Associations of paternal postpartum depressive symptoms and infant development in a Chinese longitudinal study.

Although fathers actively provide infant care and support to their partners in modern societies, data on fathers' difficulties and mental health problems is still limited. This study examined paternal postpartum depression and its adverse impact on infants, and the possible mediating role of father-infant attachment in the link between fathers' depressive symptoms and infants' outcomes. Pregnant women and their partners were recruited from the antenatal clinics of two public hospitals in Hong Kong. Information about paternal and maternal depression, paternal-infant attachment, and infant development were collected at antenatal period, 6 weeks and 6 months postpartum. Linear regression was employed to examine risk factors for paternal depression symptoms, and mediation analysis was conducted to examine the mediating mechanisms. 121 couples joined the longitudinal study and completed all the assessments. Paternal postpartum depression symptoms were associated with fathers' prenatal depression symptoms, and mothers' postpartum depression symptoms. Fathers with postpartum depression symptoms experienced reduced paternal-infant attachment, which also acted as mediators between postpartum depression in fathers and adverse infants' social development. Effective assessment and interventions targeted at preventing or identifying and reducing paternal postpartum depression and improving father-infant relationship would help to lower the risk of infant disorders and poor development. Strategies improving the fathers' mental health during antenatal period and their partner's psychosocial well-being may also reduce paternal postpartum depression.

Microfluidic chip based nano liquid chromatography coupled to tandem mass spectrometry for the determination of abused drugs and metabolites in human hair.

A microfluidic chip based nano-HPLC coupled to tandem mass spectrometry (nano-HPLC-Chip-MS/MS) has been developed for simultaneous measurement of abused drugs and metabolites: cocaine, benzoylecgonine, cocaethylene, norcocaine, morphine, codeine, 6-acetylmorphine, phencyclidine, amphetamine, methamphetamine, MDMA, MDA, MDEA, and methadone in the hair of drug abusers. The microfluidic chip was fabricated by laminating polyimide films and it integrated an enrichment column, an analytical column and a nanospray tip. Drugs were extracted from hairs by sonication, and the chromatographic separation was achieved in 15 min. The drug identification and quantification criteria were fulfilled by the triple quardropule tandem mass spectrometry. The linear regression analysis was calibrated by deuterated internal standards with all of the R(2) at least over 0.993. The limit of detection (LOD) and the limit of quantification (LOQ) were from 0.1 to 0.75 and 0.2 to 1.25 pg/mg, respectively. The validation parameters including selectivity, accuracy, precision, stability, and matrix effect were also evaluated here. In conclusion, the developed sample preparation method coupled with the nano-HPLC-Chip-MS/MS method was able to reveal the presence of drugs in hairs from the drug abusers, with the enhanced sensitivity, compared with the conventional HPLC-MS/MS.

Neuregulin 1 promotes excitatory synapse development and function in GABAergic interneurons.

Neuregulin 1 (NRG1) and its receptor ErbB4 are both susceptibility genes of schizophrenia. However, little is known about the underlying mechanisms of their malfunction. Although ErbB4 is enriched in GABAergic interneurons, the role of NRG1 in excitatory synapse formation in these neurons remains poorly understood. We showed that NRG1 increased both the number and size of PSD-95 puncta and the frequency and amplitude of miniature EPSCs (mEPSCs) in GABAergic interneurons, indicating that NRG1 stimulates the formation of new synapses and strengthens existing synapses. In contrast, NRG1 treatment had no effect on either the number or size of excitatory synapses in glutamatergic neurons, suggesting its synaptogenic effect is specific to GABAergic interneurons. Ecto-ErbB4 treatment diminished both the number and size of excitatory synapses, suggesting that endogenous NRG1 may be critical for basal synapse formation. NRG1 could stimulate the stability of PSD-95 in the manner that requires tyrosine kinase activity of ErbB4. Finally, deletion of ErbB4 in parvalbumin-positive interneurons led to reduced frequency and amplitude of mEPSCs, providing in vivo evidence that ErbB4 is important in excitatory synaptogenesis in interneurons. Together, our findings suggested a novel synaptogenic role of NRG1 in excitatory synapse development, possibly via stabilizing PSD-95, and this effect is specific to GABAergic interneurons. In light of the association of the genes of both NRG1 and ErbB4 with schizophrenia and dysfunction of GABAergic system in this disorder, these results provide insight into its potential pathological mechanism.

The establishment of a highly sensitive method in detecting ketamine and norketamine simultaneously in human hairs by HPLC-Chip-MS/MS.

