Reduction in re-rupture rates following implementation of return-to-sport testing after anterior cruciate ligament reconstruction in 313 patients with a mean follow-up of 50 months.

OBJECTIVES: The objective of this study was to assess the mid-term effectiveness of a return to sport (RTS) test in relation to preventing anterior cruciate ligament (ACL) re-rupture and contralateral ACL injury following ACL reconstruction (ACLR). Furthermore, this study aimed to assess the timing of passing a, RTS-test after surgery, and the effect age has on RTS outcomes. METHODS: Patients undergoing ACLR between August 2014 and December 2018 took an RTS-test following rehabilitation. The RTS-test consisted of the Anterior Cruciate Ligament Return to Sport After Injury Scale, a single-leg hop, a single-leg triple hop, a single-leg triple cross-over hop, a box-drop vertical jump down, a single-leg 4-rep max-incline leg press, and a modified agility T test. RTS-passing criteria were ≥90% limb symmetry index in addition to defined takeoff and landing parameters. Mid-term review assessed sporting level, ACL re-injury, and contralateral ACL injury. RESULTS: A total of 352 patients underwent RTS-testing, following ACLR with 313 (89%) contactable at follow-up, a mean of 50 months (standard deviation: 11.41, range: 28-76) after surgery. The re-rupture rate was 6.6% after passing the RTS-test and 10.3% following failure (p â€‹= â€‹0.24), representing a 36% reduction. Contralateral ACL injury rate after surgery was 6% and was 19% lower in those passing the RTS test. The mean age of patients passing their first RTS-test was significantly higher than that of those who failed (p â€‹= â€‹0.0027). Re-ruptures in those who passed the RTS test first time occurred late (>34 months), compared to those who failed first time, which all occurred early (<33 months) (p â€‹= â€‹0.0015). The mean age of re-rupture was significantly less than those who did not sustain a re-rupture (p â€‹= â€‹0.025). CONCLUSION: Passing a RTS-test following ACLR reduces ACL re-rupture by 36.21% and contralateral ACL injury by 19.15% at mid-term follow-up. Younger patients are more likely to fail a RTS-test and are at higher risk of contralateral ACL rupture.

Prenatal Evaluation of Intracranial Hemorrhage on Fetal MRI: A Retrospective Review.

BACKGROUND AND PURPOSE: The evaluation and characterization of germinal matrix hemorrhages have been predominantly described on postnatal head sonography in premature neonates. However, germinal matrix hemorrhages that are seen in premature neonates can be also seen in fetuses of the same postconceptual age and are now more frequently encountered in the era of fetal MR imaging. Our aim was to examine and describe the MR imaging findings of fetuses with intracranial hemorrhage. MATERIALS AND METHODS: A retrospective review of diagnostic-quality fetal MRIs showing intracranial hemorrhage from January 2004 to May 2020 was performed. Images were reviewed by 2 radiologists, and imaging characteristics of fetal intracranial hemorrhages were documented. Corresponding postnatal imaging and clinical parameters were reviewed. RESULTS: One hundred seventy-seven fetuses with a mean gestational age of 25.73 (SD, 5.01) weeks were included. Germinal matrix hemorrhage was identified in 60.5% (107/177) and nongerminal matrix hemorrhage in 39.5% (70/177) of patients. Significantly increased ventricular size correlated with higher germinal matrix hemorrhage grade (P < .001). Fetal growth restriction was present in 21.3% (20/94) of our population, and there was no significant correlation with germinal matrix grade or type of intracranial hemorrhage. An increased incidence of neonatal death with grade III germinal matrix hemorrhages (P = .069) compared with other grades was identified; 23.2% (16/69) of the neonates required ventriculoperitoneal shunts, with an increased incidence in the nongerminal matrix hemorrhage group (P = .026). CONCLUSIONS: MR imaging has become a key tool in the diagnosis and characterization of intracranial hemorrhage in the fetus. Appropriate characterization is important for optimizing work-up, therapeutic approach, and prenatal counseling.

Mutant ubiquitin found in Alzheimer's disease causes neuritic beading of mitochondria in association with neuronal degeneration.

