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PubMed1990

Alkaline phosphatase-fast red, a new fluorescent label. Application in double labelling for cell cycle analysis.

We have observed that the red reaction product of alkaline phosphatase immuno-conjugates and certain substrate preparations produces a brilliant red fluorescence that is visible by fluorescence microscopy using both fluorescein and rhodamine filter combinations. This provides a level of sensitivity greater than that obtained with other commonly used red fluorochromes or by inspection of the reaction product under bright field illumination. Of particular value, the reaction product is unaffected by the denaturing conditions required for the detection of incorporated nuclear BrdU with FITC conjugated anti-BrdU antibody and provides a simple and robust method for the simultaneous detection of cell proliferation and cell surface markers.

Alkaline Phosphatase↗
PubMed2004

Unmodified oligodeoxynucleotides require single-strandedness to induce targeted repair of a chromosomal EGFP gene.

BACKGROUND: A number of genetic defects in humans are due to point mutations in a single, often tightly regulated gene. Genetic treatment of such defects is preferably done by correcting only the altered base pair at the endogenous locus rather than by a gene replacement strategy involving viral vectors. Promisingly high repair rates have been achieved in some systems with the non-viral approach of transfecting chimeric RNA/DNA oligonucleotides (chimeraplasts). However, since this technique does not yet perform robustly, several parameters thought to be important in oligonucleotide-mediated gene repair were examined. METHODS: A series of transgenic HEK-293 cell clones has been established harboring high or low copy numbers of a point-mutated 'enhanced green fluorescent protein' (EGFP) gene as the target. At the level of single living cells, repair efficiencies were measured by fluorescence-activated cell sorting (FACS) regarding topology (single-stranded, double-stranded), exonuclease protection (four phosphorothioate linkages at both ends), polarity (sense, antisense), and length (13mer, 19mer, 35mer, 69mer) of the oligonucleotide. RESULTS: When targeting chromosomal loci, up to 0.2% corrected cells were obtained with single-stranded unmodified oligodeoxynucleotides, whereas a chimeraplast, its DNA analogue, and double-stranded DNA fragments were practically non-functional. Correction efficiencies correlated with target gene copy numbers. Modifying exonuclease resistance, polarity or length of single-stranded oligodeoxynucleotides did not enhance repair efficacy above the sub-percentage range. CONCLUSIONS: Successful chromosomal reporter gene repair in HEK-293 cells required an oligodeoxynucleotide to be single-stranded. In concert with the gene copy number correlation, functional interaction between the repair molecule and the target site seems to be one bottleneck in targeted gene repair.

Cell Line↗
PubMed1987

Reserpine augmentation of desipramine in refractory depression: clinical and neurobiological effects.

Early studies showed dramatic improvement in some depressed patients when a brief course of parenteral reserpine was added to ineffective tricyclic antidepressant (TCA) treatment. We treated eight patients with DSM-III melancholic major depression with desipramine (DMI) greater than or equal to 2.5 mg/kg/day (plasma levels greater than 125 ng/ml) for at least 4 weeks. All patients failed to respond and received reserpine 5 mg IM b.i.d. over 2 days, in seven cases as a placebo-controlled, double-blind trial. One patient had dramatic resolution of depressive and psychotic symptoms within 48 h, but relapsed within 2 weeks; two other patients had transient hypomanic symptoms. Depression ratings did not significantly change for the sample as a whole, but plasma and cerebrospinal fluid (CSF) levels of 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG) decreased and CSF levels of homovanillic acid (HVA) and 5-hydroxy-indoleacetic acid (5-HIAA) increased. Despite robust effects on central monoamine metabolism, reserpine augmentation appears insufficiently effective for routine use in managing refractory depression.

Adult↗
PubMed2005

Interlaced Fourier transformation of ultrafast 2D NMR data.

A new protocol for processing the data arising in ultrafast 2D NMR is discussed and exemplified, based on the interlaced Fourier transformation. This approach is capable of dealing in a single, combined fashion, with the two mirror-imaged interferograms arising in this kind of experiment as a result of the acquisition of a train of magnetic field gradient echoes. By combining all the acquired data points into a common Fourier processing procedure the spectral width along the direct-acquisition domain becomes effectively doubled, giving the opportunity of employing acquisition gradients that are approximately half as strong as hitherto required. This in turn should lead to an overall enhancement in the signal-to-noise ratio of the experiment of ca. 2, as well as to improvements in the achievable digital resolution. These expectations were tested by carrying out a series of homo- and heteronuclear ultrafast 2D NMR acquisitions, and found systematically fulfilled. The robustness and conditions that allow the interlaced numerical procedure to be implemented in routine analytical applications were explored and are briefly discussed.