An effective way to reveal the history of drug abuse is to determine the parental drug and its metabolites in hair. Here, a quantitative HPLC-Chip-MS/MS method was developed for simultaneous measurement of ketamine and its metabolite norketamine in human hair. Ketamine and norketamine were extracted from hair by acid hydrolysis, and then enriched by organic solvent extraction. The chromatographic separation was achieved in 15 min, with the drug identification and quantification by a tandem mass spectrometer. The linear regression analysis was calibrated by deuterated internal standards with a R(2) of over 0.996. The limit of detection (LOD) and the limit of quantification (LOQ) for ketamine and norketamine were 0.5 and 1 pg/mg of hair, respectively. The standard curves were linear from the value of LOQ up to 100 pg/mg of hair. The validation parameters including selectivity, accuracy, precision, stability and matrix effect were also determined. In conclusion, this method was able to reveal the present of ketamine and norketamine with less hair from the drug abusers, and which had the sensitivity of ∼1000-fold higher than the conventional method. In addition, the amount of ketamine and norketamine being detected in different hair segments would be useful in revealing the historical record of ketamine uptake in the drug abusers.

ErbB4 in parvalbumin-positive interneurons is critical for neuregulin 1 regulation of long-term potentiation.

Neuregulin 1 (NRG1) is a trophic factor that acts by stimulating ErbB receptor tyrosine kinases and has been implicated in neural development and synaptic plasticity. In this study, we investigated mechanisms of its suppression of long-term potentiation (LTP) in the hippocampus. We found that NRG1 did not alter glutamatergic transmission at SC-CA1 synapses but increased the GABA(A) receptor-mediated synaptic currents in CA1 pyramidal cells via a presynaptic mechanism. Inhibition of GABA(A) receptors blocked the suppressing effect of NRG1 on LTP and prevented ecto-ErbB4 from enhancing LTP, implicating a role of GABAergic transmission. To test this hypothesis further, we generated parvalbumin (PV)-Cre;ErbB4(-/-) mice in which ErbB4, an NRG1 receptor in the brain, is ablated specifically in PV-positive interneurons. NRG1 was no longer able to increase inhibitory postsynaptic currents and to suppress LTP in PV-Cre;ErbB4(-/-) hippocampus. Accordingly, contextual fear conditioning, a hippocampus-dependent test, was impaired in PV-Cre;ErbB4(-/-) mice. In contrast, ablation of ErbB4 in pyramidal neurons had no effect on NRG1 regulation of hippocampal LTP or contextual fear conditioning. These results demonstrate a critical role of ErbB4 in PV-positive interneurons but not in pyramidal neurons in synaptic plasticity and support a working model that NRG1 suppresses LTP by enhancing GABA release. Considering that NRG1 and ErbB4 are susceptibility genes of schizophrenia, these observations contribute to a better understanding of how abnormal NRG1/ErbB4 signaling may be involved in the pathogenesis of schizophrenia.

HSP90 beta regulates rapsyn turnover and subsequent AChR cluster formation and maintenance.

Rapsyn, an acetylcholine receptor (AChR)-interacting protein, is essential for synapse formation at the neuromuscular junction (NMJ). Like many synaptic proteins, rapsyn turns over rapidly at synapses. However, little is known about molecular mechanisms that govern rapsyn stability. Using a differential mass-spectrometry approach, we identified heat-shock protein 90beta (HSP90beta) as a component in surface AChR clusters. The HSP90beta-AChR interaction required rapsyn and was stimulated by agrin. Inhibition of HSP90beta activity or expression, or disruption of its interaction with rapsyn attenuated agrin-induced formation of AChR clusters in vitro and impaired the development and maintenance of the NMJ in vivo. Finally, we showed that HSP90beta was necessary for rapsyn stabilization and regulated its proteasome-dependent degradation. Together, these results indicate a role of HSP90beta in NMJ development by regulating rapsyn turnover and subsequent AChR cluster formation and maintenance.

ErbB4-neuregulin signaling modulates synapse development and dendritic arborization through distinct mechanisms.

Perturbations in neuregulin-1 (NRG1)/ErbB4 function have been associated with schizophrenia. Affected patients exhibit altered levels of these proteins and display hypofunction of glutamatergic synapses as well as altered neuronal circuitry. However, the role of NRG1/ErbB4 in regulating synapse maturation and neuronal process formation has not been extensively examined. Here we demonstrate that ErbB4 is expressed in inhibitory interneurons at both excitatory and inhibitory postsynaptic sites. Overexpression of ErbB4 postsynaptically enhances size but not number of presynaptic inputs. Conversely, knockdown of ErbB4 using shRNA decreases the size of presynaptic inputs, demonstrating a specific role for endogenous ErbB4 in synapse maturation. Using ErbB4 mutant constructs, we demonstrate that ErbB4-mediated synapse maturation requires its extracellular domain, whereas its tyrosine kinase activity is dispensable for this process. We also demonstrate that depletion of ErbB4 decreases the number of primary neurites and that stimulation of ErbB4 using a soluble form of NRG1 results in exuberant dendritic arborization through activation of the tyrosine kinase domain of ErbB4 and the phosphoinositide 3-kinase pathway. These findings demonstrate that NRG1/ErbB4 signaling differentially regulates synapse maturation and dendritic morphology via two distinct mechanisms involving trans-synaptic signaling and tyrosine kinase activity, respectively.