A dinucleotide deletion in human ubiquitin (Ub) B messenger RNA leads to formation of polyubiquitin (UbB)+1, which has been implicated in neuronal cell death in Alzheimer's and other neurodegenerative diseases. Previous studies demonstrate that UbB+1 protein causes proteasome dysfunction. However, the molecular mechanism of UbB+1-mediated neuronal degeneration remains unknown. We now report that UbB+1 causes neuritic beading, impairment of mitochondrial movements, mitochondrial stress and neuronal degeneration in primary neurons. Transfection of UbB+1 induced a buildup of mitochondria in neurites and dysregulation of mitochondrial motor proteins, in particular, through detachment of P74, the dynein intermediate chain, from mitochondria and decreased mitochondria-microtubule interactions. Altered distribution of mitochondria was associated with activation of both the mitochondrial stress and p53 cell death pathways. These results support the hypothesis that neuritic clogging of mitochondria by UbB+1 triggers a cascade of events characterized by local activation of mitochondrial stress followed by global cell death. Furthermore, UbB+1 small interfering RNA efficiently blocked expression of UbB+1 protein, attenuated neuritic beading and preserved cellular morphology, suggesting a potential neuroprotective strategy for certain neurodegenerative disorders.

Ternary complexes and cooperative interplay between NCoA-62/Ski-interacting protein and steroid receptor coactivators in vitamin D receptor-mediated transcription.

The vitamin D receptor (VDR) is a ligand-dependent transcriptional factor that binds to vitamin D-responsive elements as a heterodimer with retinoid X receptor (RXR) to regulate target gene transcription. The steroid receptor coactivator (SRC) proteins are coactivators that interact with the AF-2 domain of VDR to augment 1,25-dihydroxyvitamin D3-dependent transcription. In contrast, NCoA-62/Ski-interacting protein (SKIP) is a distinct, activation function-2-independent coactivator for VDR. The current study examined whether these two distinct classes of coactivators impact functionally on VDR-mediated transcription. Using a ternary complex binding assay, we observed a marked preference for the direct interaction of NCoA-62/SKIP with the VDR-RXR heterodimer as compared with the VDR-VDR homodimer or VDR monomer. The liganded VDR also formed a ternary complex with NCoA-62/SKIP and SRC proteins in vitro. Competition experiments using LXXLL peptides showed that NCoA-62/SKIP and SRC coactivators contact different domains of the VDR-RXR heterodimer. Synergistic interplays were observed between NCoA-62/SKIP and SRC coactivators in VDR-mediated transcriptional assays, and protein interference assays indicated a requirement for both NCoA-62/SKIP and SRCs in VDR- mediated transcription. These studies suggest that the ligand-dependent and simultaneous interaction of NCoA-62/SKIP and SRC coactivators with distinct interaction domains within the VDR-RXR heterodimer results in cooperative interplays between coactivators in VDR-mediated transcription.

Vitamin D receptor and nuclear receptor coactivators: crucial interactions in vitamin D-mediated transcription.

The nuclear actions of 1,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] are mediated by the vitamin D receptor (VDR). Binding of ligand induces conformational changes in the VDR which promote heterodimerization with retinoid X receptor (RXR) and recruitment of a number of nuclear receptor coactivator proteins including the steroid receptor coactivator (SRC) family members, select SMAD proteins, a novel coactivator complex referred to as DRIP, and a variety of other putative factors. We recently described a novel nuclear receptor coactivator termed NCoA-62 that interacts with the VDR to enhance 1alpha,25(OH)(2)D(3)-activated transcription. NCoA-62 is unrelated to the SRC family, the DRIP complex, as well as other nuclear receptor coactivators described thus far. The molecular mechanisms involved in NCoA-62 coactivator function are poorly understood, but protein-protein interactions are likely to play an important role. The purpose of this paper is to briefly review salient features of the coactivators involved in VDR-activated transcription and to focus on our current understanding of NCoA-62 and its interplay with other nuclear receptor coactivator proteins. It is clear from the studies described here that a concerted series of interactions with multiple coactivator proteins are essential for high order transactivation by 1alpha,25(OH)(2)D(3) and the VDR.

Involvement of thyrotroph embryonic factor in calcium-mediated regulation of gene expression.

