Electrophysiological and morphological properties of prefrontal pyramidal neurons innervated by mediodorsal thalamus.

The high-order cognitive and executive functions are necessary for an individual to survive. The densely bidirectional innervations between the medial prefrontal cortex (mPFC) and the mediodorsal thalamus (MD) play a vital role in regulating high-order functions. Pyramidal neurons in mPFC have been classified into several subclasses according to their morphological and electrophysiological properties, but the properties of the input-specific pyramidal neurons in mPFC remain poorly understood. The present study aimed to profile the morphological and electrophysiological properties of mPFC pyramidal neurons innervated by MD. In the past, the studies for characterizing the morphological and electrophysiological properties of neurons mainly relied on the electrophysiological recording of a large number of neurons and their morphologic reconstructions. But, it is a low efficient method for characterizing the circuit-specific neurons. The present study combined the advantages of traditional morphological and electrophysiological methods with machine learning to address the shortcomings of the past method, to establish a classification model for the morphological and electrophysiological properties of mPFC pyramidal neurons, and to achieve more accurate and efficient identification of the properties from a small size sample of neurons. We labeled MD-innervated pyramidal neurons of mPFC using the trans-synaptic neural circuitry tracing method and obtained their morphological properties using whole-cell patch-clamp recording and morphologic reconstructions. The results showed that the classification model established in the present study could predict the electrophysiological properties of MD-innervated pyramidal neurons based on their morphology. MD-innervated pyramidal neurons exhibit larger basal dendritic length but lower apical dendrite complexity compared to non-MD-innervated neurons in the mPFC. The morphological characteristics of the two subtypes (ET-1 and ET-2) of mPFC pyramidal neurons innervated by MD are different, with the apical dendrites of ET-1 neurons being longer and more complex than those of ET-2 neurons. These results suggest that the electrophysiological properties of MD- innervated pyramidal neurons within mPFC correlate with their morphological properties, indicating that the different roles of these two subclasses in local circuits within PFC, as well as in PFC-cortical/subcortical brain region circuits.

Inhibition of GluN2B-containing NMDA receptors in early life combined with social stress in adulthood leads to alterations in prefrontal PNNs in mice.

Perineuronal nets (PNNs) are specialized extracellular matrix (ECM) structures present in the central nervous system (CNS) and have been identified as significant regulators of developmental plasticity in the developing cortex. PNNs are particularly enriched in the cortex surrounding parvalbumin-expressing (PV+) cells. A growing body of evidence suggests that the abnormalities in PV+ neurons and PNNs are associated with various neurological disorders, including schizophrenia, which is a neurodevelopmental defect disease. The N-methyl-D-aspartate receptor (NMDAR) selective antagonist is frequently employed to establish animal models of schizophrenia in laboratory settings. The crucial involvement of GluN2B-containing NMDARs in the development of CNS has been extensively established. However, the role of GluN2B in the pathophysiology of schizophrenia has yet to be thoroughly investigated. The present study inhibited GluN2B function through intraperitoneal infusion of the GluN2B selective antagonist ifenprodil into juvenile mice aged 3-4 weeks, followed by the administration of social stress when these mice reached 9 weeks of age. Then, immunofluorescence staining was employed to examine the changes in the PNNs and PV+ cells, an acoustic startle and prepulse inhibition test was used to detect activities of the PV+ cells, and Western blot was used to quantify the protein expression levels of GluN2A and GluN2B in the prefrontal cortex (PFC). The study revealed that in the PFC of mice subjected to GluN2B antagonist treatment in early life and social stress in adulthood, there was an increase in the number of PV+ cells wrapped by PNNs, and a decrease in the activation of PV+ cells during the prepulse inhibition test, which is an indicator of sensory gating functions, as well as changes in the protein expression levels of GluN2A and GluN2B, which resulted in an increase in the ratio of GluN2A to GluN2B. These aberrations in the mice are comparable to those observed in animal models and patients with schizophrenia. The findings suggest that even a transient hypofunction of GluN2B in early life poses a significant risk for the emergence of schizophrenia symptoms in adulthood.