Fourier Analysis↗
PubMed1991

Afferent regulation of locus coeruleus neurons: anatomy, physiology and pharmacology.

Tract-tracing and electrophysiology studies have revealed that major inputs to the nucleus locus coeruleus (LC) are found in two structures, the nucleus paragigantocellularis (PGi) and the perifascicular area of the nucleus prepositus hypoglossi (PrH), both located in the rostral medulla. Minor afferents to LC were found in the dorsal cap of the paraventricular hypothalamus and spinal lamina X. Recent studies have also revealed limited inputs from two areas nearby the LC, the caudal midbrain periaqueductal gray (PAG) and the ventromedial pericoerulear region. The pericoeruleus may provide a local circuit interface to LC neurons. Recent electron microscopic analyses have revealed that LC dendrites extend preferentially into the rostromedial and caudal juxtaependymal pericoerulear regions. These extracoerulear LC dendrites may receive afferents in addition to those projecting to LC proper. However, single-pulse stimulation of inputs to such dendritic regions reveals little or no effect on LC neurons. Double-labeling studies have revealed that a variety of neurotransmitters impinging on LC neurons originate in its two major afferents, PGi and PrH. The LC is innervated by PGi neurons that stain for markers of adrenalin, enkephalin or corticotropin-releasing factor. Within PrH, large proportions of LC-projecting neurons stained for GABA or met-enkephalin. Finally, in contrast to previous conclusions, the dorsal raphe does not provide the robust 5-HT innervation found in the LC. We conclude that 5-HT inputs may derive from local 5-HT neurons in the pericoerulear area. Neuropharmacology experiments revealed that the PGi provides a potent excitatory amino acid (EAA) input to the LC, acting primarily at non-NMDA receptors in the LC. Other studies indicated that this pathway mediates certain sensory responses of LC neurons. NMDA-mediated sensory responses were also revealed during local infusion of magnesium-free solutions. Finally, adrenergic inhibition of LC from PGi could also be detected in nearly every LC neuron tested when the EAA-mediated excitation is first eliminated. In contrast to PGi, the PrH potently and consistently inhibited LC neurons via a GABAergic projection acting at GABAA receptors within LC. Such PrH stimulation also potently attenuated LC sensory responses. Finally, afferents to PGi areas that also contain LC-projecting neurons were identified. Major inputs were primarily autonomic in nature, and included the caudal medullary reticular formation, the parabrachial and Kölliker-Fuse nuclei, the PAG, NTS and certain hypothalamic areas.(ABSTRACT TRUNCATED AT 400 WORDS)

Afferent Pathways↗
PubMed1999

The distribution of 3,4-methylenedioxymethamphetamine "Ecstasy"-induced c-fos expression in rat brain.

Rats were injected with 3,4-methylenedioxymethamphetamine ("Ecstasy") and assessed for changes in locomotor activity and for the expression of the immediate early gene c-fos throughout the brain. A dose-dependent increase in locomotor activity was seen with 3,4-methylenedioxymethamphetamine (0, 5 and 20 mg/kg) that continued for at least 2 h following administration. Dose-dependent increases in c-fos expression were seen in much of the cortex, forebrain, brainstem and cerebellum in rats given 3,4-methylenedioxymethamphetamine. Expression was pronounced in 5-hydroxytryptamine terminal regions including the medial prefrontal cortex, caudate-putamen, nucleus accumbens, olfactory tubercle, islands of Calleja, lateral septum, paraventricular hypothalamus and paraventricular thalamus. High levels of c-fos expression were also seen in the supraoptic and median preoptic nuclei, regions involved in the control of fluid balance and body temperature, respectively. This is potentially important since deaths in 3,4-methylenedioxymethamphetamine users have been linked to hyperthermia and hyponatremia. In the brainstem, two regions of high c-fos expression were Barrington's nucleus, which is involved in micturition, and the pontine reticular nucleus oralis, a region involved in motor control of mastication. Activation of this latter structure may partly explain the bruxism (grinding of the jaw) reported by human 3,4-methylenedioxymethamphetamine users. Robust c-fos expression was seen in the cerebellum, particularly in the flocculus, and this may explain the reported deleterious effects of 3,4-methylenedioxymethamphetamine on balance and co-ordination. Significant c-fos expression was also seen in the ventral tegmental area, amidst the cell bodies of mesolimbic and mesocortical dopamine neurons, and in the median and dorsal raphe, where the serotonergic innervation of the forebrain originates. Double-labelling of fos-positive neurons with 5-hydroxytryptamine showed that only a small number of serotonergic neurons in the raphe expressed c-fos following 3,4-methylenedioxymethamphetamine. The widespread distribution of 3,4-methylenedioxymethamphetamine-induced c-fos expression seen in this study can be linked to the profound alterations in physiological function, mood and behaviour produced by this drug.