In the present study, we used an expression cloning strategy to identify transcription factors that bind specifically to a limited region of the inducible cAMP early repressor (ICER) promoter and regulate transcription. Murine thyrotroph embryonic factor (mTEF) was isolated and was shown to bind to a site located at nucleotides -117 to -108 from the transcriptional start site. Transient expression of reporter constructs containing either a consensus TEFRE or the icerTEF binding site demonstrated that TEF-dependent transcription correlated with relative binding affinities, i.e. the consensus TEFRE bound TEF more tightly and was more responsive to TEF than the icerTEFRE. Because the icerTEFRE overlapped a cAMP response element, the responsiveness of these sequences to either cAMP or Ca(2+) was tested. Although TEF expression had no effect on the cAMP-regulated transcriptional response of the ICER promoter, TEF did confer calcium responsiveness to these sequences. Calcium also modestly increased the TEF-mediated transcription from a consensus TEFRE. Additional studies using Ca(2+)-activated kinases indicate that Ca(2+)/TEF/TEFRE-regulated transcription may be mediated through Ca(2+)/calmodulin-dependent kinase (CaMK) IV. Moreover, studies with the icerTEFRE in a CaMK IV-deficient cell line demonstrated that these cells were transcriptionally unresponsive to thapsigargin; however, responsiveness was restored by co-expression of the active CaMK IV. These studies are the first to demonstrate that TEF is a calcium-responsive transcription factor, and they suggest that there are two classes of TEF-regulated genes. One class, represented by a consensus TEFRE, is regulated by TEF in the resting cell; the second class, represented by icerTEFRE, is regulated by TEF in the calcium-activated cell.

cAMP-dependent plasticity at excitatory cholinergic synapses in Drosophila neurons: alterations in the memory mutant dunce.

It is well known that cAMP signaling plays a role in regulating functional plasticity at central glutamatergic synapses. However, in the Drosophila CNS, where acetylcholine is thought to be a primary excitatory neurotransmitter, cellular changes in neuronal communication mediated by cAMP remain unexplored. In this study we examined the effects of elevated cAMP levels on fast excitatory cholinergic synaptic transmission in cultured embryonic Drosophila neurons. We report that chronic elevation in neuronal cAMP (in dunce neurons or wild-type neurons grown in db-cAMP) results in an increase in the frequency of cholinergic miniature EPSCs (mEPSCs). The absence of alterations in mEPSC amplitude or kinetics suggests that the locus of action is presynaptic. Furthermore, a brief exposure to db-cAMP induces two distinct changes in transmission at established cholinergic synapses in wild-type neurons: a short-term increase in the frequency of spontaneous action potential-dependent synaptic currents and a long-lasting, protein synthesis-dependent increase in the mEPSC frequency. A more persistent increase in cholinergic mEPSC frequency induced by repetitive, spaced db-cAMP exposure in wild-type neurons is absent in neurons from the memory mutant dunce. These data demonstrate that interneuronal excitatory cholinergic synapses in Drosophila, like central excitatory glutamatergic synapses in other species, are sites of cAMP-dependent plasticity. In addition, the alterations in dunce neurons suggest that cAMP-dependent plasticity at cholinergic synapses could mediate changes in neuronal communication that contribute to memory formation.

HIV-1 Tat-mediated inhibition of the tyrosine hydroxylase gene expression in dopaminergic neuronal cells.

Treatment of dopaminergic rat PC12 cells with human immunodeficiency virus, type 1 (HIV-1) Tat protein or tat cDNA inhibited the expression of tyrosine hydroxylase (TH), the rate-limiting enzyme for the dopamine biosynthetic pathway, as well as the production and release of dopamine into the culture medium. Moreover, the Tat addition to PC12 cells up-regulated the expression of the inducible cAMP early repressor (ICER), a specific member of the cAMP-responsive element modulator transcription factor family, in a cAMP-dependent manner. In turn, ICER overexpression abrogated the transcription activity of the TH promoter in PC12 cells, strongly suggesting ICER involvement in Tat-mediated inhibition of TH gene expression. In vivo injection of synthetic HIV-1 Tat protein into the striatum of healthy rats induced a subclinical Parkinson's-like disease that became manifested only when the animals were treated with amphetamine. As early as one week postinjection, the histochemical examination of the rat substantia nigra showed a reduced staining of neurons expressing TH followed by a loss of TH(+) neurons at later time points. As Tat protein can be locally released into the central nervous system by HIV-1-infected microglial cells, our findings may contribute to the explanation of the pathogenesis of the motorial abnormalities often reported in HIV-1 seropositive individuals.

