Brain-derived neurotrophic factor produced long-term synaptic enhancement in the anterior cingulate cortex of adult mice.

Previous studies have demonstrated that brain-derived neurotrophic factor (BDNF) is one of the diffusible messengers for enhancing synaptic transmission in the hippocampus. Less information is available about the possible roles of BDNF in the anterior cingulate cortex (ACC). In the present study, we used 64-electrode array field recording system to investigate the effect of BDNF on ACC excitatory transmission. We found that BDNF enhanced synaptic responses in a dose-dependent manner in the ACC in C57/BL6 mice. The enhancement was long-lasting, and persisted for at least 3 h. In addition to the enhancement, BDNF also recruited inactive synaptic responses in the ACC. Bath application of the tropomyosin receptor kinase B (TrkB) receptor antagonist K252a blocked BDNF-induced enhancement. L-type voltage-gated calcium channels (L-VGCC), metabotropic glutamate receptors (mGluRs), but not NMDA receptors were required for BDNF-produced enhancement. Moreover, calcium-stimulated adenylyl cyclase subtype 1 (AC1) but not AC8 was essential for the enhancement. A selective AC1 inhibitor NB001 completely blocked the enhancement. Furthermore, BDNF-produced enhancement occluded theta burst stimulation (TBS) induced long-term potentiation (LTP), suggesting that they may share similar signaling mechanisms. Finally, the expression of BDNF-induced enhancement depends on postsynaptic incorporation of calcium-permeable AMPA receptors (CP-AMPARs) and protein kinase Mζ (PKMζ). Our results demonstrate that cortical BDNF may contribute to synaptic potentiation in the ACC.

Contribution of AMPA Receptor-Mediated LTD in LA/BLA-CeA Pathway to Comorbid Aversive and Depressive Symptoms in Neuropathic Pain.

Comorbid anxiety and depressive symptoms in chronic pain are a common health problem, but the underlying mechanisms remain unclear. Previously, we have demonstrated that sensitization of the CeA neurons via decreased GABAergic inhibition contributes to anxiety-like behaviors in neuropathic pain rats. In this study, by using male Sprague Dawley rats, we reported that the CeA plays a key role in processing both sensory and negative emotional-affective components of neuropathic pain. Bilateral electrolytic lesions of CeA, but not lateral/basolateral nucleus of the amygdala (LA/BLA), abrogated both pain hypersensitivity and aversive and depressive symptoms of neuropathic rats induced by spinal nerve ligation (SNL). Moreover, SNL rats showed structural and functional neuroplasticity manifested as reduced dendritic spines on the CeA neurons and enhanced LTD at the LA/BLA-CeA synapse. Disruption of GluA2-containing AMPAR trafficking and endocytosis from synapses using synthetic peptides, either pep2-EVKI or Tat-GluA2(3Y), restored the enhanced LTD at the LA/BLA-CeA synapse, and alleviated the mechanical allodynia and comorbid aversive and depressive symptoms in neuropathic rats, indicating that the endocytosis of GluA2-containing AMPARs from synapses is probably involved in the LTD at the LA/BLA-CeA synapse and the comorbid aversive and depressive symptoms in neuropathic pain in SNL-operated rats. These data provide a novel mechanism for elucidating comorbid aversive and depressive symptoms in neuropathic pain and highlight that structural and functional neuroplasticity in the amygdala may be important as a promising therapeutic target for comorbid negative emotional-affective disorders in chronic pain.SIGNIFICANCE STATEMENT Several studies have demonstrated the high comorbidity of negative affective disorders in patients with chronic pain. Understanding the affective aspects related to chronic pain may facilitate the development of novel therapies for more effective management. Here, we unravel that the CeA plays a key role in processing both sensory and negative emotional-affective components of neuropathic pain, and LTD at the amygdaloid LA/BLA-CeA synapse mediated by GluA2-containing AMPAR endocytosis underlies the comorbid aversive and depressive symptoms in neuropathic pain. This study provides a novel mechanism for elucidating comorbid aversive and depressive symptoms in neuropathic pain and highlights that structural and functional neuroplasticity in the amygdala may be important as a promising therapeutic target for comorbid negative emotional-affective disorders in chronic pain.

NMDAR-dependent long-term depression is associated with increased short term plasticity through autophagy mediated loss of PSD-95.

