linked to age-related alterations in signaling pathways activated by synaptic estrogen receptor alpha (ER-alpha) that potentially regulate spine formation, such as LIM-kinase (LIMK), an actin depolymerizing factor/cofilin kinase. We hypothesized that, as with ER-alpha, phospho-LIM-kinase (pLIMK) may be less abundant or responsive to E in CA1 synapses of aged female rats. To address this, cellular and subcellular distribution of pLIMK-immunoreactivity (IR) in CA1 was analyzed by light and electron microscopy in young and aged female rats that were ovariectomized and treated with either vehicle or E. pLIMK-IR was found primarily in perikarya within the pyramidal cell layer and dendritic shafts and spines in stratum radiatum (SR). While pLIMK-IR was occasionally present in terminals, post-embedding quantitative analysis of SR showed that pLIMK had a predominant post-synaptic localization and was preferentially localized within the postsynaptic density (PSD). The percentage of pLIMK-labeled synapses increased (30%) with E treatment (P<0.02) in young animals, and decreased (43%) with age (P<0.002) regardless of treatment. The pattern of distribution of pLIMK-IR within dendritic spines and synapses was unaffected by age or E treatment, with the exception of an E-induced increase in the non-synaptic core of spines in young females. These data suggest that age-related synaptic alterations similar to those seen with ER-alpha occur with signaling molecules such as pLIMK, and support the hypothesis that age-related failure of E treatment to increase synapse number in CA1 may be due to changes in the molecular profile of axospinous synapses with respect to signaling pathways linked to formation of additional spines and synapses in response to E.

Dynamometry of intrinsic hand muscles in patients with Charcot-Marie-Tooth disease.

BACKGROUND: Several problems are associated with manual muscle testing and dynamometry in the hands of patients with Charcot-Marie-Tooth (CMT) disease. OBJECTIVE: To evaluate the efficacy of the Rotterdam Intrinsic Hand Myometer (RIHM) to directly measure intrinsic hand muscle strength in CMT disease. METHODS: We measured hand muscle strength and hand function in 41 patients with CMT disease. RESULTS: RIHM measurement of intrinsic strength had excellent reliability. We found overlapping RIHM strength values in Medical Research Council grades 3 to 5. High grip and pinch strength could be found in patients with severe intrinsic muscle weakness. RIHM measurements were more strongly correlated with fine motor skills of the hand than grip and pinch strength. CONCLUSIONS: The Rotterdam Intrinsic Hand Myometer is a reliable instrument to measure intrinsic hand muscles strength in patients with Charcot-Marie-Tooth disease, providing more detailed information than manual muscle testing and a more direct assessment of intrinsic muscle loss than grip and pinch dynamometers.

Analysis and decomposition of accelerometric signals of trunk and thigh obtained during the sit-to-stand movement.

Piezoresistive accelerometer signals are frequently used in movement analysis. However, their use and interpretation are complicated by the fact that the signal is composed of different acceleration components. The aim of the study was to obtain insight into the components of accelerometer signals from the trunk and thigh segments during four different sit-to-stand (STS) movements (self-selected, slow, fast and fullflexion). Nine subjects performed at least six trials of each type of STS movement. Accelerometer signals from the trunk and thigh in the sagittal direction were decomposed using kinematic data obtained from an opto-electronic device. Each acceleration signal was decomposed into gravitational and inertial components, and the inertial component of the trunk was subsequently decomposed into rotational and translational components. The accelerometer signals could be reliably reconstructed: mean normalised root mean square (RMS) trunk: 6.5% (range 3-12%), mean RMS thigh: 3% (range 2-5%). The accelerometric signals were highly characteristic and repeatable. The influence of the inertial component was significant, especially on the timing of the specific event of maximum trunk flexion in the accelerometer signal. The effect of inertia was larger in the trunk signal than in the thigh signal and increased with higher speeds. The study provides insight into the acceleration signal, its components and the influence of the type of STS movement and supports its use in STS movement analysis.

AMPA GluR2 subunit is differentially distributed on GABAergic neurons and pyramidal cells in the macaque monkey visual cortex.

