Autism-like behaviours and germline transmission in transgenic monkeys overexpressing MeCP2.

Methyl-CpG binding protein 2 (MeCP2) has crucial roles in transcriptional regulation and microRNA processing. Mutations in the MECP2 gene are found in 90% of patients with Rett syndrome, a severe developmental disorder with autistic phenotypes. Duplications of MECP2-containing genomic segments cause the MECP2 duplication syndrome, which shares core symptoms with autism spectrum disorders. Although Mecp2-null mice recapitulate most developmental and behavioural defects seen in patients with Rett syndrome, it has been difficult to identify autism-like behaviours in the mouse model of MeCP2 overexpression. Here we report that lentivirus-based transgenic cynomolgus monkeys (Macaca fascicularis) expressing human MeCP2 in the brain exhibit autism-like behaviours and show germline transmission of the transgene. Expression of the MECP2 transgene was confirmed by western blotting and immunostaining of brain tissues of transgenic monkeys. Genomic integration sites of the transgenes were characterized by a deep-sequencing-based method. As compared to wild-type monkeys, MECP2 transgenic monkeys exhibited a higher frequency of repetitive circular locomotion and increased stress responses, as measured by the threat-related anxiety and defensive test. The transgenic monkeys showed less interaction with wild-type monkeys within the same group, and also a reduced interaction time when paired with other transgenic monkeys in social interaction tests. The cognitive functions of the transgenic monkeys were largely normal in the Wisconsin general test apparatus, although some showed signs of stereotypic cognitive behaviours. Notably, we succeeded in generating five F1 offspring of MECP2 transgenic monkeys by intracytoplasmic sperm injection with sperm from one F0 transgenic monkey, showing germline transmission and Mendelian segregation of several MECP2 transgenes in the F1 progeny. Moreover, F1 transgenic monkeys also showed reduced social interactions when tested in pairs, as compared to wild-type monkeys of similar age. Together, these results indicate the feasibility and reliability of using genetically engineered non-human primates to study brain disorders.

Spontaneous expression of mirror self-recognition in monkeys after learning precise visual-proprioceptive association for mirror images.

Mirror self-recognition (MSR) is generally considered to be an intrinsic cognitive ability found only in humans and a few species of great apes. Rhesus monkeys do not spontaneously show MSR, but they have the ability to use a mirror as an instrument to find hidden objects. The mechanism underlying the transition from simple mirror use to MSR remains unclear. Here we show that rhesus monkeys could show MSR after learning precise visual-proprioceptive association for mirror images. We trained head-fixed monkeys on a chair in front of a mirror to touch with spatiotemporal precision a laser pointer light spot on an adjacent board that could only be seen in the mirror. After several weeks of training, when the same laser pointer light was projected to the monkey's face, a location not used in training, all three trained monkeys successfully touched the face area marked by the light spot in front of a mirror. All trained monkeys passed the standard face mark test for MSR both on the monkey chair and in their home cage. Importantly, distinct from untrained control monkeys, the trained monkeys showed typical mirror-induced self-directed behaviors in their home cage, such as using the mirror to explore normally unseen body parts. Thus, bodily self-consciousness may be a cognitive ability present in many more species than previously thought, and acquisition of precise visual-proprioceptive association for the images in the mirror is critical for revealing the MSR ability of the animal.

Automatic detection and classification of marmoset vocalizations using deep and recurrent neural networks.

This paper investigates the methods to detect and classify marmoset vocalizations automatically using a large data set of marmoset vocalizations and deep learning techniques. For vocalization detection, neural networks-based methods, including deep neural network (DNN) and recurrent neural network with long short-term memory units, are designed and compared against a conventional rule-based detection method. For vocalization classification, three different classification algorithms are compared, including a support vector machine (SVM), DNN, and long short-term memory recurrent neural networks (LSTM-RNNs). A 1500-min audio data set containing recordings from four pairs of marmoset twins and manual annotations is employed for experiments. Two test sets are built according to whether the test samples are produced by the marmosets in the training set (test set I) or not (test set II). Experimental results show that the LSTM-RNN-based detection method outperformed others and achieved 0.92% and 1.67% frame error rate on these two test sets. Furthermore, the deep learning models obtained higher classification accuracy than the SVM model, which was 95.60% and 91.67% on the two test sets, respectively.