Long-term depression (LTD) of synaptic strength can take multiple forms and contribute to circuit remodeling, memory encoding or erasure. The generic term LTD encompasses various induction pathways, including activation of NMDA, mGlu or P2X receptors. However, the associated specific molecular mechanisms and effects on synaptic physiology are still unclear. We here compare how NMDAR- or P2XR-dependent LTD affect synaptic nanoscale organization and function in rodents. While both LTDs are associated with a loss and reorganization of synaptic AMPARs, only NMDAR-dependent LTD induction triggers a profound reorganization of PSD-95. This modification, which requires the autophagy machinery to remove the T19-phosphorylated form of PSD-95 from synapses, leads to an increase in AMPAR surface mobility. We demonstrate that these post-synaptic changes that occur specifically during NMDAR-dependent LTD result in an increased short-term plasticity improving neuronal responsiveness of depressed synapses. Our results establish that P2XR- and NMDAR-mediated LTD are associated to functionally distinct forms of LTD.

Cadmium activates AMPA and NMDA receptors with M3 helix cysteine substitutions.

AMPA and NMDA receptors are ligand-gated ion channels that depolarize postsynaptic neurons when activated by the neurotransmitter L-glutamate. Changes in the distribution and activity of these receptors underlie learning and memory, but excessive change is associated with an array of neurological disorders, including cognitive impairment, developmental delay, and epilepsy. All of the ionotropic glutamate receptors (iGluRs) exhibit similar tetrameric architecture, transmembrane topology, and basic framework for activation; conformational changes induced by extracellular agonist binding deform and splay open the inner helix bundle crossing that occludes ion flux through the channel. NMDA receptors require agonist binding to all four subunits, whereas AMPA and closely related kainate receptors can open with less than complete occupancy. In addition to conventional activation by agonist binding, we recently identified two locations along the inner helix of the GluK2 kainate receptor subunit where cysteine (Cys) substitution yields channels that are opened by exposure to cadmium ions, independent of agonist site occupancy. Here, we generate AMPA and NMDA receptor subunits with homologous Cys substitutions and demonstrate similar activation of the mutant receptors by Cd. Coexpression of the auxiliary subunit stargazin enhanced Cd potency for activation of Cys-substituted GluA1 and altered occlusion upon treatment with sulfhydryl-reactive MTS reagents. Mutant NMDA receptors displayed voltage-dependent Mg block of currents activated by agonist and/or Cd as well as asymmetry between Cd effects on Cys-substituted GluN1 versus GluN2 subunits. In addition, Cd activation of each Cys-substituted iGluR was inhibited by protons. These results, together with our earlier work on GluK2, reveal a novel mechanism shared among the three different iGluR subtypes for prying open the gate that controls ion entry into the pore.

Seizure-induced neuronal apoptosis is related to dysregulation of the RNA-edited GluR2 subunit in the developing mouse brain.

Ca2+-permeable AMPA receptors (AMPAR) which crucially modify maturational programs of the developing brain are involved in seizure-induced glutamate excitotoxicity and apoptosis. Regulatory effects on AMPAR subunit composition and RNA-editing in the developing brain and their significance as therapeutic targets are not well understood. Here, we analyzed acute effects of recurrent pilocarpine-induced neonatal seizures on age- and region-specific expression of AMPAR subunits and adenosine deaminases (ADAR) in the developing mouse brain (P10). After recurrent seizure activity and regeneration periods of 6-72 h cerebral mRNA levels of GluR (glutamate receptor subunit) 1, GluR2, GluR3, and GluR4 were unaffected compared to controls. However, ratio of GluR2 and GluR4 to pooled GluR1-4 mRNA concentration significantly decreased in seizure-exposed brains in comparison to controls. After a regeneration period of 24-72 h ADAR1 and ADAR2 mRNA expression was significantly lower in seizure-exposed brains than in those of controls. This was confirmed at the protein level in the hippocampal CA3 region. We observed a regionally increased apoptosis (TUNEL+ and CC3+ cells) in the hippocampus, parietal cortex and subventricular zone of seizure-exposed brains in comparison to controls. Together, present in vivo data demonstrate the maturational age-specific, functional role of RNA-edited GluR2 in seizure-induced excitotoxicity in the developing mouse brain. In response to recurrent seizure activity, we observed reduced expression of GluR2 and the GluR2 mRNA-editing enzymes ADAR1 and ADAR2 accompanied by increased apoptosis in a region-specific manner. Thus, AMPA receptor subtype-specific mRNA editing is assessed as a promising target of novel neuroprotective treatment strategies in consideration of age-related developmental mechanisms.