The cellular and synaptic distribution of the AMPA receptor subunit GluR2 was analyzed in the monkey primary visual cortex (area V1), by immunocytochemistry and postembedding immunogold methods. GluR2 immunoreactivity was widely distributed in all of the layers of area V1. A quantitative double labeling analysis in layers II and III revealed that the vast majority of GABAergic interneurons in this area also contained GluR2. Postembedding immunogold analysis revealed that GluR2 immunoreactivity was present at asymmetric synapses on both GABAergic interneurons and pyramidal cells. A quantitative study indicated that the number of GluR2 immunogold particles at asymmetric synapses on pyramidal cells was significantly higher than that on GABAergic interneurons. These results from the primate neocortex are in agreement with and extend our previous studies on the rat hippocampus and amygdala. In view of the dominant role of the GluR2 subunit in regulating calcium flux through AMPA receptors, the differential synaptic distribution of GluR2 on different neuronal types might provide a mechanism for cell-specific response properties to glutamate as well as clues to selective neuronal vulnerability and cell death mediated by calcium-dependent excitotoxic mechanisms.

Differential synaptic localization of GluR2 and EAAC1 in the macaque monkey entorhinal cortex: a postembedding immunogold study.

The synaptic distribution of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor subunit GluR2 and neuronal glutamate transporter subunit EAAC1 were studied using immunogold in layer II of the macaque monkey entorhinal cortex. Immunoreactivity for EAAC1 and GluR2 was frequent at asymmetric synapses and their associated membrane. The synaptic localization of EAAC1 differed considerably from that of GluR2, in that GluR2 immunolabelling was most commonly located within the postsynaptic density, but EAAC1 localization was more heterogeneous and was predominant at the edge of postsynaptic densities and perisynaptic zones. Since EAAC1 may play an important role in clearing glutamate from the synaptic cleft and intercellular spaces, the high perisynaptic expression of EAAC1 in these neurons could presumably offer a powerful mechanism through which high concentrations of glutamate could be efficiently removed from the synapses following release and interaction with glutamate receptors. The distribution of EAAC1 may also offer protection for these neurons against excessive glutamatergic stimuli that may occur under certain pathological conditions.

Different modes of hippocampal plasticity in response to estrogen in young and aged female rats.

Estrogen regulates hippocampal dendritic spine density and synapse number in an N-methyl-D-aspartate (NMDA) receptor-dependent manner, and these effects may be of particular importance in the context of age-related changes in endocrine status. We investigated estrogen's effects on axospinous synapse density and the synaptic distribution of the NMDA receptor subunit, NR1, within the context of aging. Although estrogen induced an increase in axospinous synapse density in young animals, it did not alter the synaptic representation of NR1, in that the amount of NR1 per synapse was equivalent across groups. Estrogen replacement in aged female rats failed to increase axospinous synapse density; however, estrogen up-regulated synaptic NR1 compared with aged animals with no estrogen. Therefore, the young and aged hippocampi react differently to estrogen replacement, with the aged animals unable to mount a plasticity response generating additional synapses, yet responsive to estrogen with respect to additional NMDA receptor content per synapse. These findings have important implications for estrogen replacement therapy in the context of aging.

Struck by stroke: a pilot study exploring quality of life and coping patterns in younger patients and spouses.

So far, research on quality of life after stroke has focused mainly on elderly patients. This study is targeted at younger stroke patients and their partners, aiming to evaluate stroke impact, as related to coping strategy. For our pilot study, eight patients who had suffered a stroke and four partners completed the Impact of Event Scale questionnaire. The mean age was 47.6 years in patients and 44.5 years in partners. The patients' level of activities of daily life was assessed using the Barthel Index. They were then interviewed to obtain information with respect to stroke impact and coping. The Schedule for the Evaluation of Individual Quality of Life procedure was carried out to measure quality of life, and stroke impact was quantified using Visual Analogue Scales. On average, patients scored 19.25 on the Barthel Index. Quality of life had deteriorated by 20.1% in patients, whereas partners did not show a decline in quality of life. However, well-being was inversely correlated among patients and partners. Accommodative coping was positively correlated with quality of life in both patients and partners. Conversely, assimilation was negatively related to quality of life in patients.

Differential synaptic localization of the glutamate transporter EAAC1 and glutamate receptor subunit GluR2 in the rat hippocampus.

