Repetitive pulsed-wave ultrasound stimulation suppresses neural activity by modulating ambient GABA levels via effects on astrocytes.

Ultrasound is highly biopermeable and can non-invasively penetrate deep into the brain. Stimulation with patterned low-intensity ultrasound can induce sustained inhibition of neural activity in humans and animals, with potential implications for research and therapeutics. Although mechanosensitive channels are involved, the cellular and molecular mechanisms underlying neuromodulation by ultrasound remain unknown. To investigate the mechanism of action of ultrasound stimulation, we studied the effects of two types of patterned ultrasound on synaptic transmission and neural network activity using whole-cell recordings in primary cultured hippocampal cells. Single-shot pulsed-wave (PW) or continuous-wave (CW) ultrasound had no effect on neural activity. By contrast, although repetitive CW stimulation also had no effect, repetitive PW stimulation persistently reduced spontaneous recurrent burst firing. This inhibitory effect was dependent on extrasynaptic-but not synaptic-GABAA receptors, and the effect was abolished under astrocyte-free conditions. Pharmacological activation of astrocytic TRPA1 channels mimicked the effects of ultrasound by increasing the tonic GABAA current induced by ambient GABA. Pharmacological blockade of TRPA1 channels abolished the inhibitory effect of ultrasound. These findings suggest that the repetitive PW low-intensity ultrasound used in our study does not have a direct effect on neural function but instead exerts its sustained neuromodulatory effect through modulation of ambient GABA levels via channels with characteristics of TRPA1, which is expressed in astrocytes.

Disturbance of the reciprocal-interaction between the OXTergic and DAergic systems in the CNS causes atypical social behavior in syntaxin 1A knockout mice.

Several studies have shown that oxytocin (OXT) modulates social behavior. Similarly, monoamines such as dopamine (DA) play a role in regulating social behavior. Previous studies have demonstrated that the soluble N-ethylmaleimide-sensitive fusion attachment protein receptor (SNARE) protein syntaxin 1A (STX1A) regulates the secretion of OXT and monoamines, and that STX1A gene knockout (STX1A KO) mice exhibit atypical social behavior, such as deficient social recognition, due to reduced OXT release. In this study, we analyzed the neural mechanism regulating social behavior by OXT and/or DA using STX1A KO mice as a model animal. We found that OXT directly induced DA release from cultured DA neurons through OXT and V1a receptors. In STX1A KO mice, the atypical social behavior was partially improved by OXT administration, which was inhibited by D1 receptor blockade. In addition, the atypical social behavior in STX1A KO mice was partially improved by facilitation of DAergic signaling with the DA reuptake inhibitor GBR12909. Moreover, the amelioration by GBR12909 was inhibited by OXTR blockade. These results suggest that the reciprocal interaction between the DAergic and OXTergic neuronal systems in the CNS may be important in regulating social behavior.

Syntaxin 1B regulates synaptic GABA release and extracellular GABA concentration, and is associated with temperature-dependent seizures.

De novo heterozygous mutations in the STX1B gene, encoding syntaxin 1B, cause a familial, fever-associated epilepsy syndrome. Syntaxin 1B is an essential component of the pre-synaptic neurotransmitter release machinery as a soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein that regulates the exocytosis of synaptic vesicles. It is also involved in regulating the functions of the SLC6 family of neurotransmitter transporters that reuptake neurotransmitters, including inhibitory neurotransmitters, such as γ-aminobutyric acid (GABA) and glycine. The purpose of the present study was to elucidate the molecular mechanisms underlying the development of febrile seizures by examining the effects of syntaxin 1B haploinsufficiency on inhibitory synaptic transmission during hyperthermia in a mouse model. Stx1b gene heterozygous knockout (Stx1b+/- ) mice showed increased susceptibility to febrile seizures and drug-induced seizures. In cultured hippocampal neurons, we examined the temperature-dependent properties of neurotransmitter release and reuptake by GABA transporter-1 (GAT-1) at GABAergic neurons using whole-cell patch-clamp recordings. The rate of spontaneous quantal GABA release was reduced in Stx1b+/- mice. The hyperthermic temperature increased the tonic GABAA current in wild-type (WT) synapses, but not in Stx1b+/- synapses. In WT neurons, recurrent bursting activities were reduced in a GABA-dependent manner at hyperthermic temperature; however, this was abolished in Stx1b+/- neurons. The blockade of GAT-1 increased the tonic GABAA current and suppressed recurrent bursting activities in Stx1b+/- neurons at the hyperthermic temperature. These data suggest that functional abnormalities associated with GABA release and reuptake in the pre-synaptic terminals of GABAergic neurons may increase the excitability of the neural circuit with hyperthermia.