Inhibition of Histone Methyltransferases EHMT1/2 Reverses Amyloid-ß-Induced Loss of AMPAR Currents in Human Stem Cell-Derived Cortical Neurons.

Emerging evidence suggests that epigenetic dysregulation of gene expression is one of the key molecular mechanisms of neurodegeneration and Alzheimer's disease (AD). However, little is known about the role of epigenetic dysregulation on synaptic dysfunction in humans, because of the difficulties of obtaining live human neurons. Here we generated mature human cortical neurons differentiated from human embryonic stem cells, and exposed them to amyloid-ß (Aß). We found that the histone methyltransferase, EHMT1, which catalyzes histone lysine 9 dimethylation (H3K9me2, a mark for gene repression), was significantly elevated in Aß-treated human stem cell-derived neurons. Aß treatment led to a significant reduction of AMPAR-mediated whole-cell current and excitatory postsynaptic current. Application of BIX01294, a selective inhibitor of EHMT1/2, restored AMPAR currents and glutamatergic synaptic transmission in Aß-treated human cortical neurons. These results suggest that inhibition of the aberrant histone methylation is a novel approach to reverse Aß-induced synaptic deficits in human neurons.

Glutamatergic Signals in the Dorsal Raphe Nucleus Regulate Maternal Aggression and Care in an Opposing Manner in Mice.

Lactating female mice nurture their pups and attack intruders in their territory. When an intruder invades a dam's territory, she needs to switch her behavior from care to aggression to protect her pups and territory. Although the neuronal mechanisms underlying each distinct behavior have been studied, it is unclear how these behaviors are displayed alternatively. The dorsal raphe nucleus (DRN) regulates both nurturing and aggressive behaviors. In the present study, we examined whether the DRN is involved in regulating alternative display of maternal care and aggression. We first examined neuronal activity in the medial prefrontal cortex (mPFC) and lateral habenula (LHb), which send glutamatergic input to the DRN, in dams by injecting Fluorogold, a retrograde tracer, into the DRN. The number of c-Fos- and Fluorogold-positive neurons in the mPFC and LHb increased in the dams that displayed biting behavior in response to an intruder, but remained unchanged in the dams that displayed nurturing behavior. Injections of N-methyl-d-aspartic acid (NMDA) receptor antagonists or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptor antagonists into the DRN inhibited biting behavior but not nurturing behavior. In contrast, injections of NMDA or AMPA into the DRN inhibited nurturing behavior. These results suggest that glutamatergic signals in the DRN, which may originate from the mPFC and/or LHb, regulate the preferential display of biting behavior over nurturing behavior in dams.

GluR1 protects hypoxic ischemic brain damage via activating Akt signaling pathway in neonatal rats.

OBJECTIVE: To investigate the role of glutamic acid receptor 1 (GluR1) in hypoxic-ischemic brain damage (HIBD) in neonatal rats and its underlying mechanism. MATERIALS AND METHODS: 7-day-old neonatal rats received right common carotid artery (CCA) ligation for the establishment of HIBD. After the operation, rats were sacrificed at different time points (0, 4, 6, 12, 24, 48, and 72 h), respectively. Meanwhile, rats in Sham group underwent similar procedures without ligation. Lentivirus-GLUR1-shRNA (LV-GLUR1 shRNA group) was constructed and then transfected into the right lateral ventricles of rats to inhibit GluR1 in vivo. Rats received LV-control injection were selected in the control group (LV-control group). After injection of Lentivirus-GLUR1-shRNA, CCA ligation was performed in rats for HIBD construction. Western blot was performed to detect the protein levels of GLUR1, Akt, p-Akt, and vascular endothelial growth factor (VEGF) in brain tissues. Cell apoptosis was measured by TUNEL staining assay. RESULTS: After hypoxic ischemia (HI), GLUR1 expression increased gradually and reached a peak at 24 h. Meanwhile, p-Akt expression increased immediately and then gradually decreased. 24 h later, p-Akt expression increased again and peaked at 48 h. VEGF expression increased at 4 h after HI and reached a peak at 12 h. The expression levels of GLUR1, p-Akt, and VEGF in the brain tissues derived from rats transfected with LV-GLUR1 shRNA significantly decreased at both 4 h and 24 h after HI. In addition, results indicated that cell apoptosis was enhanced after LV-GLUR1 shRNA administration, suggesting the role of GLUR1 in protecting against HIBD. CONCLUSIONS: GLUR1 exhibits a remarkable protective role in HIBD, which may be related to the activation of the Akt signaling pathway and the upregulation of VEGF after HI.