K+ channel reorganization and homeostatic plasticity during postembryonic development: biophysical and genetic analyses in acutely dissociated Drosophila central neurons.

Intrinsic electric activities of neurons play important roles in establishing and refining neural circuits during development. However, how the underlying ionic currents undergo postembryonic reorganizations remains largely unknown. Using acutely dissociated neurons from larval, pupal, and adult Drosophila brains, we show drastic re-assemblies and compensatory regulations of voltage-gated (IKv) and Ca2+-activated (IK(Ca)) K+ currents during postembryonic development. Larval and adult neurons displayed prominent fast-inactivating IKv, mediated by the Shaker (Sh) channel to a large extent, while in the same neurons IK(Ca) was far smaller in amplitude. In contrast, pupal neurons were characterized by large sustained IKv and prominent IK(Ca), encoded predominantly by the slowpoke (slo) gene. Surprisingly, deletion of Sh in the ShM null mutant removed inactivating, transient IKv from large portions of neurons at all stages. Interestingly, elimination of Sh currents was accompanied by upregulation of non-Sh transient IKv. In comparison, the slo1 mutation abolished the vast majority of IK(Ca), particularly at the pupal stage. Strikingly, the deficiency of IK(Ca) in slo pupae was compensated by the transient component of IKv mediated by Sh channels. Thus, IK(Ca) appears to play critical roles in pupal development and its absence induces functional compensations from a specific transient IKv current. While mutants lacking either Sh or slo currents survived normally, Sh;;slo double mutants deficient in both failed to survive through pupal metamorphosis. Together, our data highlight significant reorganizations and homeostatic compensations of K+ currents during postembryonic development and uncover previously unrecognized roles for Sh and slo in this plastic process.

Population genetics of marmosets in Asian primate research centers and loci associated with epileptic risk revealed by whole-genome sequencing.

The common marmoset ( Callithrix jacchus) has emerged as a valuable nonhuman primate model in biomedical research with the recent release of high-quality reference genome assemblies. Epileptic marmosets have been independently reported in two Asian primate research centers. Nevertheless, the population genetics within these primate centers and the specific genetic variants associated with epilepsy in marmosets have not yet been elucidated. Here, we characterized the genetic relationships and risk variants for epilepsy in 41 samples from two epileptic marmoset pedigrees using whole-genome sequencing. We identified 14 558 184 single nucleotide polymorphisms (SNPs) from the 41 samples and found higher chimerism levels in blood samples than in fingernail samples. Genetic analysis showed fourth-degree of relatedness among marmosets at the primate centers. In addition, SNP and copy number variation (CNV) analyses suggested that the WW domain-containing oxidoreductase ( WWOX) and Tyrosine-protein phosphatase nonreceptor type 21 ( PTPN21) genes may be associated with epilepsy in marmosets. Notably, KCTD18-like gene deletion was more common in epileptic marmosets than control marmosets. This study provides valuable population genomic resources for marmosets in two Asian primate centers. Genetic analyses identified a reasonable breeding strategy for genetic diversity maintenance in the two centers, while the case-control study revealed potential risk genes/variants associated with epilepsy in marmosets.

Enhanced learning and memory in GAT1 heterozygous mice.

γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system. The termination of GABA transmission is through the action of a family of membrane proteins, called GABA transporters (GAT1-4). It is well established that GABA system is involved in the modulation of memory. Our previous study showed that homozygous GAT1(-/-) mice exhibited impaired hippocampus-dependent learning and memory. To evaluate the impact of endogenous reduced GABA reuptake on mice cognitive behaviors, the ability of learning and memory of heterozygous GAT1(+/-) mice was detected by the passive avoidance paradigm and Morris water maze. The hole board paradigm was also used to measure changes in anxiety-related behavior or exploratory behavior in such mice. As one form of synaptic plasticity, long-term potentiation was recorded in the mouse hippocampal CA1 area. We found that GAT1(+/-) mice displayed increased learning and memory, decreased anxiety-like behaviors, and highest synaptic plasticity compared with wild-type and homozygous GAT1(-/-) mice. Our results suggest that a moderate reduction in GAT1 activity causes the enhancement of learning and memory in mice.