Animals↗
PubMedDate not supplied

Optimization of tryptophan production in bacteria. Design of a strategy for genetic manipulation of the tryptophan operon for tryptophan flux maximization.

In the present work we have applied the indirect optimization method (Torres, N. V. et al. Biotechnol. Bioeng. 1996, 49, 247-258) to the maximization of tryptophan biosynthesis in Escherichia coli. The optimization procedure is applied to an updated model of this biochemical system (Xiu, Z-L et al., J. Biotechnol. 1997, 58, 125-140) and thus extended to a problem that includes the processes of transcription and translation. The model representation used by these authors is first translated into the corresponding S-system version. Then, to guarantee cell viability, we impose a set of constraints on some variable and parameter values, all of which are able to be modulated by available techniques. Our results show that it is possible to attain a stable and robust steady state with a rate of tryptophan production increased more than 4 times. This is achieved by changing four key parameters related to the efflux of tryptophan, the growth rate, the inhibition constant, and the tryptophan repressor level. Moreover, it is demonstrated that we can reach this optimum state in a sequential manner, each step leading us to a better situation in relation to the previous one. Thus, only by doubling the tryptophan excretion we can triplicate the rate of tryptophan production. A further, although lesser, improvement can be attained by increasing 4-fold the rate of growth and subsequently by weakening the inhibitory feedback interaction of tryptophan on the enzymes leading to its synthesis. Finally, a significant jump in the rate of production can be obtained if the level of the trp operon could be decreased. When a second approach was considered, in which the growth rate is kept constant in the optimized profile, we found out that by modulation of the parameters it is possible to increase more than 2-fold the rate of tryptophan production.

Biotechnology↗
PubMed2005

Methylation-specific MLPA (MS-MLPA): simultaneous detection of CpG methylation and copy number changes of up to 40 sequences.

Copy number changes and CpG methylation of various genes are hallmarks of tumor development but are not yet widely used in diagnostic settings. The recently developed multiplex ligation-dependent probe amplification (MLPA) method has increased the possibilities for multiplex detection of gene copy number aberrations in a routine laboratory. Here we describe a novel robust method: the methylation-specific MLPA (MS-MLPA) that can detect changes in both CpG methylation as well as copy number of up to 40 chromosomal sequences in a simple reaction. In MS-MLPA, the ligation of MLPA probe oligonucleotides is combined with digestion of the genomic DNA-probe hybrid complexes with methylation-sensitive endonucleases. Digestion of the genomic DNA-probe complex, rather than double-stranded genomic DNA, allowed the use of DNA derived from the formalin treated paraffin-embedded tissue samples, enabling retrospective studies. To validate this novel method, we used MS-MLPA to detect aberrant methylation in DNA samples of patients with Prader-Willy syndrome, Angelman syndrome or acute myeloid leukemia.

Acute Disease↗
PubMed2006

Metallothioneins and zinc dysregulation contribute to neurodevelopmental damage in a model of perinatal viral infection.

