The immunomodulatory effect of oral NaHCO3 is mediated by the splenic nerve: multivariate impact revealed by artificial neural networks.

Stimulation of the inflammatory reflex (IR) is a promising strategy for treating systemic inflammatory disorders. Recent studies suggest oral sodium bicarbonate (NaHCO3) as a potential activator of the IR, offering a safe and cost-effective treatment approach. However, the mechanisms underlying NaHCO3-induced anti-inflammatory effects remain unclear. We investigated whether oral NaHCO3's immunomodulatory effects are mediated by the splenic nerve. Female rats received NaHCO3 or water (H2O) for four days, and splenic immune markers were assessed using flow cytometry. NaHCO3 led to a significant increase (p < 0.05, and/or partial eta squared > 0.06) in anti-inflammatory markers, including CD11bc + CD206 + (M2-like) macrophages, CD3 + CD4 + FoxP3 + cells (Tregs), and Tregs/M1-like ratio. Conversely, proinflammatory markers, such as CD11bc + CD38 + TNFα + (M1-like) macrophages, M1-like/M2-like ratio, and SSChigh/SSClow ratio of FSChighCD11bc + cells, decreased in the spleen following NaHCO3 administration. These effects were abolished in spleen-denervated rats, suggesting the necessity of the splenic nerve in mediating NaHCO3-induced immunomodulation. Artificial neural networks accurately classified NaHCO3 and H2O treatment in sham rats but failed in spleen-denervated rats, highlighting the splenic nerve's critical role. Additionally, spleen denervation independently influenced Tregs, M2-like macrophages, Tregs/M1-like ratio, and CD11bc + CD38 + cells, indicating distinct effects from both surgery and treatment. Principal component analysis (PCA) further supported the separate effects. Our findings suggest that the splenic nerve transmits oral NaHCO3-induced immunomodulatory changes to the spleen, emphasizing NaHCO3's potential as an IR activator with therapeutic implications for a wide spectrum of systemic inflammatory conditions.

Phencyclidine Disrupts Neural Coordination and Cognitive Control by Dysregulating Translation.

Background: Phencyclidine (PCP) causes psychosis, is abused with increasing frequency, and was extensively used in antipsychotic drug discovery. PCP discoordinates hippocampal ensemble action potential discharge and impairs cognitive control in rats, but how this uncompetitive NMDA receptor (NMDAR) antagonist impairs cognition remains unknown. Methods: The effects of PCP were investigated on hippocampal CA1 ensemble action potential discharge in vivo in urethane-anesthetized rats and during awake behavior in mice, on synaptic responses in ex vivo mouse hippocampus slices, in mice on a hippocampus-dependent active place avoidance task that requires cognitive control, and on activating the molecular machinery of translation in acute hippocampus slices. Mechanistic causality was assessed by comparing the PCP effects with the effects of inhibitors of protein synthesis, group I metabotropic glutamate receptors (mGluR1/5), and subunit-selective NMDARs. Results: Consistent with ionotropic actions, PCP discoordinated CA1 ensemble action potential discharge. PCP caused hyperactivity and impaired active place avoidance, despite the rodents having learned the task before PCP administration. Consistent with metabotropic actions, PCP exaggerated protein synthesis-dependent DHPG-induced mGluR1/5-stimulated long-term synaptic depression. Pretreatment with anisomycin or the mGluR1/5 antagonist MPEP, both of which repress translation, prevented PCP-induced discoordination and the cognitive and sensorimotor impairments. PCP as well as the NR2A-containing NMDAR antagonist NVP-AAM077 unbalanced translation that engages the Akt, mTOR (mechanistic target of rapamycin), and 4EBP1 translation machinery and increased protein synthesis, whereas the NR2B-containing antagonist Ro25-6981 did not. Conclusions: PCP dysregulates translation, acting through NR2A-containing NMDAR subtypes, recruiting mGluR1/5 signaling pathways, and leading to neural discoordination that is central to the cognitive and sensorimotor impairments.