Anti-AMPA receptor encephalitis associated with Medullary thyroid cancer.

AMPA receptor (AMPAR) antibodies are a group of recently discovered antibodies which target the neuronal synaptic proteins causing B-cell (immune) mediated neuronal damage, resulting in various neurologic syndromes depending on the area of central nervous system involvement. These syndromes are mostly reversible if treated early. Tumour association has been reported in up to 60% of cases in the most recent case series with lung, breast, ovarian cancers and thymomas being the most commonly associated malignancies with these antibodies. We present here the first case of AMPAR encephalitis associated with medullary thyroid cancer. Our patient presented with cognitive dysfunction and behavioural changes over a period of 3 weeks, with a full recovery after starting immunotherapy, once the diagnosis of AMPAR limbic encephalitis was established. This case highlights the importance of early diagnosis and management of AMPAR encephalitis as these patients respond well to immunotherapy and can have an almost complete recovery.

Enhanced AMPA Receptor Trafficking Mediates the Anorexigenic Effect of Endogenous Glucagon-like Peptide-1 in the Paraventricular Hypothalamus.

Glucagon-like Peptide 1 (GLP-1)-expressing neurons in the hindbrain send robust projections to the paraventricular nucleus of the hypothalamus (PVN), which is involved in the regulation of food intake. Here, we describe that stimulation of GLP-1 afferent fibers within the PVN is sufficient to suppress food intake independent of glutamate release. We also show that GLP-1 receptor (GLP-1R) activation augments excitatory synaptic strength in PVN corticotropin-releasing hormone (CRH) neurons, with GLP-1R activation promoting a protein kinase A (PKA)-dependent signaling cascade leading to phosphorylation of serine S845 on GluA1 AMPA receptors and their trafficking to the plasma membrane. Finally, we show that postnatal depletion of GLP-1R in the PVN increases food intake and causes obesity. This study provides a comprehensive multi-level (circuit, synaptic, and molecular) explanation of how food intake behavior and body weight are regulated by endogenous central GLP-1. VIDEO ABSTRACT.

Deficits in cognitive flexibility induced by chronic unpredictable stress are associated with impaired glutamate neurotransmission in the rat medial prefrontal cortex.

Deficits in cognitive flexibility, the ability to modify behavior in response to changes in the environment, contribute to the onset and maintenance of stress-related neuropsychiatric illnesses, such as depression. Cognitive flexibility depends on medial prefrontal cortex (mPFC) function, and in depressed patients, cognitive inflexibility is associated with hypoactivity and decreased glutamate receptor expression in the mPFC. Rats exposed to chronic unpredictable stress (CUS) exhibit compromised mPFC function on the extradimensional (ED) set-shifting task of the attentional set-shifting test. Moreover, CUS-induced ED deficits are associated with dendritic atrophy and decreased glutamate receptor expression in the mPFC. This evidence suggests that impaired glutamate signaling may underlie stress-induced deficits in cognitive flexibility. To test this hypothesis, we first demonstrated that blocking NMDA or AMPA receptors in the mPFC during ED replicated CUS-induced deficits in naïve rats. Secondly, we found that expression of activity-regulated cytoskeleton-associated protein (Arc) mRNA, a marker of behaviorally induced glutamate-mediated plasticity, was increased in the mPFC following ED. We then showed that CUS compromised excitatory afferent activation of the mPFC following pharmacological stimulation of the mediodorsal thalamus (MDT), indicated by a reduced induction of c-fos expression. Subsequently, in vivo recordings of evoked potentials in the mPFC indicated that CUS impaired afferent activation of the mPFC evoked by MDT stimulation, but not the ventral hippocampus. Lastly, glutamate microdialysis showed that CUS attenuated the acute stress-evoked increase in extracellular glutamate in the mPFC. Together, these results demonstrate that CUS-induced ED deficits are associated with compromised glutamate neurotransmission in the mPFC.

Ras and Rab interactor 1 controls neuronal plasticity by coordinating dendritic filopodial motility and AMPA receptor turnover.