Neonatal Borna disease (NBD) virus infection in the Lewis rat results in life-long viral persistence and causes behavioral and neurodevelopmental abnormalities. A hallmark of the disorder is progressive loss of cerebellar Purkinje and dentate gyrus granule cells. Findings of increased brain metallothionein-I and -II (MT-I/-II) mRNA expression in cDNA microarray experiments led us to investigate MT isoforms and their relationship to brain zinc metabolism, cellular toxicity, and neurodevelopmental abnormalities in this model. Real-time PCR confirmed marked induction of MT-I/-II mRNA expression in the brains of NBD rats (40.5-fold increase in cerebellum, p<0.0001; 6.8-fold increase in hippocampus, p=0.003; and 9.5-fold increase in striatum, p=0.0012), whereas a trend toward decreased MT-III mRNA was found in hippocampus (1.25-fold decrease, p=0.0841). Double label immunofluorescence revealed prominent MT-I/-II expression in astrocytes throughout the brain; MT-III protein was decreased in granule cell neurons and increased in astrocytes, with differential subcellular distribution from cytoplasmic to nuclear compartments in NBD rat hippocampus. Modified Timm staining of hippocampus revealed reduced zinc in mossy fiber projections to the hilus and CA3, accumulation of zinc in glial cells and degenerating granule cell somata, and robust mossy fiber sprouting into the inner molecular layer of the dentate gyrus. Zinc Transporter 3 (ZnT-3) mRNA expression was decreased in hippocampus (2.3-fold decrease, p= 0.0065); staining for its correlate protein was reduced in hippocampal mossy fibers. Furthermore, 2 molecules implicated in axonal pathfinding and mossy fiber sprouting, the extracellular matrix glycoprotein, tenascin-R (TN-R), and the hyaluronan receptor CD44, were increased in NBD hippocampal neuropil. Abnormal zinc metabolism and mechanisms of neuroplasticity may contribute to the pathogenesis of disease in this model, raising more general implications for neurodevelopmental damage following viral infections in early life.

Animals↗
PubMed2006

Adjuvant endocrine therapy for postmenopausal women with early breast cancer.

Results from multiple clinical trials involving aromatase inhibitors have added to the knowledge base relating to endocrine therapy of postmenopausal women with hormone receptor-positive early breast cancer. In the extended adjuvant setting, data from the Austrian Breast and Colorectal Cancer Study Group 6a trial showed an advantage for anastrozole following 5 years of tamoxifen treatment, consistent with the more robust MA.17 trial that examined letrozole versus placebo following 5 years of tamoxifen treatment. The combined analysis of the Austrian Breast and Colorectal Cancer Study Group 8 trial and the German Arimidex Nolvadex 95 trial, plus the Italian Tamoxifen Anastrozole trial, have shown the advantage of switching to anastrozole over continuing the tamoxifen to complete the full 5 years of adjuvant therapy. These trials support the previously reported larger and double-blind Intergroup Exemestane Study. The Arimidex, Tamoxifen, Alone or in Combination trial now has data out to 68 months of median follow-up showing the maintenance of a significant advantage of anastrozole over tamoxifen for disease-free survival. In this initial treatment setting, the Breast International Group 1-98 trial recently showed a significant advantage for letrozole over tamoxifen. The current debate is centered on whether the optimal strategy is to give an aromatase inhibitor initially or after several years of tamoxifen treatment. Multiple important questions remain, including the predictive value of molecular markers such as progesterone receptor, the optimal duration of aromatase inhibitor therapy, the long-term adverse effects, and the relative efficacy and toxicity of the different aromatase inhibitors.

Anastrozole↗
PubMed2005

Wearable Conductive Fiber Sensors for Multi-Axis Human Joint Angle Measurements.

BACKGROUND: The practice of continuous, long-term monitoring of human joint motion is one that finds many applications, especially in the medical and rehabilitation fields. There is a lack of acceptable devices available to perform such measurements in the field in a reliable and non-intrusive way over a long period of time. The purpose of this study was therefore to develop such a wearable joint monitoring sensor capable of continuous, day-to-day monitoring. METHODS: A novel technique of incorporating conductive fibers into flexible, skin-tight fabrics surrounding a joint is developed. Resistance changes across these conductive fibers are measured, and directly related to specific single or multi-axis joint angles through the use of a non-linear predictor after an initial, one-time calibration. Because these sensors are intended for multiple uses, an automated registration algorithm has been devised using a sensitivity template matched to an array of sensors spanning the joints of interest. In this way, a sensor array can be taken off and put back on an individual for multiple uses, with the sensors automatically calibrating themselves each time. RESULTS: The wearable sensors designed are comfortable, and acceptable for long-term wear in everyday settings. Results have shown the feasibility of this type of sensor, with accurate measurements of joint motion for both a single-axis knee joint and a double axis hip joint when compared to a standard goniometer used to measure joint angles. Self-registration of the sensors was found to be possible with only a few simple motions by the patient. CONCLUSION: After preliminary experiments involving a pants sensing garment for lower body monitoring, it has been seen that this methodology is effective for monitoring joint motion of the hip and knee. This design therefore produces a robust, comfortable, truly wearable joint monitoring device.

Journal Article↗
PubMed2004

Modeling feedback loops of the Mammalian circadian oscillator.