Spatially Resolved Transcriptomic Signatures of Hippocampal Subregions and Arc-Expressing Ensembles in Active Place Avoidance Memory.

The rodent hippocampus is a spatially organized neuronal network that supports the formation of spatial and episodic memories. We conducted bulk RNA sequencing and spatial transcriptomics experiments to measure gene expression changes in the dorsal hippocampus following the recall of active place avoidance (APA) memory. Through bulk RNA sequencing, we examined the gene expression changes following memory recall across the functionally distinct subregions of the dorsal hippocampus. We found that recall induced differentially expressed genes (DEGs) in the CA1 and CA3 hippocampal subregions were enriched with genes involved in synaptic transmission and synaptic plasticity, while DEGs in the dentate gyrus (DG) were enriched with genes involved in energy balance and ribosomal function. Through spatial transcriptomics, we examined gene expression changes following memory recall across an array of spots encompassing putative memory-associated neuronal ensembles marked by the expression of the IEGs Arc, Egr1, and c-Jun. Within samples from both trained and untrained mice, the subpopulations of spatial transcriptomic spots marked by these IEGs were transcriptomically and spatially distinct from one another. DEGs detected between Arc+ and Arc- spots exclusively in the trained mouse were enriched in several memory-related gene ontology terms, including "regulation of synaptic plasticity" and "memory." Our results suggest that APA memory recall is supported by regionalized transcriptomic profiles separating the CA1 and CA3 from the DG, transcriptionally and spatially distinct IEG expressing spatial transcriptomic spots, and biological processes related to synaptic plasticity as a defining the difference between Arc+ and Arc- spatial transcriptomic spots.

Can a basic solution activate the inflammatory reflex? A review of potential mechanisms, opportunities, and challenges.

Stimulation of the inflammatory reflex (IR) is a promising strategy to treat systemic inflammatory disorders. However, this strategy is hindered by the cost and side effects of traditional IR activators. Recently, oral intake of sodium bicarbonate (NaHCO3) has been suggested to activate the IR, providing a safe and inexpensive alternative. Critically, the mechanisms whereby NaHCO3 might achieve this effect and more broadly the pathways underlying the IR remain poorly understood. Here, we argue that the recognition of NaHCO3 as a potential IR activator presents exciting clinical and research opportunities. To aid this quest, we provide an integrative review of our current knowledge of the neural and cellular pathways mediating the IR and discuss the status of physiological models of IR activation. From this vantage point, we derive testable hypotheses on potential mechanisms whereby NaHCO3 might stimulate the IR and compare NaHCO3 with classic IR activators. Elucidation of these mechanisms will help determine the therapeutic value of NaHCO3 as an IR activator and provide new insights into the IR circuitry.

Pharmacological inhibition of the primary endocannabinoid producing enzyme, DGL-α, induces autism spectrum disorder-like and co-morbid phenotypes in adult C57BL/J mice.

Accumulating evidence links dysfunction in the endocannabinoid system (ECS) with the pathology of neurodevelopmental disorders, particularly autism spectrum disorder (ASD). Variants in ECS genes CNR1 and DAGLA are associated with neurological phenotypes in humans. The endocannabinoids (eCBs), 2-AG and AEA, which act at the primary cannabinoid receptor (CB1), mediate behaviors relevant to neurodevelopmental disorders. The overlap between these eCBs is poorly understood. Most ECS studies have focused on stress responses, anxiety, and epilepsy, however, its role in social behavior and communication has only recently come under investigation. This represents a critical gap in our understanding of the ECS and its relationship to ASD. Furthermore, the increasing prevalence of ASD and a lack of therapeutics emphasize a crucial need for novel therapeutic targets. To this aim, we used an inhibitor of the eCB producing enzyme DGL-α, DO34, and the CB1 inverse agonist, rimonabant, to evaluate the role of the primary eCB, 2-AG, in ASD. Adult male C57BL/6J mice were used in a series of behavioral paradigms which assessed social behavior, social communication, repetitive behaviors, anxiety and locomotor activity. DO34 and rimonabant increased anxiety-like behavior, while only DO34 induced hyperactivity, social deficits, and repetitive self-grooming behavior. These data indicate that reduced 2-AG bioavailability, or CB1 inhibition, each induce unique respective behavioral phenotypes relevant to neurodevelopmental disorders, particularly ASD. This suggests fundamental differences in CB1 signaling via 2-AG and the CB1 receptor itself, particularly for social behaviors, and that 2-AG signaling may represent a target for the development of novel therapeutics. LAY SUMMARY: Endocannabinoids play a critical role in the developing nervous system. Alterations in the endocannabinoid system are linked to neurodevelopmental disorders. Studies suggest these variants may play a critical role in the core symptoms of autism spectrum disorder. In this study, pharmacological inhibition of the primary endocannabinoid producing enzyme, DGL-α, induced a constellation of deficits in behavioral domains associated with autism.