EAAC1, a neuron-specific glutamate transporter, is likely to play an important role in the regulation of glutamate levels in the synaptic cleft. Ultrastructural studies have demonstrated that the glutamate receptor subunit proteins (e.g., GluR2) are frequently preferentially located at the postsynaptic density of asymmetric synapses. While the glutamate/glutamate receptor interaction is likely to be influenced by the activity and location of the transporter molecules, the spatial localization of the transporter molecules relative to the receptor molecules is not well delineated. Thus, we analyzed the cellular, ultrastructural, and synaptic distribution of EAAC1 in the context of the distribution of the AMPA receptor subunit GluR2 in the hippocampus. While GluR2 and EAAC1 are both present in hippocampal projection neurons, their intracellular distribution patterns differ. Both GluR2 and EAAC1 are present in the dendritic membranes and cytoplasm; however EAAC1 has a distinctive punctate distribution in the dendrite compared to the more diffuse labeling reflected by GluR2. Pre-embedding ultrastructural studies also revealed cytoplasmic and membrane-associated pools of EAAC1 within dendritic shafts and spines, as well as in a subset of axonal profiles and terminals. Postembedding double label immunogold localization demonstrated a similar intraneuronal distribution, but in addition showed that membrane-associated EAAC1 is not intermingled with GluR2 within the synaptic complex, but in contrast is primarily located perisynaptically, often immediately outside the synaptic specialization. In addition, there is a significant presynaptic pool of EAAC1, whereas GluR2 is essentially absent from the pre-synaptic profile. Thus, membrane-associated EAAC1 within the synaptic region is ideally situated to restrict the site of action of glutamate with respect to ionotropic receptors to the synaptic cleft, as well as regulate glutamate levels in the perisynaptic and presynaptic domains, the ultrastructural sites that have been associated with metabotropic receptor localization.

Differential synaptic distribution of the AMPA-GluR2 subunit on GABAergic and non-GABAergic neurons in the basolateral amygdala.

The cellular and ultrastructural distribution patterns of the AMPA glutamate receptor subunit, GluR2, were determined in the rat basolateral amygdala. GluR2 immunoreactivity was widely and uniformly distributed in the basolateral nucleus, with both pyramidal and non-pyramidal neurons labelled. In fact, double label immunohistochemical analyses demonstrated that over 90% of the GABAergic interneurons were labelled for GluR2. Electron microscopic analyses further confirmed the presence of GluR2 in the soma and dendrites of GABAergic interneurons as well as in the soma, spines and dendritic shafts of pyramidal cells. As in our parallel study in the rat hippocampus, immunogold analyses revealed that GluR2 immunoreactivity was frequently preferentially located at asymmetric synapses on both pyramidal cell spines and shafts, as well as the dendritic processes and soma of GABAergic interneurons. However, the number of immunogold particles per labelled synapse on GABAergic neurons was significantly lower than at similar labelled asymmetric synapses on spines of presumed pyramidal cells. Given that the presence of GluR2 within the AMPA receptor complex decreases calcium flux, these data indicate that GABAergic local circuit neurons might possess AMPA receptors with higher calcium permeability on average than pyramidal cells, as has been suggested for hippocampus. Such cell class-specific differences in the subunit representation and resultant channel properties of AMPA receptors have implications for response properties as well as selective vulnerability of neurons within the basolateral nucleus of the amygdala.

Synaptic coexistence of AMPA and NMDA receptors in the rat hippocampus: a postembedding immunogold study.

Synaptic distributions of N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methylisoxazolepropionic acid (AMPA) receptor subunits, NMDAR1 and GluR2, respectively, were examined by electron microscopy with the high spatial resolution of postembedding immunogold localization. We provide direct evidence for colocalization at individual axodendritic asymmetric synapses within the CA1 subfield of rat hippocampus. AMPA/NMDA receptor colocalization was found both in gamma-aminobutyric acid (GABA)ergic dendrites and non-GABAergic dendritic shafts, as well as dendritic spines. Some asymmetric synapses were found to contain only NMDAR1 or GluR2; however, most immunopositive synapses contained both subunits. Many NMDAR1 and/or GluR2 immunopositive profiles received GABAergic innervation at an adjacent synapse, providing a substrate for GABAergic modulation of both GluR classes. These data suggest that excitatory neuronal transmission in CA1 neurons may generally involve activation of both NMDA and AMPA receptor subunits at a single synapse, however, they also offer ultrastructural evidence for NMDAR1-only synapses that might represent silent synapses.