Presynaptic HCN channels constrain GABAergic synaptic transmission in pyramidal cells of the medial prefrontal cortex.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are widely expressed in neurons in the central nervous system. It has been documented that HCN channels regulate the intrinsic excitability of pyramidal cells in the medial prefrontal cortex (mPFC) of rodents. Here, we report that HCN channels limited GABAergic transmission onto pyramidal cells in rat mPFC. The pharmacological blockade of HCN channels resulted in a significant increase in the frequency of both spontaneous and miniature inhibitory postsynaptic currents (IPSCs) in mPFC pyramidal cells, whereas potentiation of HCN channels reversely decreases the frequency of mIPSCs. Furthermore, such facilitation effect on mIPSC frequency required presynaptic Ca2+ influx. Immunofluorescence staining showed that HCN channels expressed in presynaptic GABAergic terminals, as well as in both soma and neurite of parvalbumin-expressing (PV-expressing) basket cells in mPFC. The present results indicate that HCN channels in GABAergic interneurons, most likely PV-expressing basket cells, constrain inhibitory control over layer 5-6 pyramidal cells by restricting presynaptic Ca2+ entry.

Connexin43 hemichannels contribute to working memory and excitatory synaptic transmission of pyramidal neurons in the prefrontal cortex of rats.

The gap junction is essential for the communication between astrocytes and neurons by various connexins. Connexin43 hemichannels (Cx43 HCs), one of important subunits of gap junction protein, is highly expressed in astrocytes. It has been demonstrated that Cx43 HCs is involved in synaptic plasticity and learning and memory. However, whether the role of Cx43 HCs in the prefrontal cortex (PFC), a key brain region mediating cognitive and executive functions including working memory, still remains unclear. Here, we investigate that the role of Cx43 HCs in working memory through pharmacological inhibition of Cx43 HCs in the PFC. Gap26, a specific hemichannels blocker for Cx43 HCs, was bilaterally infused into the prelimbic (PrL) area of the PFC and then spatial working memory was examined in delayed alternation task in T-maze. Furthermore, the effect of Gap26 on synaptic transmission of prefrontal pyramidal neurons was examined using whole-cell patch recording in slice containing PFC. The demonstrate that inhibition of prefrontal cortex Cx43 HCs impairs the working memory and excitatory synaptic transmission of PFC neurons, suggesting that Cx43 HCs in the PFC contributes to working memory and excitatory synaptic transmission of neurons in rats.

Ankylosing spondylitis complicating Turner syndrome: Two case reports and a literature review.

RATIONALE: Turner syndrome (TS) is an anomaly caused by loss of part of or all the X chromosomes. Ankylosing spondylitis (AS) is an HLA-B27-associated autoimmune disease with a male predominance. It is widely accepted that TS patients are at higher risk of autoimmune diseases, but AS in TS patients has only rarely been reported. PATIENT CONCERNS: A 13-year-old TS patient presented with intermittent pain in both hip joints, and a 27-year-old TS patient presented with thoracic kyphosis and a history of AS. DIAGNOSES: Both patients were diagnosed with AS according to their symptoms, laboratory results, and imaging. INTERVENTIONS: The first patient was treated with tocilizumab for 8 months, whereas the second patient was treated with diclofenac initially with subsequent surgery for thoracic kyphosis. OUTCOMES: Treatment relieved the symptoms of both patients and laboratory parameters improved. LESSONS: Even though AS has a male predominance, clinicians should be aware that AS and TS may co-exist and that the clinical features are atypical in TS patients with AS.

Prefrontal AMPA receptors are involved in the effect of methylphenidate on response inhibition in rats.

Response inhibition is a critical executive control function in many species. Deficits in response inhibition have been observed in many disorders, eg, attention deficit/hyperactivity disorder (ADHD). The stop-signal task (SST) is a unique behavior task for evaluating response inhibition via measuring the covert latency of a stop process, and it is widely used in studies of humans, nonhuman primates and rodents. Methylphenidate (MPH; Ritalin®) is a psychostimulant that is widely used for the treatment of ADHD and that effectively improves response inhibition in individuals with ADHD and normal subjects. However, its mechanism of improving response inhibition remains unknown. In this study we adopted a rodent nose-poking version of the SST to examine response inhibition by estimating the stop signal reaction time (SSRT) in rats. Administration of MPH (1 mg/kg, sc) 25 min before the SST test exerted a baseline-dependent effect of MPH on response inhibition, ie, it shortened the SSRTs only in the rats with larger baseline SSRTs, thereby improving response inhibition in these rats. The effect of MPH on response inhibition remained 3 h after MPH administration. Co-administration of PP2 (1 mg/kg, sc), a Src-protein tyrosine kinase (Src-PTKs) inhibitor that inhibited the upregulation of glutamate receptor expression on the plasma membrane of the prefrontal cortex (PFC), abolished the MPH-caused improvement in response inhibition. Furthermore, intra-PFC infusion of a selective AMPAR antagonist.NASPM (0.3 mmol/L, per side) via stainless guide cannulas implanted earlier abolished the effect of MPH on SSRT. These results suggest that AMPA receptors in the PFC are involved in the effect of MPH on response inhibition in rats.