Persistent increases of PKMζ in memory-activated neurons trace LTP maintenance during spatial long-term memory storage.

PKMζ is an autonomously active PKC isoform crucial for the maintenance of synaptic long-term potentiation (LTP) and long-term memory. Unlike other kinases that are transiently stimulated by second messengers, PKMζ is persistently activated through sustained increases in protein expression of the kinase. Therefore, visualizing increases in PKMζ expression during long-term memory storage might reveal the sites of its persistent action and thus the location of memory-associated LTP maintenance in the brain. Using quantitative immunohistochemistry validated by the lack of staining in PKMζ-null mice, we examined the amount and distribution of PKMζ in subregions of the hippocampal formation of wild-type mice during LTP maintenance and spatial long-term memory storage. During LTP maintenance in hippocampal slices, PKMζ increases in the pyramidal cell body and stimulated dendritic layers of CA1 for at least 2 hr. During spatial memory storage, PKMζ increases in CA1 pyramidal cells for at least 1 month, paralleling the persistence of the memory. During the initial expression of the memory, we tagged principal cells with immediate-early gene Arc promoter-driven transcription of fluorescent proteins. The subset of memory-tagged CA1 cells selectively increases expression of PKMζ during memory storage, and the increase persists in dendritic compartments within stratum radiatum for 1 month, indicating long-term storage of information in the CA3-to-CA1 pathway. We conclude that persistent increases in PKMζ trace the molecular mechanism of LTP maintenance and thus the sites of information storage within brain circuitry during long-term memory.

Noncanonical cytoplasmic poly(A) polymerases regulate RNA levels, alternative RNA processing, and synaptic plasticity but not hippocampal-dependent behaviours.

Noncanonical poly(A) polymerases are frequently tethered to mRNA 3' untranslated regions and regulate poly(A) tail length and resulting translation. In the brain, one such poly(A) polymerase is Gld2, which is anchored to mRNA by the RNA-binding protein CPEB1 to control local translation at postsynaptic regions. Depletion of CPEB1 or Gld2 from the mouse hippocampus results in a deficit in long-term potentiation (LTP), but only depletion of CPEB1 alters animal behaviour. To test whether a related enzyme, Gld4, compensates for the lack of Gld2, we separately or simultaneously depleted both proteins from hippocampal area CA1 and again found little change in animal behaviour, but observed a deficit in LTP as well as an increase in long-term depression (LTD), two forms of protein synthesis-dependent synaptic plasticity. RNA-seq data from Gld2, Gld4, and Gld2/Gld4-depleted hippocampus show widespread changes in steady state RNA levels, alternative splicing, and alternative poly(A) site selection. Many of the RNAs subject to these alterations encode proteins that mediate synaptic function, suggesting a molecular foundation for impaired synaptic plasticity.

Hippocampal transcriptomic responses to enzyme-mediated cellular dissociation.