Light and electron microscopic distribution of the AMPA receptor subunit, GluR2, in the spinal cord of control and G86R mutant superoxide dismutase transgenic mice.

Excitotoxicity has been hypothesized to contribute to amyotrophic lateral sclerosis (ALS) neurodegeneration. The similar pattern of vulnerability in the spinal cord of mutant superoxide dismutase (SOD-1) transgenic mice and mice treated with excitotoxins supports a role for excitotoxicity in the mechanism of degeneration. The distribution of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) class of glutamate receptors (GluRs) with different calcium permeabilities has been proposed as an explanation for this differential vulnerability. GluR2 appears to be the dominant determinant of calcium permeability for AMPA receptors; thus, it is critical for their contribution to excitotoxic mechanisms. In this study, we investigate the distribution of GluR2 immunoreactivity in the spinal cord of control and SOD-1 transgenic mice. GluR2 immunoreactivity is present equally within vulnerable neurons (i.e., motor neurons and calretinin-immunoreactive neurons) as well as nonvulnerable neurons (i.e., calbindin-immunoreactive neurons and dorsal horn neurons). In addition, postembedding immunoelectron microscopy reveals that GluR2 is present in synapses of dorsal and ventral horn neurons and that the percentage of labeled synapses and numbers of immunogold particles per synapse do not vary between these spinal cord regions. Comparing control mice with SOD-1 transgenic mice, at both the light and the electron microscopic levels, the distribution and intensity of GluR2-immunoreactivity do not appear to be altered. These results suggest that the cellular and synaptic distribution of GluR2 is not a determinant of the selective vulnerability observed in SOD-1 transgenic mice or in ALS patients.

Time course of neuropathology in the spinal cord of G86R superoxide dismutase transgenic mice.

Transgenic mice with a G86R mutation in the mouse superoxide dismutase (SOD-1) gene, which corresponds to a mutation observed in familial amyotrophic lateral sclerosis (ALS), display progressive motor dysfunction leading to paralysis and premature death. In endstage SOD-1 transgenic mice, there is marked loss of spinal motor neurons and interneurons, accumulation of phosphorylated neurofilament inclusions, and reactive astrocytosis. The present study details the time course and ultrastructural appearance of these pathologic changes and correlates the timing of these events with the behavioral symptoms. There is no significant reduction in the number of total neurons, motor neurons, or interneurons in the ventral spinal cord of presymptomatic mice, as compared to age-matched control mice. In contrast, there is a significant reduction in the number of total neurons (-23.5%), motor neurons (-28.9%), and interneurons (-23.5%) in symptomatic SOD-1 transgenic mice. This neuron loss correlates temporally with the onset of reactive astrocytosis and the appearance of phosphorylated neurofilament inclusions. The identical timing of motor neuron and interneuron degeneration in this model of ALS strongly suggests that degeneration in the spinal cord of patients with ALS is not specifically directed at motor neurons, but rather more generally at several populations of neurons in the spinal cord. In addition, the late onset and rapid progression of neuron loss suggest that a toxic property is accumulating while the SOD-1 transgenic mice are presymptomatic, and that this toxic property must reach a threshold level before the onset of neuronal degeneration.

Synaptic distribution of GluR2 in hippocampal GABAergic interneurons and pyramidal cells: a double-label immunogold analysis.