[Effect of methylphenidate on c-Fos expression in parvalbumin interneurons of juvenile rat frontal cortex].

The etiology of attention-deficit hyperactivity disorder (ADHD) has been generally linked to the decrease in cortex activity, as well as to the reduction in dopamine (DA) and norepinephrine (NE) levels. Methylphenidate (MPH; Ritalin) is the most commonly prescribed medication for ADHD. It has been determined that MPH acts primarily on the dopaminergic and noradrenergic systems through blockade of DA and NE transporters, thereby increasing the concentrations of these neurotransmitters in the brain to correct the attention deficits and hyperactivity. In addition, MPH has been proposed to increase the excitability of pyramidal neurons and the overall activity of cortex. However, the effect of MPH on the activity of interneurons is lack of investigation. Here, by using immunohistochemistry technique, we examined c-Fos expression in parvalbumin (PV)-expressing interneurons of frontal cortex of rats (28-day-old) at 1 h after a single MPH infusion (1 or 8 mg/kg; s.c.). We analyzed the c-Fos expression in the medial orbitofrontal cortex (MO), ventral orbitofrontal cortex (VO), and lateral orbitofrontal cortex (LO) subregions of orbitofrontal cortex (OFC), as well as the prelimbic cortex (PrL) and infralimbic cortex (IL) subregions of the prefrontal cortex (PFC) and anterior cingulate cortex (ACC) after MPH infusion. Our data showed that MPH increased c-Fos expression in MO, VO and LO, and the c-Fos expression in PV-expressing interneurons elevated significantly in MO, VO, but not in LO. Meanwhile, the increases of c-Fos expression in PrL and IL, as well as in PV-expressing interneurons of these two regions, were only induced by 1 mg/kg MPH, but not 8 mg/kg. Both 1 and 8 mg/kg MPH dramatically increased c-Fos expression in ACC, especially, in PV-expressing interneurons of ACC as well. In conclusion, acute systemic injection of MPH significantly increases the c-Fos expression in PV-expressing interneurons of the OFC, PFC and ACC.

Surface expression of hippocampal NMDA GluN2B receptors regulated by fear conditioning determines its contribution to memory consolidation in adult rats.

The number and subtype composition of N-methyl-d-aspartate receptor (NMDAR) at synapses determines their functional properties and role in learning and memory. Genetically increased or decreased amount of GluN2B affects hippocampus-dependent memory in the adult brain. But in some experimental conditions (e.g., memory elicited by a single conditioning trial (1 CS-US)), GluN2B is not a necessary factor, which indicates that the precise role of GluN2B in memory formation requires further exploration. Here, we examined the role of GluN2B in the consolidation of fear memory using two training paradigms. We found that GluN2B was only required for the consolidation of memory elicited by five conditioning trials (5 CS-US), not by 1 CS-US. Strikingly, the expression of membrane GluN2B in CA1was training-strength-dependently increased after conditioning, and that the amount of membrane GluN2B determined its involvement in memory consolidation. Additionally, we demonstrated the increases in the activities of cAMP, ERK, and CREB in the CA1 after conditioning, as well as the enhanced intrinsic excitability and synaptic efficacy in CA1 neurons. Up-regulation of membrane GluN2B contributed to these enhancements. These studies uncover a novel mechanism for the involvement of GluN2B in memory consolidation by its accumulation at the cell surface in response to behavioral training.

Distribution of D1 and D2-dopamine receptors in calcium-binding-protein expressing interneurons in rat anterior cingulate cortex.