A natural marmoset model of genetic generalized epilepsy.

Epilepsy has been extensively studied as a common neurological disease. Efforts have been made on rodent and other animal models to reveal the pathogenic mechanisms of epilepsy and develop new drugs for treatment. However, the features of current epilepsy models cannot fully mimic different types of epilepsy in humans, hence non-human primate models of epilepsy are required. The common marmoset (Callithrix jacchus) is a New World monkey that is widely used to study brain function. Here, we present a natural marmoset model of generalized epilepsy. In this unique marmoset family, generalized epilepsy was successfully induced by handling operations in some individuals. We mapped the marmoset family with handling-sensitive epilepsy and found that the epileptic phenotype can be inherited. These marmosets were more sensitive to the epilepsy inducers pentylenetetrazol. Using electrocorticogram (ECoG) recordings, we detected epileptiform discharge in marmosets with a history of seizures. In summary, we report a family of marmosets with generalized seizures induced by handling operations. This epileptic marmoset family provides insights to better understand the mechanism of generalized epilepsy and helps to develop new therapeutic methods.

Powerful beneficial effects of benfotiamine on cognitive impairment and beta-amyloid deposition in amyloid precursor protein/presenilin-1 transgenic mice.

Reduction of glucose metabolism in brain is one of the main features of Alzheimer's disease. Thiamine (vitamin B1)-dependent processes are critical in glucose metabolism and have been found to be impaired in brains from patients with Alzheimer's disease. However, thiamine treatment exerts little beneficial effect in these patients. Here, we tested the effect of benfotiamine, a thiamine derivative with better bioavailability than thiamine, on cognitive impairment and pathology alterations in a mouse model of Alzheimer's disease, the amyloid precursor protein/presenilin-1 transgenic mouse. We show that after a chronic 8 week treatment, benfotiamine dose-dependently enhanced the spatial memory of amyloid precursor protein/presenilin-1 mice in the Morris water maze test. Furthermore, benfotiamine effectively reduced both amyloid plaque numbers and phosphorylated tau levels in cortical areas of the transgenic mice brains. Unexpectedly, these effects were not mimicked by another lipophilic thiamine derivative, fursultiamine, although both benfotiamine and fursultiamine were effective in increasing the levels of free thiamine in the brain. Most notably, benfotiamine, but not fursultiamine, significantly elevated the phosphorylation level of glycogen synthase kinase-3alpha and -3beta, and reduced their enzymatic activities in the amyloid precursor protein/presenilin-1 transgenic brain. Therefore, in the animal Alzheimer's disease model, benfotiamine appears to improve the cognitive function and reduce amyloid deposition via thiamine-independent mechanisms, which are likely to include the suppression of glycogen synthase kinase-3 activities. These results suggest that, unlike many other thiamine-related drugs, benfotiamine may be beneficial for clinical Alzheimer's disease treatment.

Neural presbycusis at ultra-high frequency in aged common marmosets and rhesus monkeys.

The aging of the population and environmental noise have contributed to high rates of presbycusis, also known as age-related hearing loss (ARHL). Because mice have a relatively short life span, murine models have not been suitable for determining the mechanism of presbycusis development and methods of diagnosis. Although the common marmoset, a non-human primate (NHP), is an ideal animal model for studying age-related diseases, its auditory spectrum has not been systematically studied. Auditory brainstem responses (ABRs) from 38 marmosets of different ages demonstrated that auditory function correlated with age. Hearing loss in geriatric common marmosets started at ultra-high frequency (>16 kHz), then extended to lower frequencies. Despite age-related deterioration of ABR threshold and amplitude in marmosets, outer hair cell (OHC) function remained stable at all ages. Spiral ganglion neurons (SGNs), which are the first auditory neurons in the auditory system, were found to degenerate distinctly in aged common marmosets, indicating that neural degeneration caused presbycusis in these animals. Similarly, age-associated ABR deterioration without loss of OHC function was observed in another NHP, rhesus monkeys. Audiometry results from these two species of NHP suggested that NHPs were ideal for studying ARHL and that neural presbycusis at high frequency may be prevalent in primates.

Distinct neuron populations for simple and compound calls in the primary auditory cortex of awake marmosets.