GluR2 is the regulatory subunit in the AMPA family of glutamate receptors (GluRs) in that its presence inhibits calcium flux and dominates the current/ voltage characteristics of AMPA receptors. Studies from other laboratories have shown that GABAergic interneurons have a lower ratio of GluR2/GluR1 mRNA than pyramidal cells as well as possessing AMPA receptors that have a higher relative permeability to calcium. We hypothesized that such differences might be related to differences in the subunit stoichiometry at the AMPA synapses in each cell class, and used a GluR2-specific monoclonal antibody in a double-label immunogold protocol with anti-GABA and anti-CaM kinase II to compare the GluR2 representation at asymmetric synapses in GABA neurons to that of pyramidal cells in rat CA1. Virtually all CA1 pyramidal cells as well as the majority of GABAergic interneurons were GluR2 positive. EM immunogold labeling also showed that GABAergic interneurons had distinctive ultrastructural features and contained GluR2 in both their soma and their dendrites, as did the spines and shafts of pyramidal cells. GluR2 immunoreactivity was frequently preferentially located at asymmetric synapses on both pyramidal cell spines and shafts as well as the dendritic processes and soma of GABAergic interneurons. However, the labeled synapses on GABAergic neurons had a significantly lower number of immunogold particles than those on pyramidal cells. In fact, 90% of the labeled asymmetric synapses on GABAergic cells had one to three gold particles, whereas greater than 70% of the labeled asymmetric synapses on pyramidal cells had four or more gold particles associated with the synapse. These data suggest that while both cell classes contain GluR2, they differ in the relative representation of GluR2 at their AMPA synapses, such that GABAergic neurons might possess AMPA receptors with higher calcium permeability on average than pyramidal cells. Such differences in subunit representation at AMPA-receptor-mediated synapses would not only lead to differences in calcium permeability and functional characteristics across these two cell classes, but might also be relevant to the hippocampal patterns of selective vulnerability with respect to excitotoxicity and neurodegeneration.

Expression of dopamine D3 receptor dimers and tetramers in brain and in transfected cells.

The expression and characteristics of the dopamine D3 receptor protein were studied in brain and in stably transfected GH3 cells. Monoclonal antibodies were used for immunoprecipitation and immunoblot experiments. Immunoprecipitates obtained from primate and rodent brain tissues contain a low molecular weight D3 protein and one or two larger protein species whose molecular mass are integral multiples of the low molecular weight protein and thus appear to have resulted from dimerization and tetramerization of a D3 monomer. Whereas D3 receptor multimers were found to be abundantly expressed in brain, the major D3 immunoreactivity expressed in stable D3-expressing rat GH3 cells was found to be a monomer. However, multimeric D3 receptor species with electrophoretic mobilities similar to those expressed in brain were also seen in D3-expressing GH3 cells when a truncated D3-like protein (named D3nf) was co-expressed in these cells. Furthermore, results from immunoprecipitation experiments with D3- and D3nf-specific antibodies show that the higher-order D3 proteins extracted from brain and D3/D3nf double transfectants also contain D3nf immunoreactivity, and immunocytochemical studies show that the expression of D3 and D3nf immunoreactivities overlaps substantially in monkey and rat cortical neurons. Altogether, these data show oligomeric D3 receptor protein expression in vivo and they suggest that at least some of these oligomers are heteroligomeric protein complexes containing D3 and the truncated D3nf protein.

Synaptic distribution of the AMPA-GluR2 subunit and its colocalization with calcium-binding proteins in rat cerebral cortex: an immunohistochemical study using a GluR2-specific monoclonal antibody.

Due to its role as the dominant AMPA receptor subunit in respect to regulation of calcium permeability, information on the neuronal localization of GluR2 is of particular importance, yet has been hampered by the lack of a GluR2-specific antibody. Monoclonal antibodies were raised against the putative N-terminal portion (amino acids 175--430) of GluR2, using the fusion protein linked to trpE as an antigen. Western blot analysis and immunocytochemistry of transiently transfected human embryonic kidney 293 cells unambiguously confirmed the specificity of monoclonal antibody 6C4 for GluR2, which did not recognize or cross-react with any other AMPA/Kainate GluR subunits expressed. 6C4 was used in immunohistochemical studies to characterize the regional, cellular, and subcellular distribution of the GluR2 subunit at the light and electron microscopic levels in rat hippocampus and somatosensory cortex and in colocalization studies with the three calcium-binding proteins: parvalbumin, calbindin, and calretinin. GluR2 was widely distributed in both pyramidal cells and interneurons. Asymmetric synapses were labeled on both spines and small dendritic shafts. In contrast to previous reports, our double labeling studies using monoclonal antibody 6C4 with polyclonal antisera against calcium-binding proteins demonstrated that 84--97% of parvalbumin and calbindin-immunoreactive and 45--66% of the calretinin-immunoreactive interneurons in CA1 and somatosensory cortex also contain GluR2. These data have important implications regarding heterogeneity in calcium permeability of AMPA receptors across cell types in neocortex and hippocampus, as well as for differential vulnerability to excitotoxic injury.