Dopamine plays an important role in cognitive functions including decision making, attention, learning and memory in the anterior cingulate cortex (ACC). However, little is known about dopamine receptors (DAR) expression patterns in ACC neurons, especially GABAergic interneurons. The aim of the present study was to investigate the expression of the most abundant DAR subtypes, D1 receptors (D1Rs) and D2 receptors (D2Rs), in major types of GABAergic interneurons in rat ACC, including parvalbumin (PV)-, calretinin (CR)-, and calbindin D-28k (CB)-containing interneurons. Double immunofluorescence staining and confocal scanning were used to detect protein expression in rat brain sections. The results showed a high proportion of PV-containing interneurons express D1Rs and D2Rs, while a low proportion of CR-positive interneurons express D1Rs and D2Rs. D1R- and D2R-expressing PV interneurons are more prevalently distributed in deep layers than superficial layers of ACC. Moreover, we found the proportion of D2Rs expressed in CR cells is much greater than that of D1Rs. These regional and interneuron type-specific differences of D1Rs and D2Rs indicate functionally distinct roles for dopamine in modulating ACC activities via stimulating D1Rs and D2Rs.

The expression of α2A-adrenoceptors in the calcium-binding protein immunoreactive interneurons in rat prefrontal cortex.

The α2A adrenoceptors (α2A-ARs) are the most common adrenergic receptor subtype found in the prefrontal cortex (PFC). It is generally accepted that stimulation of postsynaptic α2A-ARs on pyramidal neurons are key to PFC functions, such as working memory. However, the expression of α2A-ARs in interneurons is largely unknown. In the present study using double-labeling immunofluorencence technique, we investigated the expression of α2A-ARs in major types of rat PFC interneurons expressing calcium-binding proteins parvalbumin (PV), calretinin (CR), and calbindin (CB). Our data demonstrated that α2A-ARs are highly expressed in calcium-binding protein immunoreactive interneurons of rat PFC, suggesting that stimulation of α2A-ARs may alter neural networks comprising pyramidal neurons and interneurons, thereby exerting a beneficial effect on PFC cognitive functions. The present study provides the morphological basis for a potential mechanism by which stimulation of α2A-ARs induces cognitive improvement.

Contribution of dopamine d1/5 receptor modulation of post-spike/burst afterhyperpolarization to enhance neuronal excitability of layer v pyramidal neurons in prepubertal rat prefrontal cortex.

Dopamine (DA) receptors in the prefrontal cortex (PFC) modulate both synaptic and intrinsic plasticity that may contribute to cognitive processing. However, the ionic basis underlying DA actions to enhance neuronal plasticity in PFC remains ill-defined. Using whole-cell patch-clamp recordings in layer V-VI pyramidal cells in prepubertal rat PFC, we showed that DA, via activation of D1/5, but not D2/3/4, receptors suppress a Ca(2+)-dependent, apamin-sensitive K(+) channel that mediates post-spike/burst afterhyperpolarization (AHP) to enhance neuronal excitability of PFC neurons. This inhibition is not dependent on HCN channels. The D1/5 receptor activation also enhanced an afterdepolarizing potential (ADP) that follows the AHP. Additional single-spike analyses revealed that DA or D1/5 receptor activation suppressed the apamin-sensitive post-spike mAHP, further contributing to the increase in evoked spike firing to enhance the neuronal excitability. Taken together, the D1/5 receptor modulates intrinsic mechanisms that amplify a long depolarizing input to sustain spike firing outputs in pyramidal PFC neurons.

Development of a loop-mediated isothermal amplification assay for the detection of porcine hokovirus.

Hokoviruses have recently been detected as pathogens belonging to the family Parvoviridae, which comprises porcine hokovirus (PHoV) and bovine hokovirus (BHoV). In this study, we developed a loop-mediated isothermal amplification (LAMP) assay for the rapid, specific and sensitive detection of PHoV. A set of four primers specific for six regions within the PHoV VP1/2 genes was designed using online software. The reaction temperature and time were optimized at 65°C and 60 min, respectively. LAMP products were detected by agarose gel electrophoresis or by visual inspection of a color change caused by a fluorescent dye. The method was highly specific for PHoV, and no cross-reaction was observed with porcine circovirus type 2 (PCV2), porcine parvovirus (PPV), porcine bocavirus (PBoV), pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), classic swine fever virus (CSFV), or Japanese encephalitis virus (JEV). The detection limit was approximately 10 copies per reaction, which was 10 times more sensitive than conventional PCR. Furthermore, the efficiency of detection of PHoV in clinical samples was comparable to that of PCR and sequencing. These results show that the LAMP assay is a simple, rapid, sensitive and specific method for detecting PHoV. It does not require specialized equipment and can be used to detect PHoV both in the laboratory and in the field.