Syntaxin 1B contributes to regulation of the dopaminergic system through GABA transmission in the CNS.

In neuronal plasma membrane, two syntaxin isoforms, HPC-1/syntaxin 1A (STX1A) and syntaxin 1B (STX1B), are predominantly expressed as soluble N-ethylmaleimide-sensitive fusion attachment protein receptors, also known as t-SNAREs. We previously reported that glutamatergic and GABAergic synaptic transmissions are impaired in Stx1b null mutant (Stx1b-/- ) mice but are almost normal in Stx1a null mutant (Stx1a-/- ) mice. These observations suggested that STX1A and STX1B have distinct functions in fast synaptic transmission in the central nervous system (CNS). Interestingly, recent studies indicated that Stx1a-/- or Stx1a+/- mice exhibit disruption in the monoaminergic system in the CNS, causing unusual behaviour that is similar to neuropsychological alterations observed in psychiatric patients. Here, we studied whether STX1B contributes to the regulation of monoaminergic system and if STX1B is related to neuropsychological properties in human neuropsychological disorders similar to STX1A. We found that monoamine release in vitro was normal in Stx1b+/- mice unlike Stx1a-/- or Stx1a+/- mice, but the basal extracellular dopamine (DA) concentration in the ventral striatum was increased. DA secretion in the ventral striatum is regulated by GABAergic neurons, and Stx1b+/- mice exhibited reduced GABA release both in vitro and in vivo, disrupting the DAergic system in the CNS of these mice. We also found that Stx1b+/- mice exhibited reduced pre-pulse inhibition (PPI), which is believed to represent one of the prominent schizotypal behavioural profiles of human psychiatric patients. The reduction in PPI was rescued by DA receptor antagonists. These observations indicated that STX1B contributes to excess activity of the DAergic system through regulation of GABAergic transmission.

A part of patients with autism spectrum disorder has haploidy of HPC-1/syntaxin1A gene that possibly causes behavioral disturbance as in experimentally gene ablated mice.

Autism spectrum disorder (ASD) is highly heritable and encompasses a various set of neuropsychiatric disorders with a wide-ranging presentation. HPC-1/syntaxin1A (STX1A) encodes a neuronal plasma membrane protein that regulates the secretion of neurotransmitters and neuromodulators. STX1A gene ablated mice (null and heterozygote mutant) exhibit abnormal behavioral profiles similar to human autistic symptoms, accompanied by reduction of monoamine secretion. To determine whether copy number variation of STX1A gene and the change of its expression correlate with ASD as in STX1A gene ablated mice, we performed copy number assay and real-time quantitative RT-PCR using blood or saliva samples from ASD patients. We found that some ASD patients were haploid for the STX1A gene similar to STX1A heterozygote mutant mice. However, copy number of STX1A gene was normal in the parents and siblings of ASD patients with STX1A gene haploidy. In ASD patients with gene haploidy, STX1A mRNA expression was reduced to about half of their parents. Thus, a part of ASD patients had haploidy of STX1A gene and lower STX1A gene expression.

Unusual social behavior in HPC-1/syntaxin1A knockout mice is caused by disruption of the oxytocinergic neural system.