Ras and Rab interactor 1 (RIN1) is predominantly expressed in the nervous system. RIN1-knockout animals have deficits in latent inhibition and fear extinction in the amygdala, suggesting a critical role for RIN1 in preventing the persistence of unpleasant memories. At the molecular level, RIN1 signals through Rab5 GTPases that control endocytosis of cell-surface receptors and Abl nonreceptor tyrosine kinases that participate in actin cytoskeleton remodeling. Here we report that RIN1 controls the plasticity of cultured mouse hippocampal neurons. Our results show that RIN1 affects the morphology of dendritic protrusions and accelerates dendritic filopodial motility through an Abl kinase-dependent pathway. Lack of RIN1 results in enhanced mEPSC amplitudes, indicating an increase in surface AMPA receptor levels compared with wild-type neurons. We further provide evidence that the Rab5 GEF activity of RIN1 regulates surface GluA1 subunit endocytosis. Consequently loss of RIN1 blocks surface AMPA receptor down-regulation evoked by chemically induced long-term depression. Our findings indicate that RIN1 destabilizes synaptic connections and is a key player in postsynaptic AMPA receptor endocytosis, providing multiple ways of negatively regulating memory stabilization during neuronal plasticity.

GABA and Endocannabinoids Mediate Depotentiation of Schaffer Collateral Synapses Induced by Stimulation of Temperoammonic Inputs.

Long-term potentiation (LTP) of Schaffer collateral (SC) synapses in the hippocampus is thought to play a key role in episodic memory formation. Because the hippocampus is a shorter-term, limited capacity storage system, repeated bouts of learning and synaptic plasticity require that SC synapses reset to baseline at some point following LTP. We previously showed that repeated low frequency activation of temperoammonic (TA) inputs to the CA1 region depotentiates SC LTP without persistently altering basal transmission. This heterosynaptic depotentiation involves adenosine A1 receptors but not N-methyl-D-aspartate receptors, metabotropic glutamate receptors or L-type calcium channels. In the present study, we used rat hippocampal slices to explore other messengers contributing to TA-induced SC depotentiation, and provide evidence for the involvement of cannabinoid-1 and γ-aminobutyric acid (GABA) type-A receptors as more proximal signaling events leading to synaptic resetting, with A1 receptor activation serving as a downstream event. Surprisingly, we found that TA-induced SC depotentiation is independent of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate glutamate receptors. We also examined the involvement of mitogen-activated protein kinases (MAPKs), and found a role for extracellular-signal related kinase 1/2 and p38 MAPK, but not c-Jun-N-terminal kinase. These results indicate that low frequency stimulation of TA inputs to CA1 activates a complex signaling network that instructs SC synaptic resetting. The involvement of GABA and endocannabinoids suggest mechanisms that could contribute to cognitive dysfunction associated with substance abuse and neuropsychiatric disorders.

Adenosine A1 Receptor-Mediated Endocytosis of AMPA Receptors Contributes to Impairments in Long-Term Potentiation (LTP) in the Middle-Aged Rat Hippocampus.

Aging causes multiple changes in the mammalian brain, including changes in synaptic signaling. Previous reports have shown increased extracellular adenosine in the aging brain, and we recently reported that activation of adenosine A1 receptors (A1Rs) induces AMPA receptor (AMPAR) internalization in rat hippocampus. This study investigated whether aging-related changes in the rat hippocampus include altered surface expression of adenosine A1 and A2A receptors, and whether these changes correspond to changes in AMPAR surface expression and altered synaptic plasticity. We found reduced A1R surface expression in middle-aged rat hippocampus, and also reduced GluA1 and GluA2 AMPAR subunit surface expression. Using a chemically-induced LTP (cLTP) experimental protocol, we recorded fEPSPs in young (1 month old) and middle-aged (7-12 month old) rat hippocampal slices. There were significant impairments in cLTP in middle-aged slices, suggesting impaired synaptic plasticity. Since we previously showed that the A1R agonist N(6)-cyclopentyladenosine (CPA) reduced both A1Rs and GluA2/GluA1 AMPARs, we hypothesized that the observed impaired synaptic plasticity in middle-aged brains is regulated by A1R-mediated AMPAR internalization by clathrin-mediated endocytosis. Following cLTP, we found a significant increase in GluA1 and GluA2 surface expression in young rats, which was blunted in middle-aged brains or in young brains pretreated with CPA. Blocking A1Rs with 8-cyclopentyl-1,3-dipropylxanthine or AMPAR endocytosis with either Tat-GluA2-3Y peptide or dynasore (dynamin inhibitor) similarly enhanced AMPAR surface expression following cLTP. These data suggest that age-dependent alteration in adenosine receptor expression contributes to increased AMPAR endocytosis and impaired synaptic plasticity in aged brains.