Signaling mechanism underlying α2A -adrenergic suppression of excitatory synaptic transmission in the medial prefrontal cortex of rats.

Stimulation of α2A -adrenoceptors (ARs) in the prefrontal cortex (PFC) produces a beneficial effect on cognitive functions such as working memory. A previous study in our laboratory showed that α2A -AR stimulation suppresses excitatory synaptic transmission in layer V-VI pyramidal cells of the rat medial PFC (mPFC). However, the intracellular mechanism underlying the α2A -AR suppression remains unclear. In the present study, we recorded evoked excitatory postsynaptic current (eEPSC) in layer V-VI pyramidal cells of the mPFC, using whole-cell patch-clamp recording. We found that the α2A -AR agonist guanfacine significantly suppresses eEPSC in mPFC pyramidal cells. The α2A -AR inhibition is mediated by the Gi-cAMP-PKA-PP1-CaMKII-AMPAR signaling pathway, as such inhibition no longer exists when each step of this pathway is blocked with NF023, Rp-cAMP, PKI5-24 or H89, tautomycin, and KN-62 or KN-93, respectively.

Activation of ß2-adrenoceptor enhances synaptic potentiation and behavioral memory via cAMP-PKA signaling in the medial prefrontal cortex of rats.

The prefrontal cortex (PFC) plays a critical role in cognitive functions, including working memory, attention regulation, behavioral inhibition, as well as memory storage. The functions of PFC are very sensitive to norepinephrine (NE), and even low levels of endogenously released NE exert a dramatic influence on the functioning of the PFC. Activation of ß-adrenoceptors (ß-ARs) facilitates synaptic potentiation and enhances memory in the hippocampus. However, little is known regarding these processes in the PFC. In the present study, we investigate the role of ß2-AR in synaptic plasticity and behavioral memory. Our results show that ß2-AR selective agonist clenbuterol facilitates spike-timing-dependent long-term potentiation (tLTP) under the physiological conditions with intact GABAergic inhibition, and such facilitation is prevented by co-application with the cAMP inhibitor Rp-cAMPS. Loading postsynaptic pyramidal cells with Rp-cAMPS, the PKA inhibitor PKI(5-24), or the G protein inhibitor GDP-ß-S significantly decreases, but does not eliminate, the effect of clenbuterol. Clenbuterol suppresses the GABAergic transmission, while blocking GABAergic transmission by the GABA(A) receptor blocker partially mimics the effect of clenbuterol. In behavioral tests, a post-training infusion of clenbuterol into mPFC enhances 24-h trace fear memory. In summary, we observed that prefrontal cortical ß2-AR activation by clenbuterol facilitates tLTP and enhances trace fear memory. The mechanism underlying tLTP facilitation involves stimulating postsynaptic cAMP-PKA signaling cascades and suppressing GABAergic circuit activities.

Delay-dependent impairment of spatial working memory with inhibition of NR2B-containing NMDA receptors in hippocampal CA1 region of rats.

Hippocampal N-methyl-D-aspartate receptor (NMDAR) is required for spatial working memory. Although evidence from genetic manipulation mice suggests an important role of hippocampal NMDAR NR2B subunits (NR2B-NMDARs) in spatial working memory, it remains unclear whether or not the requirement of hippocampal NR2B-NMDARs for spatial working memory depends on the time of spatial information maintained. Here, we investigate the contribution of hippocampal NR2B-NMDARs to spatial working memory on delayed alternation task in T-maze (DAT task) and delayed matched-to-place task in water maze (DMP task). Our data show that infusions of the NR2B-NMDAR selective antagonists, Ro25-6981 or ifenprodil, directly into the CA1 region, impair spatial working memory in DAT task with 30-s delay (not 5-s delay), but severely impair error-correction capability in both 5-s and 30-s delay task. Furthermore, intra-CA1 inhibition of NR2B-NMDARs impairs spatial working memory in DMP task with 10-min delay (not 30-s delay). Our results suggest that hippocampal NR2B-NMDARs are required for spatial working memory in long-delay task, whereas spare for spatial working memory in short-delay task. We conclude that the requirement of NR2B-NMDARs for spatial working memory is delay-dependent in the CA1 region.

