Tomoyuki Miyazaki

Disrupted cortical function underlies behavior dysfunction due to social isolation

Stressful events during early childhood can have a profound lifelong influence on emotional and cognitive behaviors. However, the mechanisms by which stress affects neonatal brain circuit formation are poorly understood. Here, we show that neonatal social isolation disrupts molecular, cellular, and circuit developmental processes, leading to behavioral dysfunction. Neonatal isolation prevented long-term potentiation and experience-dependent synaptic trafficking of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors normally occurring during circuit formation in the rodent barrel cortex. This inhibition of AMPA receptor trafficking was mediated by an increase of the stress glucocorticoid hormone and was associated with reduced calcium/calmodulin-dependent protein kinase type II (CaMKII) signaling, resulting in attenuated whisker sensitivity at the cortex. These effects led to defects in whisker-dependent behavior in juvenile animals. These results indicate that neonatal social isolation alters neuronal plasticity mechanisms and perturbs the initial establishment of a normal cortical circuit, which potentially explains the long-lasting behavioral effects of neonatal stress.

Isoflurane Impairs Learning and Hippocampal Long-term Potentiation via the Saturation of Synaptic Plasticity

Background:General anesthesia induces long-lasting cognitive and learning deficits. However, the underlying mechanism remains unknown. The GluA1 subunit of AMPAR is a key molecule for learning and synaptic plasticity, which requires trafficking of GluA1-containing AMPARs into the synapse. Methods:Adult male rats were exposed to 1.8% isoflurane for 2 h and subjected to an inhibitory avoidance task, which is a hippocampus-dependent contextual fear learning paradigm (n = 16 to 39). The in vitro extracellular field potential of hippocampal synapses between the Schaffer collateral and the CA1 was evaluated using a multielectrode recorder (n = 6 per group). GluA1 expression in the synaptoneurosome was assessed using Western blotting (n = 5 to 8). The ubiquitination level of GluA1 was evaluated using immunoprecipitation and Western blotting (n = 7 per group). Results:Seven days after exposure to 1.8% isoflurane for 2 h (Iso1.8), the inhibitory avoidance learning (control vs. Iso1.8; 294 ± 34 vs. 138 ± 28, the mean ± SEM [%]; P = 0.002) and long-term potentiation (125.7 ± 6.1 vs. 105.7 ± 3.3; P < 0.001) were impaired. Iso1.8 also temporarily increased GluA1 in the synaptoneurosomes (100 ± 9.7 vs. 138.9 ± 8.9; P = 0.012) and reduced the GluA1 ubiquitination, a main degradation pathway of GluA1 (100 ± 8.7 vs. 71.1 ± 6.1; P = 0.014). Conclusions:Isoflurane impairs hippocampal learning and modulates synaptic plasticity in the postanesthetic period. Increased GluA1 may reduce synaptic capacity for additional GluA1-containing AMPARs trafficking.

Developmental AMPA receptor subunit specificity during experience-driven synaptic plasticity in the rat barrel cortex

During early postnatal brain development, experience-driven delivery of AMPA receptors to synapses participates in the initial organization of cortical function. By combining virus-mediated in vivo gene delivery with in vitro whole cell recordings, we identified a subunit-specific developmental program of experience-driven AMPA receptor delivery to synapses in rat barrel cortex. We expressed green fluorescent protein (GFP)-tagged AMPA receptors (GFP-GluR1, or GFP-GluR4) into layer 2/3 pyramidal neurons at two distinct developmental periods, postnatal day (P)8-P10 and P12-P14. Two days after viral infection, acute brain slices were prepared, and synaptic transmission from layer 4 to layer 2/3 was analyzed by whole cell recordings. We found that whisker experience drives GluR4 but not GluR1 into these synapses early in postnatal development (P8-P10). However, at P12-14, GluR1 but not GluR4 is delivered into synapses by whisker experience. This precise developmental plan suggests unique plasticity properties endowed in different AMPA receptor subunits which shape the initial experience-driven organization of cortical function.

Social isolation perturbs experience-driven synaptic glutamate receptor subunit 4 delivery in the developing rat barrel cortex

In neonates, the stress of social isolation can alter developing neural circuits and cause mental illness. However, the molecular and cellular bases for these effects are poorly understood. Experience‐driven synaptic AMPA receptor delivery is crucial for circuit organisation during development. In the rat, whisker experience drives the delivery of glutamate receptor subunit 4 (GluA4) but not glutamate receptor subunit 1 (GluA1) to layer 4–2/3 pyramidal synapses in the barrel cortex during postnatal day (P)8–10, whereas GluA1 but not GluA4 is delivered to these synapses during P12–14. We recently reported that early social isolation disrupts experience‐driven GluA1 delivery to layer 4–2/3 pyramidal synapses during P12–14. Here, we report that neonatal isolation affects even earlier stages of development by preventing experience‐dependent synaptic GluA4 delivery. Thus, social isolation severely affects synaptic maturation throughout early postnatal development.