HPC-1/syntaxin1A (STX1A), a neuronal soluble N-ethylmaleimide-sensitive fusion attachment protein receptor, contributes to neural function in the CNS by regulating transmitter release. Recent studies reported that STX1A is associated with human neuropsychological disorders, such as autism spectrum disorder and attention deficit hyperactivity disorder. Previously, we showed that STX1A null mutant mice (STX1A KO) exhibit neuropsychological abnormalities, such as fear memory deficits, attenuation of latent inhibition, and unusual social behavior. These observations suggested that STX1A may be involved in the neuropsychological basis of these abnormalities. Here, to study the neural basis of social behavior, we analyzed the profile of unusual social behavior in STX1A KO with a social novelty preference test, which is a useful method for quantification of social behavior. Interestingly, the unusual social behavior in STX1A KO was partially rescued by intracerebroventricular administration of oxytocin (OXT). In vivo microdialysis studies revealed that the extracellular OXT concentration in the CNS of STX1A KO was significantly lower compared with wild-type mice. Furthermore, dopamine-induced OXT release was reduced in STX1A KO. These results suggested that STX1A plays an important role in social behavior through regulation of the OXTergic neural system. Dopamine (DA) release is reduced in CNS of syntaxin1A null mutant mice (STX1A KO). Unusual social behavior was observed in STX1A KO. We found that oxytocin (OXT) release, which was stimulated by DA, was reduced and was rescued the unusual social behavior in STX1A KO was rescued by OXT. These results indicated that STX1A plays an important role in promoting social behavior through regulation of DA-induced OXT release in amygdala.

HPC-1/syntaxin 1A and syntaxin 1B play distinct roles in neuronal survival.

Two types of syntaxin 1 isoforms, HPC-1/syntaxin 1A (STX1A) and syntaxin 1B (STX1B), are thought to have similar functions in exocytosis of synaptic vesicles. STX1A(-/-) mice which we generated previously develop normally, possibly because of compensation by STX1B. We produced STX1B(-/-) mice using targeted gene disruption and investigated their phenotypes. STX1B(-/-) mice were born alive, but died before postnatal day 14, unlike STX1A(-/-) mice. Morphologically, brain development in STX1B(-/-) mice was impaired. In hippocampal neuronal culture, the cell viability of STX1B(-/-) neurons was lower than that of WT or STX1A(-/-) neurons after 9 days. Interestingly, STX1B(-/-) neurons survived on WT or STX1A(-/-) glial feeder layers as well as WT neurons. However, STX1B(-/-) glial feeder layers were less effective at promoting survival of STX1B(-/-) neurons. Conditioned medium from WT or STX1A(-/-) glial cells had a similar effect on survival, but that from STX1B(-/-) did not promote survival. Furthermore, brain-derived neurotrophic factor (BDNF) or neurotrophin-3 supported survival of STX1B(-/-) neurons. BDNF localization in STX1B(-/-) glial cells was disrupted, and BDNF secretion from STX1B(-/-) glial cells was impaired. These results suggest that STX1A and STX1B may play distinct roles in supporting neuronal survival by glia. Syntaxin 1A (STX1A) and syntaxin 1B (STX1B) are thought to have similar functions as SNARE proteins. However, we found that STX1A and STX1B play distinct roles in neuronal survival using STX1A(-/-) mice and STX1B(-/-) mice. STX1B was important for neuronal survival, possibly by regulating the secretion of neurotrophic factors, such as BDNF, from glial cells.

Syntaxin 1B, but not syntaxin 1A, is necessary for the regulation of synaptic vesicle exocytosis and of the readily releasable pool at central synapses.

Two syntaxin 1 (STX1) isoforms, HPC-1/STX1A and STX1B, are coexpressed in neurons and function as neuronal target membrane (t)-SNAREs. However, little is known about their functional differences in synaptic transmission. STX1A null mutant mice develop normally and do not show abnormalities in fast synaptic transmission, but monoaminergic transmissions are impaired. In the present study, we found that STX1B null mutant mice died within 2 weeks of birth. To examine functional differences between STX1A and 1B, we analyzed the presynaptic properties of glutamatergic and GABAergic synapses in STX1B null mutant and STX1A/1B double null mutant mice. We found that the frequency of spontaneous quantal release was lower and the paired-pulse ratio of evoked postsynaptic currents was significantly greater in glutamatergic and GABAergic synapses of STX1B null neurons. Deletion of STX1B also accelerated synaptic vesicle turnover in glutamatergic synapses and decreased the size of the readily releasable pool in glutamatergic and GABAergic synapses. Moreover, STX1A/1B double null neurons showed reduced and asynchronous evoked synaptic vesicle release in glutamatergic and GABAergic synapses. Our results suggest that although STX1A and 1B share a basic function as neuronal t-SNAREs, STX1B but not STX1A is necessary for the regulation of spontaneous and evoked synaptic vesicle exocytosis in fast transmission.

