Takefumi Kofuji

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.

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.

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.

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.

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‐HT2A receptor agonist), but not by 8‐OH‐DPAT (a 5‐HT1A receptor agonist), mCPP (a 5‐HT2C receptor agonist), SKF 38393 (a D1 receptor agonist), quinpirole (a D2/D3 receptor agonist) or haloperidol (a D2/D3 receptor antagonist). Thus, attenuation of LI is mainly caused by disruption of 5‐HT‐ergic systems via 5‐HT2A 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.

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

BackgroundEnkephalins are endogenous opiates that are assumed to modulate nociceptive information by mediating synaptic transmission in the central nervous system, including the spinal dorsal horn.ResultsTo 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.ConclusionsThese 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.

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.

Dysfunction of the hypothalamic-pituitary-adrenal axis in STX1A knockout mice.

HPC‐1/syntaxin1A (STX1A) is considered to regulate exocytosis in neurones and endocrine cells. Previously, we reported that STX1A null mutant (STX1A KO) mice unexpectedly showed normal glutamatergic and GABAergic fast synaptic transmission but exhibited disturbances in monoaminergic transmission, such as serotonin, 5‐hydroxytryptamine (5‐HT), which may induce attenuation of latent inhibition. These results suggest that STX1A may contribute to dense‐core vesicle exocytosis in vivo. Thus, we hypothesised that the lack of STX1A might affect the secretion of several hormones, as also mediated by dense‐core vesicles exocytosis. In the present study, we focused on the hypothalamic‐pituitary‐adrenal (HPA) axis, which is a neuroendocrine system that regulates responses to stress stimuli and is considered to be associated with neuropsychiatric disorders. Specifically, we examined whether the HPA axis is impaired in STX1A KO mice. Interestingly, plasma concentrations of both corticosterone (CORT) and adrenocorticotrophin hormone (ACTH) during the resting condition decreased in STX1A KO mice compared to WT mice. Additionally, elevated plasma CORT, ACTH and corticotrophin‐releasing hormone (CRH) which were usually observed after acute restraint stress, were also reduced in STX1A KO mice. We also observed the suppression of 5‐HT‐induced CRH release in STX1A KO mice in vitro. Furthermore, an in vivo microdialysis study revealed that the elevation of extracellular 5‐HT in the hypothalamus, which was induced by the selective serotonin reuptake inhibitor, fluoxetine, was significantly reduced in STX1A KO mice compared to WT mice. 5‐HT elevation in the hypothalamus, which was induced by acute restraint stress, was also reduced in STX1A KO mice. Finally, STX1A KO mice showed abnormal behavioural responses after mild restraint stress. These results indicate that the lack of STX1A could induce dysfunction of the HPA axis, and the deficit may result in abnormal behavioural properties, such as unusual responses to stress stimuli.

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.

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.