Abnormal thalamocortical activity in patients with Complex Regional Pain Syndrome (CRPS) type I.

Complex Regional Pain Syndrome (CRPS) is a neuropathic disease that presents a continuing challenge in terms of pathophysiology, diagnosis, and treatment. Recent studies of neuropathic pain, in both animals and patients, have established a direct relationship between abnormal thalamic rhythmicity related to Thalamo-cortical Dysrhythmia (TCD) and the occurrence of central pain. Here, this relationship has been examined using magneto-encephalographic (MEG) imaging in CRPS Type I, characterized by the absence of nerve lesions. The study addresses spontaneous MEG activity from 13 awake, adult patients (2 men, 11 women; age 15-62), with CRPS Type I of one extremity (duration range: 3months to 10years) and from 13 control subjects. All CRPS I patients demonstrated peaks in power spectrum in the delta (<4Hz) and/or theta (4-9Hz) frequency ranges resulting in a characteristically increased spectral power in those ranges when compared to control subjects. The localization of such abnormal activity, implemented using independent component analysis (ICA) of the sensor data, showed delta and/or theta range activity localized to the somatosensory cortex corresponding to the pain localization, and to orbitofrontal-temporal cortices related to the affective pain perception. Indeed, CRPS Type I patients presented abnormal brain activity typical of TCD, which has both diagnostic value indicating a central origin for this ailment and a potential treatment interest involving pharmacological and electrical stimulation therapies.

Protein tyrosine and serine-threonine phosphatases in the sea urchin, Strongylocentrotus purpuratus: identification and potential functions.

Protein phosphatases, in coordination with protein kinases, play crucial roles in regulation of signaling pathways. To identify protein tyrosine phosphatases (PTPs) and serine-threonine (ser-thr) phosphatases in the Strongylocentrotus purpuratus genome, 179 annotated sequences were studied (122 PTPs, 57 ser-thr phosphatases). Sequence analysis identified 91 phosphatases (33 conventional PTPs, 31 dual specificity phosphatases, 1 Class III Cysteine-based PTP, 1 Asp-based PTP, and 25 ser-thr phosphatases). Using catalytic sites, levels of conservation and constraint in amino acid sequence were examined. Nine of 25 receptor PTPs (RPTPs) corresponded to human, nematode, or fly homologues. Domain structure revealed that sea urchin-specific RPTPs including two, PTPRLec and PTPRscav, may act in immune defense. Embryonic transcription of each phosphatase was recorded from a high-density oligonucleotide tiling microarray experiment. Most RPTPs are expressed at very low levels, whereas nonreceptor PTPs (NRPTPs) are generally expressed at moderate levels. High expression was detected in MAP kinase phosphatases (MKPs) and numerous ser-thr phosphatases. For several expressed NRPTPs, MKPs, and ser-thr phosphatases, morpholino antisense-mediated knockdowns were performed and phenotypes obtained. Finally, to assess roles of annotated phosphatases in endomesoderm formation, a literature review of phosphatase functions in model organisms was superimposed on sea urchin developmental pathways to predict areas of functional activity.

The Stat3/5 locus encodes novel endoplasmic reticulum and helicase-like proteins that are preferentially expressed in normal and neoplastic mammary tissue.

The signal transducers and activators of transcription (STAT) 5 and 3 are critical for mammary alveolar development during pregnancy and remodeling during involution. In the mouse, STAT3, STAT5a, and STAT5b are encoded by adjacent genes on chromosome 11 (60.5 cM). To identify additional genes in the Stat3/5 locus that may participate in normal and neoplastic development of the mammary gland, we have cloned and sequenced 500 kb and searched for genes preferentially expressed in mammary tissue. We identified six known genes and cloned two new genes, termed D11Lgp1 and D11Lgp2. Both genes are most highly expressed in normal mammary tissue and mammary tumors from several transgenic mouse models. LGP1 consists of 532 and 530 amino acids in mouse and human, respectively (88% similarity). A region in the carboxy-terminal half of LGP1 has limited homology with Arabidopsis thaliana GH3-like proteins. Immunofluorescence studies demonstrated that LGP1 is located in the nuclear envelope and the endoplasmic reticulum. LGP2 is a cytoplasmic protein of 678 amino acids.