GABA(A) receptor-mediated miniature postsynaptic currents and alpha-subunit expression in developing cortical neurons.

Previous studies have described maturational changes in GABAergic inhibitory synaptic transmission in the rodent somatosensory cortex during the early postnatal period. To determine whether alterations in the functional properties of synaptically localized GABA(A) receptors (GABA(A)Rs) contribute to development of inhibitory transmission, we used the whole cell recording technique to examine GABAergic miniature postsynaptic currents (mPSCs) in developing cortical neurons. Neurons harvested from somatosensory cortices of newborn mice showed a progressive, eightfold increase in GABAergic mPSC frequency during the first 4 wk of development in dissociated cell culture. A twofold decrease in the decay time of the GABAergic mPSCs, between 1 and 4 wk, demonstrates a functional change in the properties of GABA(A)Rs mediating synaptic transmission in cortical neurons during development in culture. A similar maturational profile observed in GABAergic mPSC frequency and decay time in cortical neurons developing in vivo (assessed in slices), suggests that these changes in synaptically localized GABA(A)Rs contribute to development of inhibition in the rodent neocortex. Pharmacological and reverse transcription-polymerase chain reaction (RT-PCR) studies were conducted to determine whether changes in subunit expression might contribute to the observed developmental alterations in synaptic GABA(A)Rs. Zolpidem (300 nM), a subunit-selective benzodiazepine agonist with high affinity for alpha1-subunits, caused a reversible slowing of the mPSC decay kinetics in cultured cortical neurons. Development was characterized by an increase in the potency of zolpidem in modulating the mPSC decay, suggesting a maturational increase in percentage of functionally active GABA(A)Rs containing alpha1 subunits. The relative expression of alpha1 versus alpha5 GABA(A)R subunit mRNA in cortical tissue, both in vivo and in vitro, also increased during this same period. Furthermore, single-cell RT-multiplex PCR analysis revealed more rapidly decaying mPSCs in individual neurons in which alpha1 versus alpha5 mRNA was amplified. Together these data suggest that changes in alpha-subunit composition of GABA(A)Rs contribute to the maturation of GABAergic mPSCs mediating inhibition in developing cortical neurons.

Functional analysis of the mouse ICER (Inducible cAMP Early Repressor) promoter: evidence for a protein that blocks calcium responsiveness of the CAREs (cAMP autoregulatory elements).

Although Ca2+ and cAMP mediate their effects through distinct pathways, both signals converge upon the phosphorylation of the cAMP response element (CRE) binding protein, CREB, thereby activating transcription of CRE-regulated genes. In WEHI7.2 thymocytes, cAMP increases the expression of the inducible cAMP early repressor (ICER) gene through CRE-like elements, known as cAMP autoregulatory elements (CAREs). Because Ca2+ -and cAMP-mediated transcription converge in WEHI7.2 thymocytes, we examined the effect of Ca2+ fluxes on the expression of the ICER gene in these cells. Despite the presence of multiple CAREs within its promoter, ICER gene transcription was not activated by Ca2+. Moreover, Ca2+ attenuated the stimulatory effect of cAMP on ICER expression. Transient expression of reporter constructs demonstrated that when these CAREs were placed in a different DNA promoter context, the elements became responsive to Ca2+. Detailed studies using chimeric promoter constructs to map the region responsible for blocking the transcriptional response to Ca2+ indicated that a small portion of the ICER promoter was necessary for the effect. Southwestern blot analysis identified a 83-kDa nuclear protein that bound specifically to that region. The relative binding activity of the factor to the ICER promoter and mutant promoter sequences correlated with an inhibition of Ca2+ -activated gene expression in WEHI7.2 cells. These data suggest that the factor functions as a putative Ca2+ -activated repressor of CREB/CRE-mediated transcription. Thus, depending on the surrounding context in which the CRE is located, CREs of individual genes can be regulated separately by Ca2+ and cAMP despite the convergence of these two signaling pathways.