Impairment of catecholamine systems during induction of long-term potentiation at hippocampal CA1 synapses in HPC-1/syntaxin 1A knock-out mice.

The membrane protein HPC-1/syntaxin 1A is believed to play a key role in synaptic vesicle exocytosis, and it was recently suggested to be required for synaptic plasticity. Despite evidence for the function of HPC-1/syntaxin 1A in synaptic plasticity, the underlying cellular mechanism is unclear. We found that although fast synaptic transmission and long-term depression were unaffected, HPC-1/syntaxin 1A knock-out (STX1A(-/-)) mice showed impaired long-term potentiation (LTP) in response to theta-burst stimulation in CA1 hippocampal slices. The impairment in LTP was rescued by the application of forskolin, an adenylyl cyclase activator, or more robust stimulation, suggesting that cAMP/protein kinase A signaling was suppressed in these mice. In addition, catecholamine release from the hippocampus was significantly reduced in STX1A(-/-) mice. Because HPC-1/syntaxin 1A regulates exocytosis of dense-core synaptic vesicles, which contain neuromodulatory transmitters such as noradrenaline, dopamine and 5-HT, we examined the effect of neuromodulatory transmitters on LTP induction. Noradrenaline and dopamine enhanced LTP induction in STX1A(-/-) mice, whereas catecholamine depletion reduced LTP induction in wild-type mice. Theses results suggest that HPC-1/syntaxin 1A regulates catecholaminergic systems via exocytosis of dense-core synaptic vesicles, and that deletion of HPC-1/syntaxin 1A causes impairment of LTP induction.

Physiological properties of enkephalin-containing neurons in the spinal dorsal horn visualized by expression of green fluorescent protein in BAC transgenic mice.

BACKGROUND: Enkephalins are endogenous opiates that are assumed to modulate nociceptive information by mediating synaptic transmission in the central nervous system, including the spinal dorsal horn. RESULTS: To develop a new tool for the identification of in vitro enkephalinergic neurons and to analyze enkephalin promoter activity, we generated transgenic mice for a bacterial artificial chromosome (BAC). Enkephalinergic neurons from these mice expressed enhanced green fluorescent protein (eGFP) under the control of the preproenkephalin (PPE) gene (penk1) promoter. eGFP-positive neurons were distributed throughout the gray matter of the spinal cord, and were primarily observed in laminae I-II and V-VII, in a pattern similar to the distribution pattern of enkephalin-containing neurons. Double immunostaining analysis using anti-enkephalin and anti-eGFP antibodies showed that all eGFP-expressing neurons contained enkephalin. Incubation in the presence of forskolin, an activator of adenylate cyclase, increased the number of eGFP-positive neurons. These results indicate that eGFP expression is controlled by the penk1 promoter, which contains cyclic AMP-responsive elements. Sections obtained from sciatic nerve-ligated mice exhibited increased eGFP-positive neurons on the ipsilateral (nerve-ligated side) compared with the contralateral (non-ligated side). These data indicate that PPE expression is affected by peripheral nerve injury. Additionally, single-neuron RT-PCR analysis showed that several eGFP positive-neurons in laminae I-II expressed glutamate decarboxylase 67 mRNA and that some expressed serotonin type 3 receptors. CONCLUSIONS: These results suggest that eGFP-positive neurons in laminae I-II coexpress enkephalin and γ-aminobutyric acid (GABA), and are activated by forskolin and in conditions of nerve injury. The penk1-eGFP BAC transgenic mouse contributes to the further characterization of enkephalinergic neurons in the transmission and modulation of nociceptive information.

HPC-1/syntaxin 1A gene knockout mice show abnormal behavior possibly related to a disruption in 5-HTergic systems.