Retrograde regulation of motoneuron differentiation by muscle beta-catenin.

Synapse formation requires proper interaction between pre- and postsynaptic cells. In anterograde signaling, neurons release factors to guide postsynaptic differentiation. However, less is known about how postsynaptic targets retrogradely regulate presynaptic differentiation or function. We found that muscle-specific conditional knockout of beta-catenin (Ctnnb1, also known as beta-cat) in mice caused both morphologic and functional defects in motoneuron terminals of neuromuscular junctions (NMJs). In the absence of muscle beta-catenin, acetylcholine receptor clusters were increased in size and distributed throughout a wider region. Primary nerve branches were mislocated, whereas secondary or intramuscular nerve branches were elongated and reduced in number. Both spontaneous and evoked neurotransmitter release was reduced at the mutant NMJs. Furthermore, short-term plasticity and calcium sensitivity of neurotransmitter release were compromised in beta-catenin-deficient muscle. In contrast, the NMJ was normal in morphology and function in motoneuron-specific beta-catenin-deficient mice. Taken together, these observations indicate a role for muscle beta-catenin in presynaptic differentiation and function, identifying a previously unknown retrograde signaling in the synapse formation and synaptic plasticity.

Calcitonin gene-related peptide induces the expression of acetylcholinesterase-associated collagen ColQ in muscle: a distinction in driving two different promoters between fast- and slow-twitch muscle fibers.

The presence of a collagenous protein (ColQ) characterizes the collagen-tailed forms of acetylcholinesterase at vertebrate neuromuscular junctions (nmjs). Two ColQ transcripts as ColQ-1 and ColQ-1a, driven by two promoters: pColQ-1 and pColQ-1a, were found in mammalian slow- and fast-twitch muscles, respectively, which have distinct expression pattern in different muscle fibers. In this study, we show the differential expression of CoQ in different muscles is triggered by calcitonin gene-related peptide (CGRP), a known motor neuron-derived factor. Application of CGRP, or dibutyryl-cAMP (Bt(2)-cAMP), in cultured myotubes induced the expression of ColQ-1a transcript and promoter activity; however, the expression of ColQ-1 transcript did not respond to CGRP or Bt(2)-cAMP. The CGRP-induced gene activation was blocked by an adenylyl cyclase inhibitor or a dominant negative mutant of cAMP-responsive element (CRE) binding protein (CREB). Two CRE sites were mapped within the ColQ-1a promoter, and mutations of the CRE sites abolished the response of CGRP or Bt(2)-cAMP. In parallel, CGRP receptor complex was dominantly expressed at the nmjs of fast muscle but not of slow muscle. These results suggested that the expression of ColQ-1a at the nmjs of fast-twitch muscle was governed by a CGRP-mediated cAMP signaling mechanism.

Transcriptional control of different acetylcholinesterase subunits in formation and maintenance of vertebrate neuromuscular junctions.

Acetylcholinesterase (AChE; EC 3.1.1.7) is a highly polymorphic enzyme (Massoulié, 2002). Asingle ACHE gene produces several types of catalytic subunits by alternative splicing, but a single splice variant, called type T (AChET), is expressed in adult mammalian muscle and brain. Catalytic subunits of AChET produce amphiphilic monomers and dimers, nonamphiphilic homotetramers, as well as heteromeric associations with anchoring proteins, ColQ (collagenous subunit) and PRiMA (proline-rich membrane anchor), which allow their functional localization in cholinergic synapses (Massoulié, 2002). ColQ characterizes the collagen-tailed forms (Aforms) of AChE and butyrylcholinesterase (BChE), which are localized in the basal lamina at neuromuscular junctions (NMJs) of vertebrates (Krejci et al., 1999); in these molecules (A4, A8, A12), one, two, or three tetramers of catalytic subunits are disulfide-linked to the strands of a triple helix of ColQ collagen. The cDNAs encoding ColQ, which have two transcripts, have been cloned: ColQ-1a predominantly in fast-twitch muscle, and ColQ-1 predominantly in slow-twitch muscle. The tetrameric globular (G4) form of AChE is characterized by linkage to PRiMA. PRiMAcDNA encodes a single-pass approximately 20-kDa type-I transmembrane protein and, similar to that of ColQ, contains a short PRAD (proline-rich attachment domain) that is able to organize AChE catalytic subunits into tetramers and anchor the enzyme at the surface of neuron and muscle (Massoulié, 2002).

Transcriptional regulation of acetylcholinesterase-associated collagen ColQ in fast- and slow-twitch muscle fibers.