Role of nerve growth factor-tyrosine kinase receptor A signaling in paclitaxel-induced peripheral neuropathy in rats

The mechanisms underlying paclitaxel-induced peripheral neuropathy remain unknown. Nerve growth factor (NGF) is a representative neurotrophic factor that maintains neuronal function, promotes survival, and mediates neuropathic pain. We investigated expression levels of NGF and its receptors in the dorsal root ganglia (DRG) and spinal dorsal horn (DH) following paclitaxel treatment. Intraperitoneal (I.P.) administration of paclitaxel induced significant mechanical hypersensitivity and cold allodynia in rats, significantly increased the expression of NGF and its receptor tyrosine kinase receptor A (trkA) in the DRG, and increased NGF expression in the DH. In contrast, paclitaxel treatment did not alter the mRNA levels of NGF or its receptors in the DRG, DH, sciatic nerve, or hindpaw skin. Moreover, expression of NEDD4-2, a negative regulator of trkA, was significantly increased in the DRG of paclitaxel-treated rats. Intrathecal (I.T.) administration of the tyrosine kinase receptor inhibitor k252a significantly alleviated mechanical hypersensitivity in paclitaxel-treated rats. Our results suggest that NGF-trkA signaling is involved in mechanical allodynia in paclitaxel-induced neuropathy.

Postanesthetic effects of isoflurane on behavioral phenotypes of adult male C57BL/6J mice.

Isoflurane was previously the major clinical anesthetic agent but is now mainly used for veterinary anesthesia. Studies have reported widespread sites of action of isoflurane, suggesting a wide array of side effects besides sedation. In the present study, we phenotyped isoflurane-treated mice to investigate the postanesthetic behavioral effects of isoflurane. We applied comprehensive behavioral test batteries comprising sensory test battery, motor test battery, anxiety test battery, depression test battery, sociability test battery, attention test battery, and learning test battery, which were started 7 days after anesthesia with 1.8% isoflurane. In addition to the control group, we included a yoked control group that was exposed to the same stress of handling as the isoflurane-treated animals before being anesthetized. Our comprehensive behavioral test batteries revealed impaired latent inhibition in the isoflurane-treated group, but the concentration of residual isoflurane in the brain was presumably negligible. The yoked control group and isoflurane-treated group exhibited higher anxiety in the elevated plus-maze test and impaired learning function in the cued fear conditioning test. No influences were observed in sensory functions, motor functions, antidepressant behaviors, and social behaviors. A number of papers have reported an effect of isoflurane on animal behaviors, but no systematic investigation has been performed. To the best of our knowledge, this study is the first to systematically investigate the general health, neurological reflexes, sensory functions, motor functions, and higher behavioral functions of mice exposed to isoflurane as adults. Our results suggest that the postanesthetic effect of isoflurane causes attention deficit in mice. Therefore, isoflurane must be used with great care in the clinical setting and veterinary anesthesia.

Intrathecally administered Sema3A protein attenuates neuropathic pain behavior in rats with chronic constriction injury of the sciatic nerve.

Semaphorins, one of the repulsive axonal guidance factors during development, are produced under pathological conditions in adult animals. In the neuropathic pain state associated with peripheral nerve injury, synaptic reorganization occurs in spinal cord dorsal horn. In the present study, we investigated the roles of intrathecal administration of Sema3A, a secreted semaphorin, in the spinal cord of chronic constriction injury (CCI) model rat. Neuropilin 1 (NPR1) and Plexin A (PlexA), co-receptors of Sema3A, were expressed in the dorsal horn of naïve rats. NPR1, and not PlexA, protein expression increased in the dorsal spinal cord of CCI rats. Recombinant Sema3A protein attenuated mechanical allodynia and heat hyperalgesia in CCI rats, whereas heat-inactivated Sema3A had no effect. Immunohistochemistry revealed that Sema3A partially restored the decrease of isolectin B4-positive unmyelinated nerve terminals in lamina II of the ipsilateral dorsal horn of CCI rats. Contrary to our expectations, Sema3A did not change the distribution of myelinated fibers in lamina II at 7 days after CCI. Those results suggested that the suppressive role for Sema3A in the development of neuropathic pain associated with peripheral nerve injury in adult rats, which seemed to be independent from prevention of the myelinated fiber sprouting into lamina II.