Marmosets are highly social non-human primates that live in families. They exhibit rich vocalization, but the neural basis underlying this complex vocal communication is largely unknown. Here we report the existence of specific neuron populations in marmoset A1 that respond selectively to distinct simple or compound calls made by conspecific marmosets. These neurons were spatially dispersed within A1 but distinct from those responsive to pure tones. Call-selective responses were markedly diminished when individual domains of the call were deleted or the domain sequence was altered, indicating the importance of the global rather than local spectral-temporal properties of the sound. Compound call-selective responses also disappeared when the sequence of the two simple-call components was reversed or their interval was extended beyond 1 s. Light anesthesia largely abolished call-selective responses. Our findings demonstrate extensive inhibitory and facilitatory interactions among call-evoked responses, and provide the basis for further study of circuit mechanisms underlying vocal communication in awake non-human primates.

GABA transporter-1 activity modulates hippocampal theta oscillation and theta burst stimulation-induced long-term potentiation.

The network oscillation and synaptic plasticity are known to be regulated by GABAergic inhibition, but how they are affected by changes in the GABA transporter activity remains unclear. Here we show that in the CA1 region of mouse hippocampus, pharmacological blockade or genetic deletion of GABA transporter-1 (GAT1) specifically impaired long-term potentiation (LTP) induced by theta burst stimulation, but had no effect on LTP induced by high-frequency stimulation or long-term depression induced by low-frequency stimulation. The extent of LTP impairment depended on the precise burst frequency, with significant impairment at 3-7 Hz that correlated with the time course of elevated GABAergic inhibition caused by GAT1 disruption. Furthermore, in vivo electrophysiological recordings showed that GAT1 gene deletion reduced the frequency of hippocampal theta oscillation. Moreover, behavioral studies showed that GAT1 knock-out mice also exhibited impaired hippocampus-dependent learning and memory. Together, these results have highlighted the important link between GABAergic inhibition and hippocampal theta oscillation, both of which are critical for synaptic plasticity and learning behaviors.

Having Infants in the Family Group Promotes Altruistic Behavior of Marmoset Monkeys.

The common marmoset (Callithrix jacchus) has attracted much attention as a useful model for studying social behaviors [1-3]. They naturally live in a monogamous family group and exhibit cooperative breeding [4], in which parents and older siblings help to carry infants less than 2 months old [5-7]. Marmoset parents also transfer foods to their offspring, a process that may help them learn the food diet [8]. Furthermore, marmosets show spontaneous altruistic behaviors, such as providing food to non-reciprocating and genetically unrelated individuals [9]. These social habits indicate that marmosets may be a useful non-human primate model for studying parenting and altruistic behaviors, as well as underlying neural mechanisms. Using a novel rescue paradigm, we found that marmoset parents and older siblings showed strong motivation to rescue trapped young infants but not juvenile marmosets beyond 2 months of age, and infant calls alone could trigger these parents' rescue behaviors. The marmoset parents showed little rescue of each other, but young infants or infant calls could also induce such parents' mutual rescue. Moreover, all these infant- and mate-rescue behaviors depended on currently having young infants in the family group. Functional MRI studies on awake adult marmosets showed that calls from young infants, but not juvenile marmosets, elicited a large-scale activation of specific brain areas including auditory and insular cortices, and such activation was absent in marmosets not living with infants. Thus, such infant-induced modification of neural activity offers a window for examining the neural basis of altruistic behaviors in marmoset monkeys.

Bell-shaped D-serine actions on hippocampal long-term depression and spatial memory retrieval.

D-Serine, the endogenous coagonist of N-methyl-D-aspartate receptors (NMDARs), is considered to be an important gliotransmitter, and is essential for the induction of long-term potentiation. However, less is known about the role of D-serine in another form of synaptic plasticity, long-term depression (LTD). In this study, we found that exogenous D-serine regulated LTD in the hippocampal CA1 region in a "bell-shaped" concentration-dependent manner through regulating the function of NMDARs in the same manner, whereas endogenous D-serine was activity-dependently released and, in turn, contributed to the induction of LTD during low-frequency stimulation. Furthermore, impairing glial functions with sodium fluoroacetate (NaFAC) reduced the magnitude of LTD, which could be restored by exogenous D-serine, indicating that endogenous D-serine is mainly glia-derived during LTD induction. More interestingly, similar to the effects on LTD, exogenous D-serine enhanced spatial memory retrieval in the Morris water maze in a bell-shaped dose-dependent manner and rescued the NaFAC-induced impairment of memory retrieval, suggesting links between LTD and spatial memory retrieval. Our study thus provides direct evidence of the bell-shaped D-serine actions on hippocampal LTD and spatial memory retrieval, and underscores the importance of D-serine in synaptic plasticity, learning, and memory.