The presence of a collagenous protein (ColQ) characterizes the collagen-tailed forms of acetylcholinesterase (AChE) and butyrylcholinesterase at vertebrate neuromuscular junctions, which is tethered in the synaptic basal lamina. ColQ subunits, differing mostly by their signal sequences, are encoded by transcripts ColQ-1 and ColQ-1a, which are differentially expressed in slow- and fast-twitch muscles in mammals, respectively. Both ColQ transcripts are derived from a single COLQ gene. Transcripts encoding ColQ increased during myogenic differentiation of C2C12 cells; the increase was in parallel with AChE catalytic subunit. Quantitative PCR analysis indicated that the increase during the myotube formation was due to the up regulation of ColQ-1 transcript instead of ColQ-1a. In order to reveal the regulatory mechanism of ColQ transcripts, two distinct promoters, pColQ-1 and pColQ-1a, were isolated from human COLQ gene. The ColQ promoters showed a muscle fiber type-specific expression pattern, and which was in line with the expression of endogenous transcript. After in vivo DNA transfection, pColQ-1 showed strong activity in slow-twitch muscle (e.g. soleus), while pColQ-1a was preferably expressed in fast-twitch muscle (e.g. tibialis). Mutation analysis of the ColQ promoters suggested that the muscle fiber type-specific expression pattern of ColQ transcripts was regulated by a slow upsteam regulatory element (SURE) and a fast intronic regulatory element (FIRE). These results explain the specific expression patterns of collagen-tailed AChE in slow and fast muscle fibers.

ATP potentiates agrin-induced AChR aggregation in cultured myotubes: activation of RhoA in P2Y1 nucleotide receptor signaling at vertebrate neuromuscular junctions.

At vertebrate neuromuscular junctions, ATP is known to stabilize acetylcholine in the synaptic vesicles and to be co-released with it. We have shown previously that a nucleotide receptor, P2Y(1) receptor, is localized at the nmjs, and we propose that this mediates a trophic role for synaptic ATP there. In cultured myotubes, the activation of P2Y(1) receptors modulated agrin-induced acetylcholine receptor (AChR) aggregation in a potentiation manner. This potentiation effect in agrin-induced AChR aggregation was reduced by antagonizing the P2Y(1) receptors. The guanosine triphosphatase RhoA was shown to be responsible for this P2Y(1)-potentiated effect. The localization of RhoA in rat and chicken skeletal muscles was restricted at the neuromuscular junctions. Application of P2Y(1) agonists in cultured myotubes induced RhoA activation, which showed an additive effect with agrin-induced RhoA activation. Over-expression of dominant-negative mutant of RhoA in cultured myotubes diminished the agrin-induced AChR aggregation, as well as the potentiation effect of P2Y(1)-specific agonist. Application of UTP in the cultures also triggered similar responses as did 2-methylthioadenosine 5'-diphosphate, suggesting the involvement of other subtypes of P2Y receptors. These results demonstrate that RhoA could serve as a downstream mediator of signaling mediated by P2Y(1) receptor and agrin, which therefore synergizes the effects of the two neuron-derived trophic factors in modulating the formation and/or maintenance of post-synaptic apparatus at the neuromuscular junctions.

Transcriptional regulation of acetylcholinesterase-associated collagen ColQ: differential expression in fast and slow twitch muscle fibers is driven by distinct promoters.

The presence of a collagenous protein (ColQ) characterizes the collagen-tailed forms of acetylcholinesterase and butyrylcholinesterase at vertebrate neuromuscular junctions which is tethered in the synaptic basal lamina. ColQ subunits, differing mostly by their signal sequences, are encoded by transcripts ColQ-1 and ColQ-1a, which are differentially expressed in slow and fast twitch muscles in mammals. Two distinct promoters, pColQ-1 and pColQ-1a, were isolated from the upstream sequences of human COLQ gene; they showed muscle-specific expression and were activated by myogenic transcriptional elements in cultured myotubes. After in vivo DNA transfection, pColQ-1 showed strong activity in slow twitch muscle (e.g. soleus), whereas pColQ-1a was preferably expressed in fast twitch muscle (e.g. tibialis). Mutation analysis of the ColQ promoters suggested that the muscle fiber type-specific expression pattern of ColQ transcripts were regulated by a slow upsteam regulatory element (SURE) and a fast intronic regulatory element (FIRE). These regulatory elements were responsive to a calcium ionophore and to calcineurin inhibition by cyclosporine A. The slow fiber type-specific expression of ColQ-1 was abolished by the mutation of an NFAT element in pColQ-1. Moreover, both the ColQ promoters contained N-box element that was responsible for the synapse-specific expression of ColQ transcripts. These results explain the specific expression patterns of collagen-tailed acetylcholinesterase in slow and fast muscle fibers.