Conditional deletion of the bcl-x gene from mouse mammary epithelium results in accelerated apoptosis during involution but does not compromise cell function during lactation.

In the mammary gland Bcl-x is the most abundant cell survival factor from the Bcl-2 family. Since Bcl-x null mice die around day 12 of embryogenesis, the relevance of this protein in organ development and function is poorly understood. In erythroid cells bcl-x gene expression is controlled by cytokines and the transcription factor Stat5 (signal transducer and activator of transcription). However, we identified that bcl-x RNA levels in mammary tissue from prolactin receptor- and Stat5-null mice were indistinguishable from wild type mice. We have proposed that Bcl-x might control the survival of mammary epithelial cells throughout pregnancy, lactation, and the early stages of involution, and we have now tested this hypothesis through the conditional deletion of the bcl-x gene from mouse mammary epithelium. Conditional (floxed) bcl-x alleles were excised from alveolar cells during pregnancy using a Cre transgene under the control of the whey acidic protein gene promoter. Deletion of the bcl-x gene from the entire epithelial compartment (ducts and alveoli) was achieved by expressing Cre-recombinase under control of the mouse mammary tumor virus long terminal repeat. The absence of Bcl-x did not compromise proliferation and differentiation of mammary ductal and alveolar epithelial cells in virgin mice and during pregnancy and lactation. However, epithelial cell death and tissue remodeling were accelerated in the bcl-x conditional knockout mice during the first stage of involution. Concomitant deletion of the bax gene did not significantly modify the Bcl-x phenotype. Our results suggest that Bcl-x is not essential during mammopoiesis, but is critical for controlled apoptosis during the first phase of involution.

Space flight affects magnocellular supraoptic neurons of young prepuberal rats: transient and permanent effects.

Effects of microgravity on postural control and volume of extracellular fluids as well as stress associated with space flight may affect the function of hypothalamic neurosecretory neurons. Since environmental modifications in young animals may result in permanent alterations in neuroendocrine function, the present study was designed to determine the effect of a space flight on oxytocinergic and vasopressinergic magnocellular hypothalamic neurons of prepuberal rats. Fifteen-day-old Sprague-Dawley female rats were flown aboard the Space Shuttle Columbia (STS-90, Neurolab mission, experiment 150) for 16 days. Age-matched litters remained on the ground in cages similar to those of the flight animals. Six animals from each group were killed on the day of landing and eight animals from each group were maintained under standard vivarium conditions and killed 18 weeks after landing. Several signs of enhanced transcriptional and biosynthetic activity were observed in magnocellular supraoptic neurons of flight animals on the day of landing compared to control animals. These include increased c-Fos expression, larger nucleoli and cytoplasm, and higher volume occupied in the neuronal perikaryon by mitochondriae, endoplasmic reticulum, Golgi apparatus, lysosomes and cytoplasmic inclusions known as nematosomes. In contrast, the volume occupied by neurosecretory vesicles in the supraoptic neuronal perikarya was significantly decreased in flight rats. This decrease was associated with a significant decrease in oxytocin and vasopressin immunoreactive levels, suggestive of an increased hormonal release. Vasopressin levels, cytoplasmic volume and c-Fos expression returned to control levels by 18 weeks after landing. These reversible effects were probably associated to osmotic stimuli resulting from modifications in the volume and distribution of extracellular fluids and plasma during flight and landing. However, oxytocin levels were still reduced at 18 weeks after landing in flight animals compared to controls. This indicates that space flight during prepuberal age may induce irreversible modifications in the regulation of oxytocinergic neurons, which in turn may result in permanent endocrine and behavioral impairments.

The microarray explorer tool for data mining of cDNA microarrays: application for the mammary gland.