Glycine uptake regulates hippocampal network activity via glycine receptor-mediated tonic inhibition.

Functional glycine receptors (GlyRs) are enriched in the hippocampus, but their role in hippocampal function remains unclear. Since the concentration of ambient glycine is determined by the presence of powerful glycine transporter (GlyT), we blocked the reuptake of glycine in hippocampal slices to examine the role of GlyRs. Antagonists of GlyT type 1 (GlyT1) but not that of GlyT type 2 (GlyT2) induced excitatory postsynaptic potential (EPSP)-spike depression, which was reversed by the specific GlyR antagonist strychnine. Moreover, endogenously elevating the glycine concentration with the GlyT1 antagonists facilitated NMDA receptor-dependent long-term potentiation induction, and elicited a strychnine-sensitive chloride current. In addition, impairment of glial function with fluoroacetate blocked the effect of GlyT1 antagonists on the EPSP-spike curve. Furthermore, pretreatment with sarcosine was effective in controlling pentylenetetrazol-induced seizures. These results indicate an essential role of GlyTs in fine-tuning tonic activation of GlyRs and suggest a potential role of GlyR-dependent EPSP-spike depression in hippocampal network stability.

The aspirin metabolite salicylate enhances neuronal excitation in rat hippocampal CA1 area through reducing GABAergic inhibition.

Salicylate is the major metabolite and active component of aspirin (acetylsalicylic acid), which is widely used in clinical medicine for treating inflammation, pain syndromes and cardiovascular disorders. The well-known mechanism underlying salicylate's action mainly involves the inhibition of cyclooxygenase and subsequent decrease in prostaglandin production. Recent evidence suggests that salicylate also affects neuronal function through interaction with specific membrane channels/receptors. However, the effect of salicylate on synaptic and neural network function remains largely unknown. In this study, we investigated the effect of sodium salicylate on the synaptic transmission and neuronal excitation in the hippocampal CA1 area of rats, a key structure for many complex brain functions. With electrophysiological recordings in hippocampal slices, we found that sodium salicylate significantly enhanced neuronal excitation through reducing inhibitory GABAergic transmission without affecting the basal excitatory synaptic transmission. Salicylate significantly inhibited the amplitudes of both evoked and miniature inhibitory postsynaptic currents, and directly reduced gamma-aminobutyric acid type A (GABA(A)) receptor-mediated responses in cultured rat hippocampal neurons. Together, our results suggest that the widely used aspirin might impair hippocampal synaptic and neural network functions through its actions on GABAergic neurotransmission. Given the capability of aspirin to penetrate the blood-brain barrier, the present data imply that aspirin intake may cause network hyperactivity and be potentially harmful in susceptible subpopulations.

Differential effects of thiopental and pentobarbital on spinal GABA(A) receptors.

General anesthetics thiopental and pentobarbital possess very similar chemical structures whereas their clinical potency is quite different. The underlying molecular mechanism is not fully understood. This study was designed to assess the differential effects of thiopental and pentobarbital on GABA(A) receptors of mechanically dissociated rat spinal dorsal horn neurons by using whole-cell patch-clamp technique. Pentobarbital, at a concentration of 30 microM, which markedly enhanced sub-saturated GABA-induced current (I(GABA)), had no effect on thiopental-induced maximal current. Similarly, the pentobarbital-induced maximal current was also not affected by 30 microM thiopental. Moreover, a linear summation of thiopental-induced maximal current and pentobarbital-induced sub-maximal current was observed. In addition, pentobarbital failed to further enhance I(GABA) in the presence of thiopental at a concentration with maximal modulatory effects on I(GABA), and vice versa. Our results thus suggest that thiopental and pentobarbital might exert the GABA mimetic effects independently, but share a common mechanism to produce the GABA modulatory effects.

