Impaired synaptic plasticity in an animal model of autism exhibiting early hippocampal GABAergic-BDNF/TrkB signaling alterations.

In Neurodevelopmental Disorders, alterations of synaptic plasticity may trigger structural changes in neuronal circuits involved in cognitive functions. This hypothesis was tested in mice carrying the human R451C mutation of Nlgn3 gene (NLG3R451C KI), found in some families with autistic children. To this aim, the spike time dependent plasticity (STDP) protocol was applied to immature GABAergic Mossy Fibers (MF)-CA3 connections in hippocampal slices from NLG3R451C KI mice. These animals failed to exhibit STD-LTP, an effect that persisted in adulthood when these synapses became glutamatergic. Similar results were obtained in mice lacking the Nlgn3 gene (NLG3 KO mice), suggesting a loss of function. The loss of STD-LTP was associated with a premature shift of GABA from the depolarizing to the hyperpolarizing direction, a reduced BDNF availability and TrkB phosphorylation at potentiated synapses. These effects may constitute a general mechanism underlying cognitive deficits in those forms of Autism caused by synaptic dysfunctions.

The Effect of Limosilactobacillus reuteri on Social Behavior Is Independent of the Adaptive Immune System.

Gut microbes can modulate almost all aspects of host physiology throughout life. As a result, specific microbial interventions are attracting considerable attention as potential therapeutic strategies for treating a variety of conditions. Nonetheless, little is known about the mechanisms through which many of these microbes work. Recently, we and others have found that the commensal bacterium Limosilactobacillus reuteri (formerly Lactobacillus reuteri) reverses social deficits in several mouse models (genetic, environmental, and idiopathic) for neurodevelopmental disorders in a vagus nerve-, oxytocin-, and biopterin-dependent manner. Given that gut microbes can signal to the brain through the immune system and L. reuteri promotes wound healing via the adaptive immune response, we sought to determine whether the prosocial effect mediated by L. reuteri also depends on adaptive immunity. Here, we found that the effects of L. reuteri on social behavior and related changes in synaptic function are independent of the mature adaptive immune system. Interestingly, these findings indicate that the same microbe (L. reuteri) can affect different host phenotypes through distinct mechanisms. IMPORTANCE Because preclinical animal studies support the idea that gut microbes could represent novel therapeutics for brain disorders, it is essential to fully understand the mechanisms by which gut microbes affect their host's physiology. Previously, we discovered that treatment with Limosilactobacillus reuteri selectively improves social behavior in different mouse models for autism spectrum disorder through the vagus nerve, oxytocin reward signaling in the brain, and biopterin metabolites (BH4) in the gut. However, given that (i) the immune system remains a key pathway for host-microbe interactions and that (ii) L. reuteri has been shown to facilitate wound healing through the adaptive immune system, we examined here whether the prosocial effects of L. reuteri require immune signaling. Unexpectedly, we found that the mature adaptive immune system (i.e., conventional B and T cells) is not required for L. reuteri to reverse social deficits and related changes in synaptic function. Overall, these findings add new insight into the mechanism through which L. reuteri modulates brain function and behavior. More importantly, they highlight that a given bacterial species can modulate different phenotypes (e.g., wound healing versus social behavior) through separate mechanisms.

Dissecting the contribution of host genetics and the microbiome in complex behaviors.

The core symptoms of many neurological disorders have traditionally been thought to be caused by genetic variants affecting brain development and function. However, the gut microbiome, another important source of variation, can also influence specific behaviors. Thus, it is critical to unravel the contributions of host genetic variation, the microbiome, and their interactions to complex behaviors. Unexpectedly, we discovered that different maladaptive behaviors are interdependently regulated by the microbiome and host genes in the Cntnap2-/- model for neurodevelopmental disorders. The hyperactivity phenotype of Cntnap2-/- mice is caused by host genetics, whereas the social-behavior phenotype is mediated by the gut microbiome. Interestingly, specific microbial intervention selectively rescued the social deficits in Cntnap2-/- mice through upregulation of metabolites in the tetrahydrobiopterin synthesis pathway. Our findings that behavioral abnormalities could have distinct origins (host genetic versus microbial) may change the way we think about neurological disorders and how to treat them.

Therapeutic inhibition of mTORC2 rescues the behavioral and neurophysiological abnormalities associated with Pten-deficiency.