Fast excitatory synaptic transmission mediated by nicotinic acetylcholine receptors in Drosophila neurons.

Difficulty in recording from single neurons in vivo has precluded functional analyses of transmission at central synapses in Drosophila, where the neurotransmitters and receptors mediating fast synaptic transmission have yet to be identified. Here we demonstrate that spontaneously active synaptic connections form between cultured neurons prepared from wild-type embryos and provide the first direct evidence that both acetylcholine and GABA mediate fast interneuronal synaptic transmission in Drosophila. The predominant type of fast excitatory transmission between cultured neurons is mediated by nicotinic acetylcholine receptors (nAChRs). Detailed analysis of cholinergic transmission reveals that spontaneous EPSCs (sEPSCs) are composed of both evoked and action potential-independent [miniature EPSC (mEPSC)] components. The mEPSCs are characterized by a broad, positively skewed amplitude histogram in which the variance is likely to reflect differences in the currents induced by single quanta. Biophysical characteristics of the cholinergic mEPSCs include a rapid rise time (0.6 msec) and decay (tau = 2 msec). Regulation of mEPSC frequency by external calcium and cobalt suggests that calcium influx through voltage-gated channels influences the probability of ACh release. In addition, brief depolarization of the cultures with KCl can induce a calcium-dependent increase in sEPSC frequency that persists for up to 3 hr after termination of the stimulus, illustrating one form of plasticity at these cholinergic synapses. These data demonstrate that cultured embryonic neurons, amenable to both genetic and biochemical manipulations, present a unique opportunity to define genes/signal transduction cascades involved in functional regulation of fast excitatory transmission at interneuronal cholinergic synapses in Drosophila.

Formation of functional synaptic connections between cultured cortical neurons from agrin-deficient mice.

Numerous studies suggest that the extracellular matrix protein agrin directs the formation of the postsynaptic apparatus at the neuromuscular junction (NMJ). Strong support for this hypothesis comes from the observation that the high density of acetylcholine receptors (AChR) normally present at the neuromuscular junction fails to form in muscle of embryonic agrin mutant mice. Agrin is expressed by many populations of neurons in the central nervous system (CNS), suggesting that this molecule may also play a role in neuron-neuron synapse formation. To test this hypothesis, we examined synapse formation between cultured cortical neurons isolated from agrin-deficient mouse embryos. Our data show that glutamate receptors accumulate at synaptic sites on agrin-deficient neurons. Moreover, electrophysiological analysis demonstrates that functional glutamatergic and gamma-aminobutyric acid (GABA)ergic synapses form between mutant neurons. The frequency and amplitude of miniature postsynaptic glutamatergic and GABAergic currents are similar in mutant and age-matched wild-type neurons during the first 3 weeks in culture. These results demonstrate that neuron-specific agrin is not required for formation and early development of functional synaptic contacts between CNS neurons, and suggest that mechanisms of interneuronal synaptogenesis are distinct from those regulating synapse formation at the neuromuscular junction.

Regulation of alpha7 nicotinic acetylcholine receptors in mouse somatosensory cortex following whisker removal at birth.

Previous studies in postnatal mouse demonstrating high levels of alpha7 nicotinic acetylcholine receptors on layer IV somatosensory cortical neurons coincident with the onset of functional synaptic transmission led us to investigate whether the number and/or the localization of these receptors could be regulated by activity. Accordingly, we examined alpha-bungarotoxin binding in mouse somatosensory cortex following removal of all of the vibrissae on one side of the face, either by vibrissal follicle cauterization or daily plucking beginning on the day of birth. Following vibrissa plucking, the levels of [125I]alpha-bungarotoxin binding on postnatal day 6 were significantly higher (23 +/- 7%) in the denervated cortex (contralateral to the peripheral manipulation) than the intact cortex. Cauterization also resulted in significantly higher (14 +/- 3%) [125I]alpha-bungarotoxin binding in the contralateral vs. the ipsilateral cortex. In contrast, there was no difference in [125I]alpha-bungarotoxin binding in the left and right cortices of unoperated control animals. At postnatal day 14, levels of [125I]alpha-bungarotoxin binding in layer IV were very low in control animals as well as in animals subjected to whisker plucking or cautery. These findings suggest that reducing activity in the somatosensory pathway regulates the density of alpha7 nicotinic acetylcholine receptors during the first postnatal week. However, the normal decrease in receptor density that is seen during the second postnatal week of development proceeds despite altered sensory activity.