Single-neuron gene expression studies may be especially important for understanding nervous system structure and function because of the neuron-specific functionality and plasticity that defines functional neural circuits. Cellular dissociation is a prerequisite technical manipulation for single-cell and single cell-population studies, but the extent to which the cellular dissociation process affects neural gene expression has not been determined. This information is necessary for interpreting the results of experimental manipulations that affect neural function such as learning and memory. The goal of this research was to determine the impact of cellular dissociation on brain transcriptomes. We compared gene expression of microdissected samples from the dentate gyrus (DG), CA3, and CA1 subfields of the mouse hippocampus either prepared by a standard tissue homogenization protocol or subjected to enzymatic digestion used to dissociate cells within tissues. We report that compared to homogenization, enzymatic dissociation alters about 350 genes or 2% of the hippocampal transcriptome. While only a few genes canonically implicated in long-term potentiation and fear memory change expression levels in response to the dissociation procedure, these data indicate that sample preparation can affect gene expression profiles, which might confound interpretation of results depending on the research question. This study is important for the investigation of any complex tissues as research effort moves from subfield level analysis to single cell analysis of gene expression.

Normal CA1 Place Fields but Discoordinated Network Discharge in a Fmr1-Null Mouse Model of Fragile X Syndrome.

Silence of FMR1 causes loss of fragile X mental retardation protein (FMRP) and dysregulated translation at synapses, resulting in the intellectual disability and autistic symptoms of fragile X syndrome (FXS). Synaptic dysfunction hypotheses for how intellectual disabilities like cognitive inflexibility arise in FXS predict impaired neural coding in the absence of FMRP. We tested the prediction by comparing hippocampus place cells in wild-type and FXS-model mice. Experience-driven CA1 synaptic function and synaptic plasticity changes are excessive in Fmr1-null mice, but CA1 place fields are normal. However, Fmr1-null discharge relationships to local field potential oscillations are abnormally weak, stereotyped, and homogeneous; also, discharge coordination within Fmr1-null place cell networks is weaker and less reliable than wild-type. Rather than disruption of single-cell neural codes, these findings point to invariant tuning of single-cell responses and inadequate discharge coordination within neural ensembles as a pathophysiological basis of cognitive inflexibility in FXS. VIDEO ABSTRACT.

Effects of regulatory BC1 RNA deletion on synaptic plasticity, learning, and memory.

Nonprotein coding dendritic BC1 RNA regulates translation of mRNAs in neurons. We examined two lines of BC1 knockout mice and report that loss of BC1 RNA exaggerates group I mGluR-stimulated LTD of the Schaffer collateral synapse, with one of the lines showing a much more enhanced DHPG-induced LTD than the other. When the animals were given the hippocampus-synaptic plasticity-dependent active place avoidance task, learning and memory were impaired in the BC1-KO line with the more severely altered DHPG-induced LTD. These findings indicate a role for BC1 RNA control of mGluR-dependent synaptic function in hippocampus and associated cognitive ability.

Persistent modifications of hippocampal synaptic function during remote spatial memory.

A widely accepted notion for a process underlying memory formation is that learning changes the efficacy of synapses by the mechanism of synaptic plasticity. While there is compelling evidence of changes in synaptic efficacy observed after learning, demonstration of persistent synaptic changes accompanying memory has been elusive. We report that acquisition of a hippocampus and long-term potentiation dependent place memory persistently changes the function of CA1 synapses. Using extracellular recordings we measured CA3-CA1 and EC-CA1 synaptic responses and found robust changes in the CA3-CA1 pathway after memory training. Crucially, these changes in synaptic function lasted at least a month and coincided with the persistence of long-term place memories; the changes were only observed in animals that expressed robust memory, and not in animals with poor memory recall. Interestingly, our findings were observed at the level of populations of synapses; suggesting that memory formation recruits widespread synaptic circuits and persistently reorganizes their function to store information.

Learning-induced and stathmin-dependent changes in microtubule stability are critical for memory and disrupted in ageing.