The Microarray Explorer (MAExplorer) is a versatile Java-based data mining bioinformatic tool for analyzing quantitative cDNA expression profiles across multiple microarray platforms and DNA labeling systems. It may be run as either a stand-alone application or as a Web browser applet over the Internet. With this program it is possible to (i) analyze the expression of individual genes, (ii) analyze the expression of gene families and clusters, (iii) compare expression patterns and (iv) directly access other genomic databases for clones of interest. Data may be downloaded as required from a Web server or in the case of the stand-alone version, reside on the user's computer. Analyses are performed in real-time and may be viewed and directly manipulated in images, reports, scatter plots, histograms, expression profile plots and cluster analyses plots. A key feature is the clone data filter for constraining a working set of clones to those passing a variety of user-specified logical and statistical tests. Reports may be generated with hypertext Web access to UniGene, GenBank and other Internet databases for sets of clones found to be of interest. Users may save their explorations on the Web server or local computer and later recall or share them with other scientists in this groupware Web environment. The emphasis on direct manipulation of clones and sets of clones in graphics and tables provides a high level of interaction with the data, making it easier for investigators to test ideas when looking for patterns. We have used the MAExplorer to profile gene expression patterns of 1500 duplicated genes isolated from mouse mammary tissue. We have identified genes that are preferentially expressed during pregnancy and during lactation. One gene we identified, carbonic anhydrase III, is highly expressed in mammary tissue from virgin and pregnant mice and in gene knock-out mice with underdeveloped mammary epithelium. Other genes, which include those encoding milk proteins, are preferentially expressed during lactation.

Transcription originating in the long terminal repeats of the endogenous mouse mammary tumor virus MTV-3 is activated in Stat5a-null mice and picks Up hitchhiking exons.

The enhancer within the long terminal repeats (LTRs) of acquired somatic mouse mammary tumor viruses (MMTV) can activate juxtaposed genes and induce mammary tumors. In contrast, germ line proviral MMTV genomes are integrated in the host genome and considered to be genetically confined transcription units. Here we demonstrate that transcription initiated in an MMTV provirus proceeds into flanking host sequences. We discovered multiple polyadenylated transcripts which are induced in Stat5a null mice. These range from 1.5 kb to more than 8 kb and are specifically expressed in mammary tissue from pregnant and lactating mice from the 129 but not C57BL/6 strain. The RNAs emanate from both LTRs of the endogenous MTV-3 provirus on chromosome 11 and proceed at least 10 kb into the juxtaposed genomic territory. Transcripts originating in the 5' LTR splice from the native splice site within the MMTV envelope gene into at least six exons, three of which contain functional internal splice sites. The combination of alternative splicing and the use of several polyadenylation sites ensure the generation of multiple transcripts. To date no significant open reading frame has been discovered. Furthermore, we demonstrate that transcription from the MMTV 5' LTR is highly active in the absence of Stat5a, a transcription factor that had been shown previously to be required for transcription from the MMTV LTR.

Identification of a critical period for motor development in neonatal rats.

Manipulation of the developing nervous system has provided valuable insights into nervous system function. One important concept to arise from this type of study has been the identification of specific "critical periods" for the development of various functions. A critical period has been most clearly shown for the visual system where monocular eye closure for a few weeks led to functionally significant changes in visually guided behaviors and the connectivity of the visual cortex. Critical periods have also been defined for other sensory systems. Although studies of the effect of manipulating sensory systems during development are sometimes difficult to interpret (e.g. Ref. 7), this difficulty is compounded in the case of the motor system. Problems arise because manipulations of the postnatal motor system are difficult to implement and usually require invasive procedures such as tenotomy, neurotomy, and nerve crush (for review, see Ref. 17). We have approached the problem of manipulating the motor environment by adapting a paradigm widely used to study the experimental effects of simulated weightlessness in adult rats: namely, tail suspension. This method has several advantages for manipulating the motor system: (i) because it is noninvasive, it is less discomforting than neurotomy, tenotomy or nerve crush; (ii) it does not immobilize the animals, they move about the cage and extend and flex their hindlimbs; and (iii) it specifically examines the importance of load-bearing on the development of antigravity muscles and their neuronal circuits.(ABSTRACT TRUNCATED AT 250 WORDS)

Postnatal changes in motoneurone electrotonic coupling studied in the in vitro rat lumbar spinal cord.