HPC-1/syntaxin 1A (STX1A) is thought to regulate the exocytosis of synaptic vesicles in neurons. In recent human genetic studies, STX1A has been implicated in neuropsychological disorders. To examine whether STX1A gene ablation is responsible for abnormal neuropsychological profiles observed in human psychiatric patients, we analysed the behavioral phenotype of STX1A knockout mice. Abnormal behavior was observed in both homozygotes (STX1A(-/-)) and heterozygotes (STX1A(+/-)) in a social interaction test, a novel object exploring test and a latent inhibition (LI) test, but not in a pre-pulse inhibition test. Interestingly, attenuation of LI, which is closely related to human schizotypic symptoms, was restored by administration of the selective serotonin reuptake inhibitor, fluoxetine, but not by the dopamine reuptake inhibitor, GBR12935, or the noradrenalin reuptake inhibitor, desipramine. We also observed that LI attenuation was restored by DOI (a 5-HT(2A) receptor agonist), but not by 8-OH-DPAT (a 5-HT(1A) receptor agonist), mCPP (a 5-HT(2C) receptor agonist), SKF 38393 (a D(1) receptor agonist), quinpirole (a D(2)/D(3) receptor agonist) or haloperidol (a D(2)/D(3) receptor antagonist). Thus, attenuation of LI is mainly caused by disruption of 5-HT-ergic systems via 5-HT(2A) receptors. In addition, 5-HT release from hippocampal and hypothalamic slices was significantly reduced. Therefore, ablation of STX1A may cause disruption of 5-HT-ergic transmission and induce abnormal behavior.

Analysis of knock-out mice to determine the role of HPC-1/syntaxin 1A in expressing synaptic plasticity.

The protein HPC-1/syntaxin 1A is abundantly expressed in neurons and localized in the neuronal plasma membrane. It forms a complex with SNAP-25 (25 kDa synaptosomal-associated protein) and VAMP-2 (vesicle-associated membrane protein)/synaptobrevin called SNARE (a soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) complex, which is considered essential for synaptic vesicle exocytosis; thus, HPC-1/syntaxin 1A is considered crucial for synaptic transmission. To examine the physiological function of HPC-1/syntaxin 1A in vivo, we produced knock-out (KO) mice by targeted gene disruption. Although HPC-1/syntaxin 1A expression was completely depleted without any effect on the expression of other SNARE proteins, the KO mice were viable. They grew normally, were fertile, and displayed no difference in appearance compared with control littermate. In cultured hippocampal neurons derived from the KO mice, the basic synaptic transmission in vitro was normal. However, the mutant mice had impaired long-term potentiation in the hippocampal slice. Also, although KO mice exhibited normal spatial memory in the hidden platform test, consolidation of conditioned fear memory was impaired. Interestingly, the KO mice had impaired conditioned fear memory extinction. These observations suggest that HPC-1/syntaxin 1A may be closely related to synaptic plasticity.

Differentiation of smooth muscle cells in chicken gizzard. Self-catalytic mechanism for the production of smooth muscle cells.

During development of the chicken gizzard, a thick layer of undifferentiated cells (mesenchymal cells) is constructed, and the cells differentiate into smooth muscle cells or connective tissues. We found that the differentiation of smooth muscle cells occurred first near the outer surface of the gizzard and the differentiated area spread to the inside of the gizzard. Therefore, we assumed that the differentiation of most of the smooth muscle cells in the gizzard is induced by differentiated smooth muscle itself. When undifferentiated cells from gizzard of 7-day-old embryo (Hamburger and Hamilton's stages 26-27) were cultured on a coverglass coated with extract of gizzard that contained differentiated smooth muscle cells, the cells attached to the coverglass and differentiated into smooth muscle cells. On the other hand, extract of gizzard from 7-day-old embryo did not induce the differentiation of smooth muscle cells, though it induced the attachment of cells. We found that activity for the differentiation of smooth muscle cells appeared when differentiated smooth muscle cells appeared in developing gizzard. Gizzard contained higher activity for the differentiation of smooth muscle cells than the other tissues. Transforming growth factor-beta (TGF-beta), which induces the differentiation of vascular smooth muscle cells, did not induce the differentiation of smooth muscle cells in gizzard, though extract of aorta induced the differentiation of smooth muscle cells in gizzard. The results obtained here support evidence that the differentiation of most of the smooth muscle cells in gizzard is induced by a self-catalytic mechanism in which differentiated smooth muscle itself induces the differentiation of smooth muscle cells.