Changes in the stability of microtubules regulate many biological processes, but their role in memory remains unclear. Here we show that learning causes biphasic changes in the microtubule-associated network in the hippocampus. In the early phase, stathmin is dephosphorylated, enhancing its microtubule-destabilizing activity by promoting stathmin-tubulin binding, whereas in the late phase these processes are reversed leading to an increase in microtubule/KIF5-mediated localization of the GluA2 subunit of AMPA receptors at synaptic sites. A microtubule stabilizer paclitaxel decreases or increases memory when applied at the early or late phases, respectively. Stathmin mutations disrupt changes in microtubule stability, GluA2 localization, synaptic plasticity and memory. Aged wild-type mice show impairments in stathmin levels, changes in microtubule stability and GluA2 localization. Blocking GluA2 endocytosis rescues memory deficits in stathmin mutant and aged wild-type mice. These findings demonstrate a role for microtubules in memory in young adult and aged individuals.

Genetic and acute CPEB1 depletion ameliorate fragile X pathophysiology.

Fragile X syndrome (FXS), the most common cause of inherited mental retardation and autism, is caused by transcriptional silencing of FMR1, which encodes the translational repressor fragile X mental retardation protein (FMRP). FMRP and cytoplasmic polyadenylation element-binding protein (CPEB), an activator of translation, are present in neuronal dendrites, are predicted to bind many of the same mRNAs and may mediate a translational homeostasis that, when imbalanced, results in FXS. Consistent with this possibility, Fmr1(-/y); Cpeb1(-/-) double-knockout mice displayed amelioration of biochemical, morphological, electrophysiological and behavioral phenotypes associated with FXS. Acute depletion of CPEB1 in the hippocampus of adult Fmr1(-/y) mice rescued working memory deficits, demonstrating reversal of this FXS phenotype. Finally, we find that FMRP and CPEB1 balance translation at the level of polypeptide elongation. Our results suggest that disruption of translational homeostasis is causal for FXS and that the maintenance of this homeostasis by FMRP and CPEB1 is necessary for normal neurologic function.

Interaction between long-term potentiation and depression in CA1 synapses: temporal constrains, functional compartmentalization and protein synthesis.

Information arriving at a neuron via anatomically defined pathways undergoes spatial and temporal encoding. A proposed mechanism by which temporally and spatially segregated information is encoded at the cellular level is based on the interactive properties of synapses located within and across functional dendritic compartments. We examined cooperative and interfering interactions between long-term synaptic potentiation (LTP) and depression (LTD), two forms of synaptic plasticity thought to be key in the encoding of information in the brain. Two approaches were used in CA1 pyramidal neurons of the mouse hippocampus: (1) induction of LTP and LTD in two separate synaptic pathways within the same apical dendritic compartment and across the basal and apical dendritic compartments; (2) induction of LTP and LTD separated by various time intervals (0-90 min). Expression of LTP/LTD interactions was spatially and temporally regulated. While they were largely restricted within the same dendritic compartment (compartmentalized), the nature of the interaction (cooperation or interference) depended on the time interval between inductions. New protein synthesis was found to regulate the expression of the LTP/LTD interference. We speculate that mechanisms for compartmentalization and protein synthesis confer the spatial and temporal modulation by which neurons encode multiplex information in plastic synapses.

A molecular circuit composed of CPEB-1 and c-Jun controls growth hormone-mediated synaptic plasticity in the mouse hippocampus.