1. Electrotonic coupling between motoneurones innervating ankle flexor and extensor muscles, as well as between unidentified lumbar motoneurones, was studied using intracellular recordings in an in vitro spinal cord-hindlimb preparation isolated from rats between birth (P0) and 13 days (P13). 2. Graded ventral root stimulation could elicit graded, short latency depolarizations (SLD) which preceded, coincided with, or followed the antidromic action potential. These SLDs were identified as electrotonic junctional potentials by their latency, relative insensitivity to changes in membrane potential and their resistance to one or more of the following: (1) high-frequency stimulation, (2) collision with a somatofugal action potential, (3) removal of Ca2+ from the bathing solution. 3. SLDs were studied in 162 neurones and were identified in 77.2% of the cells in preparations from P0 to P3 rats (n = 57), but only in 30.8% at P8 to P13 (n = 39). 4. SLDs were largest in the youngest animals (P0 to P3), decreasing from a mean of 1.31 mV (+/- 0.17, n = 34) to 0.56 mV (+/- 0.10, n = 7) at P8 to P13. The SLDs comprised two to eight (4.3 +/- 0.36) all-or-none components as determined from twenty collision experiments. 5. Electrotonic coupling between motoneurones was specific. SLDs could be elicited in given motoneurones by stimulation of their homonymous but never of their antagonistic muscle nerves. 6. These results indicate that electrotonic coupling between lumbar motoneurones in neonatal animals exhibits a high degree of specificity and that its significance, as judged by the amplitude and frequency of occurrence of SLDs, decreases postnatally at a rate that can be correlated with the functional maturity of the motoneurones and the muscular system.

The effectiveness of different isomers of octanol as blockers of harmaline-induced tremor.

Intracellular recording in the guinea-pig brainstem slice has demonstrated that high molecular weight alcohols block the low threshold calcium channel (LTCC) in the inferior olive (IO). These alcohols thus provide a tool for understanding the function of the pacemaking cellular networks of the olivo-cerebellar system, since the LTCC has been implicated in the oscillatory behavior of these neurons. Aspects of normal and pathological tremor are also believed to be mediated by these circuits, and thus development of effective ways of blocking the LTCC in vivo may eventually lead to novel treatments for essential tremor. The present experiments evaluated the effectiveness of the isomers of octanol in decreasing harmaline-induced tremor in vivo in the rat. Harmaline was used in this study because its tremorgenic action is mediated at the level of IO; octanol was found to be a potent antagonist of harmaline-induced tremor. Significant differences between the isomers further suggested conformational differences. This, taken in conjunction with the lack of effect of octanol in both IO lesioned rats and oxotremorine-induced tremor, implied that the action of the alcohol may be mediated at a specific binding site. These findings thus support the conclusions that the antagonism of harmaline-induced tremor by octanol occurs in the IO, and, in view of the previously reported in vitro data, that octanol may be an effective blocker of the LTCC in vivo.

Activity-related extracellular potassium transients in the neonatal rat spinal cord: an in vitro study.

Transient increases and decreases in extracellular potassium (delta[K+]o) were recorded from the gray matter of hemisected, neonatal rat spinal cords isolated from 3, 4, 9- and 10-day-old pups. delta[K+]o were evoked in both the ventral and dorsal regions of the gray matter by electrical stimulation. In the ventral horn, repetitive stimulation of the ventral root was required to elicit detectable delta[K+]o. By contrast, single dorsal root stimuli evoked clear delta[K+]o. In the dorsal horn, single orthodromic stimuli elicited delta[K+]o as large as 4-5 mM from a baseline of 4.5 mM. With repetitive stimulation the [K+]o reached, but never exceeded, a ceiling of 10-11 mM. Undershoots were seen only after repetitive stimulation. Spontaneous delta[K+]o were observed in the ventral horn and were well correlated with ventral root activity. Spontaneous delta[K+]o were rare in the dorsal cord, but were recorded after bath application of apamin or tetraethylammonium. The magnitude and distribution of evoked K+ transients and postsynaptic components of the evoked field potential were well correlated in both the dorsal and the ventral gray matter. delta[K+]o were reversibly blocked by 1 mM CdCl2 in the bath and diminished by 1 mM BaCl2. Bath application of mephenesin, apamin or tetraethylammonium diminished evoked delta[K+]o in a stimulus-dependent manner. In apamin and tetraethylammonium, decreases from control responses were largest with high intensity stimulation, the opposite was the case with mephenesin. These results are interpreted in terms of the spinal circuits activated by high- and low-intensity electrical stimulation. We conclude that activity-related delta[K+]o in neonatal spinal cord are large enough to modulate neuronal electrical activity and the [K+]o is well regulated compared to other immature CNS regions studied. Thus, local increases in [K+]o may, by modulating neuronal activity, play a role in neonatal spinal cord developmental processes.