Methylphenidate enhances NMDA-receptor response in medial prefrontal cortex via sigma-1 receptor: a novel mechanism for methylphenidate action.

Methylphenidate (MPH), commercially called Ritalin or Concerta, has been widely used as a drug for Attention Deficit Hyperactivity Disorder (ADHD). Noteworthily, growing numbers of young people using prescribed MPH improperly for pleasurable enhancement, take high risk of addiction. Thus, understanding the mechanism underlying high level of MPH action in the brain becomes an important goal nowadays. As a blocker of catecholamine transporters, its therapeutic effect is explained as being due to proper modulation of D1 and α2A receptor. Here we showed that higher dose of MPH facilitates NMDA-receptor mediated synaptic transmission via a catecholamine-independent mechanism, in layer V∼VI pyramidal cells of the rat medial prefrontal cortex (PFC). To indicate its postsynaptic action, we next found that MPH facilitates NMDA-induced current and such facilitation could be blocked by σ1 but not D1/5 and α2 receptor antagonists. And this MPH eliciting enhancement of NMDA-receptor activity involves PLC, PKC and IP3 receptor mediated intracellular Ca(2+) increase, but does not require PKA and extracellular Ca(2+) influx. Our additional pharmacological studies confirmed that higher dose of MPH increases locomotor activity via interacting with σ1 receptor. Together, the present study demonstrates for the first time that MPH facilitates NMDA-receptor mediated synaptic transmission via σ1 receptor, and such facilitation requires PLC/IP3/PKC signaling pathway. This novel mechanism possibly explains the underlying mechanism for MPH induced addictive potential and other psychiatric side effects.

Development of a loop-mediated isothermal amplification method for rapid detection of porcine boca-like virus.

The porcine boca-like virus (Pbo-likeV) was recently discovered in Swedish pigs with post-weaning multisystemic wasting syndrome (PMWS). In this study, a loop-mediated isothermal amplification (LAMP) assay was developed for rapid, specific and sensitive detection of Pbo-likeV. A set of four primers specific for six regions of Pbo-likeV VP1/2 genes was designed with the online software. The reaction temperature and time were optimized to 65 °C and 60 min, respectively. LAMP products were detected by agarose gel electrophoresis or by visual inspection of a color change due to addition of fluorescent dye. The developed method was highly specific for detection of Pbo-likeV, and no cross-reaction was observed with other swine viruses, such as porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), porcine parvovirus (PPV) and classic swine fever virus (CSFV) found commonly in China. The lower detection limit of the LAMP assay was approximately 10 copies per reaction, and it was 100 times more sensitive than that of conventional PCR. Furthermore, the efficiency of LAMP for detection Pbo-likeV in clinical samples was comparable to PCR and sequencing. These results showed that the LAMP assay is a simple, rapid, sensitive and specific technique for detection of Pbo-likeV, and the procedure of LAMP does not rely on any special equipment. It has capacity for the detection of Pbo-likeV both in the laboratory and on farms.

Subcutaneous and intranasal immunization with Stx2B-Tir-Stx1B-Zot reduces colonization and shedding of Escherichia coli O157:H7 in mice.