Dysregulation of the mammalian target of rapamycin (mTOR) signaling, which is mediated by two structurally and functionally distinct complexes, mTORC1 and mTORC2, has been implicated in several neurological disorders1-3. Individuals carrying loss-of-function mutations in the phosphatase and tensin homolog (PTEN) gene, a negative regulator of mTOR signaling, are prone to developing macrocephaly, autism spectrum disorder (ASD), seizures and intellectual disability2,4,5. It is generally believed that the neurological symptoms associated with loss of PTEN and other mTORopathies (for example, mutations in the tuberous sclerosis genes TSC1 or TSC2) are due to hyperactivation of mTORC1-mediated protein synthesis1,2,4,6,7. Using molecular genetics, we unexpectedly found that genetic deletion of mTORC2 (but not mTORC1) activity prolonged lifespan, suppressed seizures, rescued ASD-like behaviors and long-term memory, and normalized metabolic changes in the brain of mice lacking Pten. In a more therapeutically oriented approach, we found that administration of an antisense oligonucleotide (ASO) targeting mTORC2's defining component Rictor specifically inhibits mTORC2 activity and reverses the behavioral and neurophysiological abnormalities in adolescent Pten-deficient mice. Collectively, our findings indicate that mTORC2 is the major driver underlying the neuropathophysiology associated with Pten-deficiency, and its therapeutic reduction could represent a promising and broadly effective translational therapy for neurological disorders where mTOR signaling is dysregulated.

Mechanisms Underlying Microbial-Mediated Changes in Social Behavior in Mouse Models of Autism Spectrum Disorder.

Currently, there are no medications that effectively treat the core symptoms of Autism Spectrum Disorder (ASD). We recently found that the bacterial species Lactobacillus (L.) reuteri reverses social deficits in maternal high-fat-diet offspring. However, whether the effect of L. reuteri on social behavior is generalizable to other ASD models and its mechanism(s) of action remains unknown. Here, we found that treatment with L. reuteri selectively rescues social deficits in genetic, environmental, and idiopathic ASD models. Interestingly, the effects of L. reuteri on social behavior are not mediated by restoring the composition of the host's gut microbiome, which is altered in all of these ASD models. Instead, L. reuteri acts in a vagus nerve-dependent manner and rescues social interaction-induced synaptic plasticity in the ventral tegmental area of ASD mice, but not in oxytocin receptor-deficient mice. Collectively, treatment with L. reuteri emerges as promising non-invasive microbial-based avenue to combat ASD-related social dysfunction.

Single Neuron Optimization as a Basis for Accurate Biophysical Modeling: The Case of Cerebellar Granule Cells.

In realistic neuronal modeling, once the ionic channel complement has been defined, the maximum ionic conductance (Gi-max) values need to be tuned in order to match the firing pattern revealed by electrophysiological recordings. Recently, selection/mutation genetic algorithms have been proposed to efficiently and automatically tune these parameters. Nonetheless, since similar firing patterns can be achieved through different combinations of Gi-max values, it is not clear how well these algorithms approximate the corresponding properties of real cells. Here we have evaluated the issue by exploiting a unique opportunity offered by the cerebellar granule cell (GrC), which is electrotonically compact and has therefore allowed the direct experimental measurement of ionic currents. Previous models were constructed using empirical tuning of Gi-max values to match the original data set. Here, by using repetitive discharge patterns as a template, the optimization procedure yielded models that closely approximated the experimental Gi-max values. These models, in addition to repetitive firing, captured additional features, including inward rectification, near-threshold oscillations, and resonance, which were not used as features. Thus, parameter optimization using genetic algorithms provided an efficient modeling strategy for reconstructing the biophysical properties of neurons and for the subsequent reconstruction of large-scale neuronal network models.

Hebbian Spike-Timing Dependent Plasticity at the Cerebellar Input Stage.

Spike-timing-dependent plasticity (STDP) is a form of long-term synaptic plasticity exploiting the time relationship between postsynaptic action potentials (APs) and EPSPs. Surprisingly enough, very little was known about STDP in the cerebellum, although it is thought to play a critical role for learning appropriate timing of actions. We speculated that low-frequency oscillations observed in the granular layer may provide a reference for repetitive EPSP/AP phase coupling. Here we show that EPSP-spike pairing at 6 Hz can optimally induce STDP at the mossy fiber-granule cell synapse in rats. Spike timing-dependent long-term potentiation and depression (st-LTP and st-LTD) were confined to a ±25 ms time-window. Because EPSPs led APs in st-LTP while APs led EPSPs in st-LTD, STDP was Hebbian in nature. STDP occurred at 6-10 Hz but vanished >50 Hz or <1 Hz (where only LTP or LTD occurred). STDP disappeared with randomized EPSP/AP pairing or high intracellular Ca2+ buffering, and its sign was inverted by GABA-A receptor activation. Both st-LTP and st-LTD required NMDA receptors, but st-LTP also required reinforcing signals mediated by mGluRs and intracellular calcium stores. Importantly, st-LTP and st-LTD were significantly larger than LTP and LTD obtained by modulating the frequency and duration of mossy fiber bursts, probably because STDP expression involved postsynaptic in addition to presynaptic mechanisms. These results thus show that a Hebbian form of STDP occurs at the cerebellum input stage, providing the substrate for phase-dependent binding of mossy fiber spikes to repetitive theta-frequency cycles of granule cell activity.SIGNIFICANCE STATEMENT Long-term synaptic plasticity is a fundamental property of the brain, causing persistent modifications of neuronal communication thought to provide the cellular basis of learning and memory. The cerebellum is critical for learning the appropriate timing of sensorimotor behaviors, but whether and how appropriate spike patterns could drive long-term synaptic plasticity remained unknown. Here, we show that this can actually occur through a form of spike-timing-dependent plasticity (STDP) at the cerebellar inputs stage. Pairing presynaptic and postsynaptic spikes at 6-10 Hz reliably induced STDP at the mossy fiber-granule cell synapse, with potentiation and depression symmetrically distributed within a ±25 ms time window. Thus, STDP can bind plasticity to the mossy fiber burst phase with high temporal precision.