The suprachiasmatic nucleus governs daily variations of physiology and behavior in mammals. Within single neurons, interlocked transcriptional/translational feedback loops generate circadian rhythms on the molecular level. We present a mathematical model that reflects the essential features of the mammalian circadian oscillator to characterize the differential roles of negative and positive feedback loops. The oscillations that are obtained have a 24-h period and are robust toward parameter variations even when the positive feedback is replaced by a constantly expressed activator. This demonstrates the crucial role of the negative feedback for rhythm generation. Moreover, it explains the rhythmic phenotype of Rev-erbalpha-/- mutant mice, where a positive feedback is missing. The interplay of negative and positive feedback reveals a complex dynamics. In particular, the model explains the unexpected rescue of circadian oscillations in Per2Brdm1/Cry2-/- double-mutant mice (Per2Brdm1 single-mutant mice are arrhythmic). Here, a decrease of positive feedback strength associated with mutating the Per2 gene is compensated by the Cry2-/- mutation that simultaneously decreases the negative feedback strength. Finally, this model leads us to a testable prediction of a molecular and behavioral phenotype: circadian oscillations should be rescued when arrhythmic Per2Brdm1 mutant mice are crossed with Rev- erbalpha -/- mutant mice.

Adaptation, Physiological↗
PubMed2001

fMRI of auditory stimulation with intermolecular double-quantum coherences (iDQCs) at 1.5T.

An intermolecular double-quantum coherence (iDQC) imaging technique was used to study auditory activation in the human brain at 1.5T with a dual temporal lobe surface phased array coil and a quadrature head coil. Preliminary results demonstrate that it is feasible to obtain auditory activation maps using iDQC imaging at 1.5T, both in individual subjects using the surface coil array and with multisubject averaging of data using the head coil. The most robust activation map was obtained when a spin-echo (SE) acquisition was combined with an iDQC excitation. Since SE with conventional single quantum coherence (SQC) and similar parameters showed much reduced activation in spite of its higher signal-to-noise ratio (SNR), it was determined that activation resulting from the SE-iDQC acquisition almost entirely originates from iDQCs. In addition, the fact that the robust activation was obtained using signals at an evolution time more sensitive to changes in magnetic susceptibilities also suggests the sensitivity of iDQCs to the BOLD effect upon activation. iDQCs provide a novel MRI method which is potentially more sensitive to the BOLD effect traditionally measured with SQC. Magn Reson Med 45:356-364, 2001.

Acoustic Stimulation↗
PubMed2005

Real-time 3-D dark-field microscopy for the validation of the cross-linking process of alginate microcapsules.

Alginate-based microencapsulation is a promising method for long-term maintenance of cellular and membrane function of the cells and tissue fragments required for in vitro and in vivo biosensors, for tissue engineering and particularly for immunoisolation of non-autologous transplants. Microcapsules of high mechanical strength and optimum permeability can be produced by injection of BaCl2 crystals into alginate droplets before they come into contact with external Ba2+. A key requirement is that the system parameters (number of crystals, speed of the crystal stream etc.) are properly adjusted according to the mannuronic and guluronic acid ratio and the average molecular mass of the alginate as well as to the diameter of the microcapsules. Robust, reliable, rapid and low-cost validation tools are, therefore, needed for assurance of the microcapsule quality. Here, we describe a novel three-dimensional (3-D) dark-field microscopy that allows the real-time measurement of the number and spatial distribution of the injected Ba2+ ions throughout the microcapsules after treatment with sulphate. This novel method requires only a conventional microscope equipped with three polarising filters and a double aperture stop. In contrast to confocal laser scanning microscopy images, peripherally attached BaSO4 precipitates can clearly be distinguished from internal ones. The data also demonstrate that several steps of the alginate gelling process must be improved before such immunoisolation can be used in patients.

Alginates↗
PubMed2002

Comparative study with double-exposure digital holographic interferometry and a shack-hartmann sensor to characterize transparent materials.

We compare wave-front measurements using double-exposure digital holography and a Shack-Hartmann sensor. A voltage-driven liquid-crystal wedge modulates the optical wave front and provides a refractive-index gradient typical of interesting transparent materials. Measurement accuracy and reliability are similar for both methods. In our opinion, digital holographic interferometry has several advantages for both laboratory and field environments. When compared with Shack-Hartmann methods, these advantages include hardware simplicity and robustness, relative insensitivity to sample dynamic range, and less computational demanding and more straightforward data evaluation algorithms. We believe that digital holography provides the methodology of choice for field studies of transparent materials such as microgravity protein crystal growth experiments.