The type III secretion system of Escherichia coli O157:H7 is involved in colonization of mammalian hosts by the organism. The translocated intimin receptor (Tir) is inserted into the mammalian host cell plasma membrane in a hairpin loop topology with the central loop of the molecule exposed to the host cell surface and accessible for interaction with an LEE-encoded bacterial outer membrane adhesin called intimin. Shiga toxin type 1 and 2 produced by E. coli O157:H7 are responsible for hemolytic uremic syndrome and able to promote intestinal colonization. Zonula occludens toxin (Zot) is a single polypeptide chain encoded by the filamentous bacteriophage CTXφ of Vibrio cholerae. Zot binds a receptor on intestinal epithelial cells and increases mucosal permeability by affecting the structure of epithelial tight junctions. Because of these properties, Zot is a promising tool for mucosal drug and antigen (Ag) delivery. In the current study, we constructed a novel fusion protein carrying both of the immunogenic B subunits derived from the two toxins, Tir and Zot, designated Stx2B-Tir-Stx1B-Zot, expressed in the E. coli BL21 and harvested the purified protein by a simple GST·Bind Resin chromatography method. We used a streptomycin-treated mouse model to evaluate the efficacy of subcutaneous vs. intranasal administration of the vaccine. Following immunization, mice were infected with E. coli O157:H7 and feces were monitored for shedding. Immune responses against Stx2B-Tir-Stx1B-Zot, Stx2B-Tir-Stx1B and control agent (GST/PBS) were also monitored. Subcutaneous immunization of mice with Stx2B-Tir-Stx1B-Zot induced significant Stx2B-Tir-Stx1B-Zot-specific serum IgG antibodies but did not significantly induce any antigen-specific IgA in feces, whereas intranasal immunization elicited significant Stx2B-Tir-Stx1B-Zot-specific serum IgG antibodies with some animals developing antigen-specific IgA in feces. Mice that were immunized intranasally with Stx2B-Tir-Stx1B-Zot showed dramatically decreased E. coli O157:H7 shedding compared to those of Stx2B-Tir-Stx1B and control agent following experimental infection. Mice immunized subcutaneously with Stx2B-Tir-Stx1B-Zot or Stx2B-Tir-Stx1B both showed reduced shedding in feces, moreover, Stx2B-Tir-Stx1B-Zot did better. These results demonstrate the perspective for the use of Stx2B-Tir-Stx1B-Zot to prevent colonization and shedding of E. coli O157:H7.

Identification and characterization of inosine 5-monophosphate dehydrogenase in Streptococcus suis type 2.

Streptococcus suis type 2 is a swine pathogen responsible for diverse diseases. Although many virulent factors have been identified and studied, relatively little is known about the pathogenic mechanisms of type 2. The aim of the study was to identify and understand the characterization of Inosine 5-monophosphate dehydrogenase (IMPDH). A 957-bp gene, impdh, was identified in the virulent S. suis serotype 2 (SS2), and analysis of the predicted IMPDH sequence revealed IMP dehydrogenase/GMP reductase domain. The gene encoding for the IMPDH of S. suis was cloned and sequenced. The DNA sequence contained an open reading frame encoding for a 318 amino acid polypeptide exhibiting 23% sequence identity with the IMPDH from Streptococcus pyogenes (YP281355) and Streptococcus pneumoniae (ZP00404150). Using the pET(32) expression plasmid, the impdh gene was inducibly overexpressed in Escherichia coli to produce IMPDH with a hexahistidyl N-terminus to permit its purification. The (His)6 IMPDH protein was found to possess functional IMPDH enzymatic activity after the purification. The impdh-knockout SS2 mutant ( Delta IMPDH) constructed in this study was slower in growth and one pH unit higher than SS2-H after 6 h of culturing, and found to be attenuated in mouse models of infection for 2.5 times and not be capable of causing death in porcine models of infection in contrast with the parent SS2-H.

NMDA NR2A and NR2B receptors in the rostral anterior cingulate cortex contribute to pain-related aversion in male rats.

NMDA receptors, which are implicated in pain processing, are highly expressed in forebrain areas including the anterior cingulate cortex (ACC). The ACC has been implicated in the affective response to noxious stimuli. Using a combination of immunohistochemical staining, Western blot, electrophysiological recording and formalin-induced conditioned place avoidance (F-CPA) rat behavioral model that directly reflects the affective component of pain, the present study examined formalin nociceptive conditioning-induced changes in the expressions of NMDA receptor subunits NR1, NR2A, and NR2B in the rostral ACC (rACC) and its possible functional significance. We found that unilateral intraplantar (i.pl.) injection of dilute formalin with or without contextual conditioning exposure markedly increased the expressions of NMDA receptor subunits NR2A and NR2B but not of NR1 in the bilateral rACC. NMDA-evoked currents in rACC neurons were significantly greater in formalin-injected rats than in naïve or normal saline-injected rats. Selectively blocking either NR2A or NR2B subunit in the rACC abolished the acquisition of F-CPA and formalin nociceptive conditioning-induced Fos expression, but it did not affect formalin acute nociceptive behaviors and non-nociceptive fear stimulus-induced CPA. These results suggest that both NMDA receptor subunits NR2A and NR2B in the rACC are critically involved in pain-related aversion. Thus, a new strategy targeted at NMDA NR2A or NR2B subunit might be raised for the prevention of pain-related emotional disturbance.