Cytoplasmic polyadenylation element binding protein 1 (CPEB-1) resides at postsynaptic sites in hippocampal neurons in which it controls polyadenylation-induced translation. CPEB-1 knock-out (KO) mice display defects in some forms of synaptic plasticity and hippocampal-dependent memories. To identify CPEB-1-regulated mRNAs, we used proteomics to compare polypeptides in wild-type (WT) and CPEB-1 KO hippocampus. Growth hormone (GH) was reduced in the KO hippocampus, as were the GH signaling molecules phospho-JAK2 and phospho-STAT3. GH mRNA and pre-mRNA were reduced in the KO hippocampus, suggesting that CPEB-1 controls GH transcription. The transcription factor c-Jun, which binds the GH promoter, was also reduced in the KO hippocampus, as was its ability to coimmunoprecipitate chromatin containing the GH promoter. CPEB-1 binds c-Jun 3' untranslated region CPEs in vitro and coimmunoprecipitates c-Jun RNA in vivo. GH induces long-term potentiation (LTP) when applied to hippocampal slices from WT and CPEB-1 KO mice, but the magnitude of LTP induced by GH in KO mice is reduced. Pretreatment with GH did not reverse the LTP deficit observed in KO mice after theta-burst stimulation (TBS). Cordycepin, an inhibitor of polyadenylation, disrupted LTP induced by either GH application or TBS. Finally, GH application to hippocampal slices induced JAK2 phosphorylation in WT but not KO animals. These results indicate that CPEB-1 control of c-Jun mRNA translation regulates GH gene expression and resulting downstream signaling events (e.g., synaptic plasticity) in the mouse hippocampus.

Transgenic mice lacking NMDAR-dependent LTD exhibit deficits in behavioral flexibility.

While most studies have focused on the role of long-term potentiation in behavior, far less is known about the role of long-term depression (LTD). To examine the potential involvement of LTD in learning and memory, we generated transgenic mice that express a fragment of the SV40 small t antigen known to inhibit protein phosphatase 2A (PP2A). Small t antigen expression blocked both stimulus-induced and chemically induced NMDAR-dependent LTD at Schaffer collateral synapses but did not affect potentiation, depotentiation, or mGluR-dependent LTD. This physiological phenotype was associated with deficits in behavioral flexibility in both the Morris water maze and a delayed nonmatch to place T-maze task, suggesting that NMDAR-dependent LTD is required for behavioral flexibility and may act by weakening previously encoded memory traces when new information is learned.

A role in learning for SRF: deletion in the adult forebrain disrupts LTD and the formation of an immediate memory of a novel context.

Whereas significant insight exists as to how LTP-related changes can contribute to the formation of long-term memory, little is known about the role of hippocampal LTD-like changes in learning and memory storage. We describe a mouse lacking the transcription factor SRF in the adult forebrain. This mouse could not acquire a hippocampus-based immediate memory for a novel context even across a few minute timespan, which led to a profound but selective deficit in explicit spatial memory. These animals were also impaired in the induction of LTD, including LTD triggered by a cholinergic agonist. Moreover, genes regulating two processes essential for LTD-calcium release from intracellular stores and phosphatase activation-were abnormally expressed in knockouts. These findings suggest that for the hippocampus to form associative spatial memories through LTP-like processes, it must first undergo learning of the context per se through exploration and the learning of familiarity, which requires LTD-like processes.

Ion channel formation by Alzheimer's disease amyloid beta-peptide (Abeta40) in unilamellar liposomes is determined by anionic phospholipids.

Incorporation of Alzheimer's disease amyloid beta-proteins (AbetaPs) across natural and artificial bilayer membranes leads to the formation of cation-selective channels. To study the peptide-membrane interactions involved in channel formation, we used cation reporter dyes to measure AbetaP-induced influx of Na+, Ca2+, and K+ into liposomes formed from phosphatidylserine (PS), phosphatidylinositol (PI) and phosphatidylcholine (PC). We found that Abeta40, but not Abeta40-1 or Abeta28, caused a dose-dependent increase in the concentration of each cation in the lumen of liposomes formed from the acidic phospholipids PS and PI. The Abeta40-induced changes in cation concentration, which we attribute to ion entry through Abeta40 channels, were not observed when using liposomes formed from the neutral phospholipid PC. Using mixtures of phospholipids, the magnitude of the AbetaP40-induced ion entry increased with the acidic phospholipid content of the liposomes, with entry being observed with as little as 5% PS or PI. Thus, while negatively charged phospholipids are required for formation of cation-permeable channels by Abeta40, a small amount is sufficient to support the process. These results have implications for the mechanisms of AbetaP cytotoxicity, suggesting that even a small amount of externalized negative charge could render cells susceptible to the deleterious effects of unregulated ion influx through AbetaP channels.