Journal Article↗
PubMed2003

Efficacy of cervical endplay assessment as an indicator for spinal manipulation.

STUDY DESIGN: Double-blind, randomized, placebo-controlled trial. OBJECTIVES: To evaluate the effect of manual endplay assessment on neck pain and stiffness outcomes in neck pain patients receiving spinal manipulation. SUMMARY OF THE BACKGROUND DATA: There have been no studies on the efficacy of palpation used as an indicator for manipulation in the management of back and neck pain. METHODS: Neck pain patients (n = 104) were randomly assigned to two groups. The study group received manipulation targeted to individual cervical vertebrae according to endplay restriction noted by the examining clinician. The control group received manipulation determined by sham, computer-generated examination findings; endplay examination was ignored and served as a placebo assessment. Treatment was rendered on a single occasion by a chiropractor. Outcomes were neck pain and stiffness assessed before and after manipulation and at least 5 hours following treatment. RESULTS: The study and control groups showed clinically important improvement in neck pain and stiffness. However, there were no clinically important or statistically significant differences between the study and control groups in terms of pain or stiffness outcomes. Findings were robust across patient, complaint, and treatment characteristics. CONCLUSIONS: Endplay assessment in and of itself did not contribute to the same-day pain and stiffness relief observed in neck pain patients receiving spinal manipulation. The impact on a longer course of treatment remains to be investigated. The data suggest that pain modulation may not be limited to mechanisms associated with manipulation of putative motion restrictions.

Adult↗
PubMed2000

dADAR, a Drosophila double-stranded RNA-specific adenosine deaminase is highly developmentally regulated and is itself a target for RNA editing.

We have identified a homolog of the ADAR (adenosine deaminases that act on RNA) class of RNA editases from Drosophila, dADAR. The dADAR locus has been localized to the 2B6-7 region of the X chromosome and the complete genomic sequence organization is reported here. dADAR is most homologous to the mammalian RNA editing enzyme ADAR2, the enzyme that specifically edits the Q/R site in the pre-mRNA encoding the glutamate receptor subunit GluR-B. Partially purified dADAR expressed in Pichia pastoris has robust nonspecific A-to-I deaminase activity on synthetic dsRNA substrates. Transcripts of the dADAR locus originate from two regulated promoters. In addition, alternative splicing generates at least four major dADAR isoforms that differ at their amino-termini as well as altering the spacing between their dsRNA binding motifs. dADAR is expressed in the developing nervous system, making it a candidate for the editase that acts on para voltage-gated Na+ channel transcripts in the central nervous system. Surprisingly, dADAR itself undergoes developmentally regulated RNA editing that changes a conserved residue in the catalytic domain. Taken together, these findings show that both transcription and processing of dADAR transcripts are under strict developmental control and suggest that the process of RNA editing in Drosophila is dynamically regulated.

Adenosine Deaminase↗
PubMed1995

D3 and D2 dopamine receptors: visualization of cellular expression patterns in motor and limbic structures.

The distribution of the D3 and D2 dopamine receptor subtypes in forebrain regions of the basal ganglia and mesocorticolimbic system was determined. This was assessed through combined fluorescent visualization of subtype selective anti-peptide antibodies for these cloned receptors and detection of their ligand recognition sites using the D2 subfamily antagonist,N-(p-aminophenethyl) spiperone (NAPS fluoroprobe). The double-labeling technique enabled direct comparison of the cloned receptor proteins and NAPS fluoroprobe binding in vitro. The application of these two methods together produced results comparable to single-labeling paradigms. Functional D3 receptors, defined as the coincident fluorescence of the D3 receptor antisera and fluoroprobe binding, were detected in the core region of the nucleus accumbens and exhibited a laminated expression pattern in the frontal cortex. D3 receptor protein was expressed robustly in neurons of the dorsolateral striatum, but showed an intense neuropil reaction in the globus pallidus. Functional D2 receptors, defined as the coincident fluorescence of the D2 receptor antisera and fluoroprobe binding, were detected in the frontal cortex and the medial shell of the nucleus accumbens. Thus, heterogeneities occurred in the cellular expression of functional D3 and D2 receptors in forebrain dopaminoceptive areas. D3 appears more related to basal ganglia and structures involved with motoric behavior, while D2 was associated with regions associated with cognitive/affective functions.

Animals↗