eIF2α-mediated translational control regulates the persistence of cocaine-induced LTP in midbrain dopamine neurons.

Recreational drug use leads to compulsive substance abuse in some individuals. Studies on animal models of drug addiction indicate that persistent long-term potentiation (LTP) of excitatory synaptic transmission onto ventral tegmental area (VTA) dopamine (DA) neurons is a critical component of sustained drug seeking. However, little is known about the mechanism regulating such long-lasting changes in synaptic strength. Previously, we identified that translational control by eIF2α phosphorylation (p-eIF2α) regulates cocaine-induced LTP in the VTA (Huang et al., 2016). Here we report that in mice with reduced p-eIF2α-mediated translation, cocaine induces persistent LTP in VTA DA neurons. Moreover, selectively inhibiting eIF2α-mediated translational control with a small molecule ISRIB, or knocking down oligophrenin-1-an mRNA whose translation is controlled by p-eIF2α-in the VTA also prolongs cocaine-induced LTP. This persistent LTP is mediated by the insertion of GluR2-lacking AMPARs. Collectively, our findings suggest that eIF2α-mediated translational control regulates the progression from transient to persistent cocaine-induced LTP.

Presynaptic BK channels control transmitter release: physiological relevance and potential therapeutic implications.

BK channels are large conductance potassium channels characterized by four pore-forming α subunits, often co-assembled with auxiliary ß and γ subunits to regulate Ca(2+) sensitivity, voltage dependence and gating properties. Abundantly expressed in the CNS, they have the peculiar characteristic of being activated by both voltage and intracellular calcium rise. The increase in intracellular calcium via voltage-dependent calcium channels (Cav ) during spiking triggers conformational changes and BK channel opening. This narrows the action potential and induces a fast after-hyperpolarization that shuts calcium channels. The tight coupling between BK and Cav channels at presynaptic active zones makes them particularly suitable for regulating calcium entry and neurotransmitter release. While in most synapses, BK channels exert a negative control on transmitter release under basal conditions, in others they do so only under pathological conditions, serving as an emergency brake to protect against hyperactivity. In particular cases, by interacting with other channels (i.e. limiting the activation of the delayed rectifier and the inactivation of Na(+) channels), BK channels induce spike shortening, increase in firing rate and transmitter release. Changes in transmitter release following BK channel dysfunction have been implicated in several neurological disorders including epilepsy, schizophrenia, fragile X syndrome, mental retardation and autism. In particular, two mutations, one in the α and one in the ß3 subunit, resulting in a gain of function have been associated with epilepsy. Hence, these discoveries have allowed identification of BK channels as new drug targets for therapeutic intervention.

Anatomical investigation of potential contacts between climbing fibers and cerebellar Golgi cells in the mouse.

Climbing fibers (CFs) originating in the inferior olive (IO) constitute one of the main inputs to the cerebellum. In the mammalian cerebellar cortex each of them climbs into the dendritic tree of up to 10 Purkinje cells (PCs) where they make hundreds of synaptic contacts and elicit the so-called all-or-none complex spikes controlling the output. While it has been proven that CFs contact molecular layer interneurons (MLIs) via spillover mechanisms, it remains to be elucidated to what extent CFs contact the main type of interneuron in the granular layer, i.e., the Golgi cells (GoCs). This issue is particularly relevant, because direct contacts would imply that CFs can also control computations at the input stage of the cerebellar cortical network. Here, we performed a systematic morphological investigation of labeled CFs and GoCs at the light microscopic level following their path and localization through the neuropil in both the granular and molecular layer. Whereas in the molecular layer the appositions of CFs to PCs and MLIs were prominent and numerous, those to cell-bodies and dendrites of GoCs in both the granular layer and molecular layer were virtually absent. Our results argue against the functional significance of direct synaptic contacts between CFs and interneurons at the input stage, but support those at the output stage.