Comparison of esomeprazole enteric-coated capsules vs esomeprazole magnesium in the treatment of active duodenal ulcer: a randomized, double-blind, controlled study.

AIM: To evaluate the efficacy and tolerability of two different preparations of esomeprazole in healing duodenal ulcers. METHODS: A total of 60 patients with active duodenal ulcers were enrolled and randomized to receive esomeprazole enteric-coated capsules (40 mg) or esomeprazole magnesium (40 mg), once daily, for 4 consecutive wk, with ulcer healing being monitored by endoscopy. Safety and tolerability were also assessed. RESULTS: Fifty seven patients completed the whole trial. The ulcer healing rates at the end of wk 2 were 86.7% and 85.2% in the esomeprazole enteric-coated capsules and esomeprazole magnesium groups, respectively (P = 0.8410), and reached 100% at the end of wk 4 in both groups. Symptom relief at the end of wk 2 was 90.8% in the esomeprazole enteric-coated capsules group and 86.7% in the esomeprazole magnesium group (P = 0.5406); at the end of wk 4 symptom relief was 95.2% and 93.2%, respectively (P = 0.5786). Adverse events occurred in 16.7% of the esomeprazole enteric-coated capsules group and 14.8% of the esomeprazole magnesium group (P = 1.0000). CONCLUSION: The efficacies of esomeprazole enteric-coated capsules and esomeprazole magnesium in healing duodenal ulcer lesions and relieving gastrointestinal symptoms are equivalent. The tolerability and safety of both drugs were comparable.

Changes of K+ -Cl- cotransporter 2 (KCC2) and circuit activity in propofol-induced impairment of long-term potentiation in rat hippocampal slices.

Enhancing inhibition via gamma-aminobutyric acid type A (GABA(A)) receptors contributes to anesthetic-induced impairment of long-term potentiation (LTP) of excitatory synaptic transmission, which may account for general anesthesia-associated memory impairment (amnesia). The neuron-specific K+ -Cl- cotransporter 2 (KCC2) is necessary for fast synaptic inhibition via maintaining the low intracellular chloride concentration required for the hyperpolarizing actions of GABA via GABA(A) receptors. To explore a possible role of KCC2-dependent inhibition in anesthetic-induced impairment of LTP, we used field excitatory postsynaptic potentials (fEPSP) recording and immunoblotting to study the effect of propofol on LTP maintenance and KCC2 expression in CA1 region of rat hippocampal slices. We found that propofol (30 microM) not only impaired LTP expression but also prevented LTP-accompanied downregulation of KCC2 without affecting the basal transmission of glutamatergic synapses. Moreover, the recurrent inhibition in hippocampal slices was enhanced by propofol. These propofol-induced effects were completely abolished by picrotoxin, a specific GABA(A) receptor-chloride channel blocker. Thus, enhancement of GABAergic inhibition and suppression of neuronal excitability may account for the sustained expression of KCC2 and the impairment of LTP by propofol. Together, this study supports a novel role for KCC2 in LTP expression and gives hints to a molecular mechanism, by which anesthetics might cause impairment of LTP.

Inhibitors of GlyT1 and GlyT2 differentially modulate inhibitory transmission.

The chronic effects of glycine transporter 1 and 2 inhibitors (sarcosine and ALX-1393, respectively) on miniature inhibitory postsynaptic currents were studied in cultured spinal neurons. We found that sarcosine increased the frequency of overall miniature inhibitory postsynaptic currents without affecting the ratio of glycinergic, mixed and GABAergic miniature inhibitory postsynaptic currents, whereas ALX-1393 changed the ratio by increasing the proportions of GABAergic and mixed miniature inhibitory postsynaptic currents without affecting overall mIPSC frequency. We propose that inhibition of glycine transporter 1 by sarcosine increased overall mIPSC frequency via the activation of presynaptic glycine receptors, while inhibition of glycine transporter 2 by ALX-1393 changed the ratio of glycinergic, mixed and GABAergic miniature inhibitory postsynaptic currents by shifting the balance of inhibitory transmitters in vesicles towards gamma-aminobutyric acid.