Assessment of interlaboratory performance in external proficiency testing programs with a direct HDL-cholesterol assay.

Direct assays for the determination of HDL-cholesterol (HDL-C) have recently become available. The methods are precise, require small sample volume, and appear to be less affected by increased triglycerides than traditional precipitation methods. In this study, we describe the inter- and intralaboratory variability of the Boehringer Mannheim Corporation direct HDL-C assay and its performance in external proficiency testing surveys. A comparison study among three laboratories, using different analyzers and 85 serum specimens, showed a correlation coefficient (r) of 0.99. The direct HDL-C assay also showed good agreement with the ultracentrifugation-dextran sulfate-Mg2+ method (r = 0.98) and the Cholesterol Reference Method Laboratory Network-Designated Comparison Method (a = 0.98x + 4.75 mg/L, r = 0.98). Total error at medical decision levels ranged from -0.8% to +11.1%. Furthermore, this assay performed adequately in the College of American Pathologists and the ALERT surveys as well as the CDC Lipid Standardization Program and met all performance criteria of regulatory agencies.

Differential regulation and transcriptional control of immediate early gene expression in forskolin-treated WEHI7.2 thymoma cells.

Agents that increase intracellular cAMP are frequently growth inhibitory for lymphocytes and induce apoptosis in cortical thymocytes by regulating gene expression. In the present study, immediate early gene expression was examined in WEHI7.2 thymoma cells undergoing cAMP-mediated apoptosis. Temporal differences in c-fos, junB, and inducible cAMP early repressor (ICER) steady-state mRNA levels were observed after forskolin exposure. Maximal induction of c-fos and junB occurred within 1 h, returning to basal levels by 3.5 h. In contrast, a 1.5-h time lag was observed before ICER transcript levels increased, reaching maximal levels after 3.5 h. This rise in expression, correlating with the decrease in c-fos and junB levels, preceded apoptotic DNA fragmentation by 1.5 h. Transient expression of ICER promoter constructs demonstrated that cAMP responsiveness occurred through cAMP-autoregulatory response element (CARE)3/4, two of the four proposed response elements in the ICER promoter. In contrast to the cAMP-responsive cell line JEG-3, CARE1/2 was not functional for cAMP-activated transcription in WEHI7.2 cells. An observed differential binding pattern of WEHI and JEG nuclear extracts to these elements may account for the cell-specific differences in expression patterns. To determine the role of endogenous ICER in regulating gene expression, cells were treated with two sequential doses of forskolin after which ICER and c-fos mRNA levels were measured. The high levels of cAMP-induced ICER expression dramatically reduced a second induction of c-fos. These data suggest that ICER expression may function as an antioncogene to attenuate the expression of certain protooncogenes, thereby preventing transformation and oncogenesis due to continuous overexpression. Moreover, inhibition of growth-stimulatory genes may be required for the activation of the cell death machinery in specific cells.

Leaf domatia and foliar mite abundance in broadleaf deciduous forest of north Asia.

Plant morphology may be shaped, in part, by the third trophic level. Leaf domatia, minute enclosures usually in vein axils on the leaf underside, may provide the basis for protective mutualism between plants and mites. Domatia are particularly frequent among species of trees, shrubs, and vines in the temperate broadleaf deciduous forests in north Asia where they may be important in determining the distribution and abundance of mites in the forest canopy. In lowland and montane broadleaf deciduous forests at Kwangn;akung and Chumbongsan in Korea, we found that approximately half of all woody species in all forest strata, including many dominant trees, have leaf domatia. Pooling across 24 plant species at the two sites, mites occupied a mode of 60% (range 20-100%) of domatia and used them for shelter, egg-laying, and development. On average, 70% of all active mites and 85% of mite eggs on leaves were found in domatia; over three-quarters of these were potentially beneficial to their hosts. Further, mite abundance and reproduction (expressed as the proportion of mites at the egg stage) were significantly greater on leaves of species with domatia than those without domatia in both forests. Effects of domatia on mite abundance were significant only for predaceous and fungivorous mite taxa; herbivore numbers did not differ significantly between leaves of species with and without domatia. Comparable patterns in broadleaf deciduous forest in North America and other biogeographic regions suggest that the effect of leaf domatia on foliar mite abundance is general. These results are consistent with several predictions of mutualism between plants and mites, and indicate that protective mutualisms may be frequent in the temperate zone.