Comprehensive behavioral study and proteomic analyses of CRMP2‐deficient mice

Collapsin response mediator protein 2 (CRMP2) plays a key role in axon guidance, dendritic morphogenesis and cell polarization. CRMP2 is implicated in various neurological and psychiatric disorders. However, in vivo functions of CRMP2 remain unknown. We generated CRMP2 gene‐deficient (crmp2−/−) mice and examined their behavioral phenotypes. During 24‐h home cage monitoring, the activity level during the dark phase of crmp2−/− mice was significantly higher than that of wild‐type (WT) mice. Moreover, the time during the open arm of an elevated plus maze was longer for crmp2−/− mice than for WT mice. The duration of social interaction was shorter for crmp2−/− mice than for WT mice. Crmp2−/− mice also showed mild impaired contextual learning. We then examined the methamphetamine‐induced behavioral change of crmp2−/− mice. Crmp2−/− mice showed increased methamphetamine‐induced ambulatory activity and serotonin release. Crmp2−/− mice also showed altered expression of proteins involved in GABAergic synapse, glutamatergic synapse and neurotrophin signaling pathways. In addition, SNAP25, RAB18, FABP5, ARF5 and LDHA, which are related genes to schizophrenia and methamphetamine sensitization, are also decreased in crmp2−/− mice. Our study implies that dysregulation of CRMP2 may be involved in pathophysiology of neuropsychiatric disorders.

Neonatal isolation augments social dominance by altering actin dynamics in the medial prefrontal cortex

Significance Social separation early in life can lead to the development of impaired interpersonal relationships and profound social disorders. However, the underlying cellular and molecular mechanisms involved are largely unknown. In a rat model of neonatal isolation, we examined social dominance in juveniles. We further investigated the relationship between actin dynamics and glutamate synaptic AMPA receptor delivery in spines of the medial prefrontal cortex (mPFC) of isolated animals. Here, we report that neonatal isolation alters spines in the mPFC by reducing actin dynamics, leading to the decrease of synaptic AMPA receptor delivery and altered social behavior later in life. Our study provides molecular and cellular mechanisms underlying the influence of social separation early in life on later social behaviors. Social separation early in life can lead to the development of impaired interpersonal relationships and profound social disorders. However, the underlying cellular and molecular mechanisms involved are largely unknown. Here, we found that isolation of neonatal rats induced glucocorticoid-dependent social dominance over nonisolated control rats in juveniles from the same litter. Furthermore, neonatal isolation inactivated the actin-depolymerizing factor (ADF)/cofilin in the juvenile medial prefrontal cortex (mPFC). Isolation-induced inactivation of ADF/cofilin increased stable actin fractions at dendritic spines in the juvenile mPFC, decreasing glutamate synaptic AMPA receptors. Expression of constitutively active ADF/cofilin in the mPFC rescued the effect of isolation on social dominance. Thus, neonatal isolation affects spines in the mPFC by reducing actin dynamics, leading to altered social behavior later in life.

Bumetanide, an Inhibitor of Cation-chloride Cotransporter Isoform 1, Inhibits γ-Aminobutyric Acidergic Excitatory Actions and Enhances Sedative Actions of Midazolam in Neonatal Rats

Background: It has been shown that &ggr;-aminobutyric acid exerts excitatory actions on the immature brain due to the increased expression of Na+–K+–2Cl− cotransporter isoform 1. The authors sought to clarify whether midazolam, a &ggr;-aminobutyric acid–mimetic hypnotic agent, causes neuronal excitation that can be blocked by bumetanide, a selective inhibitor of Na+–K+–2Cl− cotransporter isoform 1. Furthermore, the authors examined whether bumetanide potentiates the sedative effects of midazolam in neonatal rats. Methods: The authors measured the effects of midazolam with or without bumetanide on the cytosolic Ca2+ concentration ([Ca]2+i) in hippocampal slices (n = 3 in each condition) from rats at postnatal days 4, 7, and 28 (P4, P7, and P28) using fura-2 microfluorometry. Neuronal activity in the hippocampus and thalamus after intraperitoneal administration of midazolam with or without bumetanide was estimated by immunostaining of phosphorylated cyclic adenosine monophosphate–response element–binding protein (n = 12 in each condition). Furthermore, the authors assessed effects of bumetanide on the sedative effect of midazolam by measuring righting reflex latency (n = 6 in each condition). Results: Midazolam significantly increased [Ca]2+i in the CA3 area at P4 and P7 but not at P28. Bumetanide inhibited midazolam-induced increase in [Ca]2+i. Midazolam significantly up-regulated phosphorylated cyclic adenosine monophosphate–response element–binding protein expression in a bumetanide-sensitive manner in the hippocampus at P7 but not P28. Bumetanide enhanced the sedative effects of midazolam in P4 and P7 but not P28 rats. Conclusion: These results suggest that &ggr;-aminobutyric acid A receptor–mediated excitation plays an important role in attenuated sedative effects of midazolam in immature rats.