Heterogeneous expression of multiple putative patterning genes by single cells from the chick hindbrain.

The metameric organization of the vertebrate hindbrain into rhombomeres appears to result from the patterned expression of several transcription factors and putative signaling molecules. We have applied a refined single-cell reverse transcription-polymerase chain reaction strategy to examine the molecular logic proposed to pattern the hindbrain at the single-cell level. This technique allows analysis of the concurrent expression of several genes within an individual cell at higher sensitivity than by in situ hybridization. Our results demonstrate that cells in rhombomere (r) 4 and r5 are heterogeneous in their expression of Hoxa-3, Hoxb-2, Sek-1, and Krox-20, suggesting that single cells are dynamically regulating their rhombomere-specific gene-expression profiles. Furthermore, the strong correlation between Sek-1 and Krox-20 expression at stage 12 was greatly diminished by stage 16, suggesting that the proposed interdependence of these two genes is present only at early stages of hindbrain development.

Agrin gene expression in mouse somatosensory cortical neurons during development in vivo and in cell culture.

Agrin is an extracellular matrix protein involved in the formation of the postsynaptic apparatus of the neuromuscular junction. In addition to spinal motor neurons, agrin is expressed by many other neuronal populations throughout the nervous system. Agrin's role outside of the neuromuscular junction, however, is poorly understood. Here we use the polymerase chain reaction to examine expression and alternative splicing of agrin in mouse somatosensory cortex during early postnatal development in vivo and in dissociated cell culture. Peak levels of agrin gene expression in developing cortex coincide with ingrowth of thalamic afferent fibres and formation of thalamocortical and intracortical synapses. Analysis of alternatively spliced agrin messenger RNA variants shows that greater than 95% of all agrin in developing and adult somatosensory cortex originates in neurons, including isoforms that have little or no activity in acetylcholine receptor aggregation assays. The levels of expression of "active" and "inactive" isoforms, however, are regulated during development. A similar pattern of agrin gene expression is also observed during a period when new synapses are being formed between somatosensory neurons growing in dissociated cell culture. Changes in agrin gene expression, observed both in vivo and in vitro, are consistent with a role for agrin in synapse formation in the central nervous system.

Differential expression of K4-AP currents and Kv3.1 potassium channel transcripts in cortical neurons that develop distinct firing phenotypes.

Maturation of electrical excitability during early postnatal development is critical to formation of functional neural circuitry in the mammalian neocortex. Little is known, however, about the changes in gene expression underlying the development of firing properties that characterize different classes of cortical neurons. Here we describe the development of cortical neurons with two distinct firing phenotypes, regular-spiking (RS) and fast-spiking (FS), that appear to emerge from a population of immature multiple-spiking (IMS) neurons during the first two postnatal weeks, both in vivo (within layer IV) and in vitro. We report the expression of a slowly inactivating, 4-AP-sensitive potassium current (K4-AP) at significantly higher density in FS compared with RS neurons. The same current is expressed at intermediate levels in IMS neurons. The kinetic, voltage-dependent, and pharmacological properties of the K4-AP current are similar to those observed by heterologous expression of Kv3.1 potassium channel mRNA. Single-cell RT-PCR analysis demonstrates that PCR products representing Kv3.1 transcripts are amplified more frequently from FS than RS neurons, with an intermediate frequency of Kv3.1 detection in neurons with immature firing properties. Taken together, these data suggest that the Kv3.1 gene encodes the K4-AP current and that expression of this gene is regulated in a cell-specific manner during development. Analysis of the effects of 4-AP on firing properties suggests that the K4-AP current is important for rapid action potential repolarization, fast after-hyperpolarization, brief refractory period, and high firing frequency characteristic of FS GABAergic interneurons.