Rapid increases in immature synapses parallel estrogen-induced hippocampal learning enhancements.

Dramatic increases in hippocampal spine synapse density are known to occur within minutes of estrogen exposure. Until now, it has been assumed that enhanced spinogenesis increased excitatory input received by the CA1 pyramidal neurons, but how this facilitated learning and memory was unclear. Delivery of 17ß-estradiol or an estrogen receptor (ER)-α (but not ER-ß) agonist into the dorsal hippocampus rapidly improved general discrimination learning in female mice. The same treatments increased CA1 dendritic spines in hippocampal sections over a time course consistent with the learning acquisition phase. Surprisingly, estrogen-activated spinogenesis was associated with a decrease in CA1 hippocampal excitatory input, rapidly and transiently reducing CA1 AMPA activity via a mechanism likely reflecting AMPA receptor internalization and creation of silent or immature synapses. We propose that estrogens promote hippocampally mediated learning via a mechanism resembling some of the broad features of normal development, an initial overproduction of functionally immature connections being subsequently "pruned" by experience.

Microglia-dependent alteration of glutamatergic synaptic transmission and plasticity in the hippocampus during peripheral inflammation.

Peripheral inflammatory diseases are often associated with behavioral comorbidities including anxiety, depression, and cognitive dysfunction, but the mechanism for these is not well understood. Changes in the neuronal and synaptic functions associated with neuroinflammation may underlie these behavioral abnormalities. We have used a model of colonic inflammation induced by 2,4,6-trinitrobenzenesulfonic acid in Sprague Dawley rats to identify inflammation-induced changes in hippocampal synaptic transmission. Hippocampal slices obtained 4 d after the induction of inflammation revealed enhanced Schaffer collateral-induced excitatory field potentials in CA1 stratum radiatum. This was associated with larger-amplitude mEPSCs, but unchanged mEPSC frequencies and paired-pulse ratios, suggesting altered postsynaptic effects. Both AMPA- and NMDA-mediated synaptic currents were enhanced, and analysis of AMPA-mediated currents revealed increased contributions of GluR2-lacking receptors. In keeping with this, both transcripts and protein levels of the GluR2 subunit were reduced in hippocampus. Both long-term potentiation (LTP) and depression (LTD) were significantly reduced in hippocampal slices taken from inflamed animals. Chronic administration of the microglial/macrophage activation inhibitor minocycline to the inflamed animals both lowered the level of the cytokine tumor necrosis factor α in the hippocampus and completely abolished the effect of peripheral inflammation on the field potentials and synaptic plasticity (LTP and LTD). Our results reveal profound synaptic changes caused by a mirror microglia-mediated inflammatory response in hippocampus during peripheral organ inflammation. These synaptic changes may underlie the behavioral comorbidities seen in patients.

Prenatal nicotine is associated with reduced AMPA and NMDA receptor-mediated rises in calcium within the laterodorsal tegmentum: a pontine nucleus involved in addiction processes.

Despite huge efforts from public sectors to educate society as to the deleterious physiological consequences of smoking while pregnant, 12-25% of all babies worldwide are born to mothers who smoked during their pregnancies. Chief among the negative legacies bestowed to the exposed individual is an enhanced proclivity postnatally to addict to drugs of abuse, which suggests that the drug exposure during gestation changed the developing brain in such a way that biased it towards addiction. Glutamate signalling has been shown to be altered by prenatal nicotine exposure (PNE) and glutamate is the major excitatory neurotransmitter within the laterodorsal tegmental nucleus (LDT), which is a brainstem region importantly involved in responding to motivational stimuli and critical in development of drug addiction-associated behaviours, however, it is unknown whether PNE alters glutamate signalling within this nucleus. Accordingly, we used calcium imaging, to evaluate AMPA and NMDA receptor-mediated calcium responses in LDT brain slices from control and PNE mice. We also investigated whether the positive AMPA receptor modulator cyclothiazide (CYZ) had differential actions on calcium in the LDT following PNE. Our data indicated that PNE significantly decreased AMPA receptor-mediated calcium responses, and altered the neuronal calcium response to consecutive NMDA applications within the LDT. Furthermore, CYZ strongly potentiated AMPA-induced responses, however, this action was significantly reduced in the LDT of PNE mice when compared with enhancements in responses in control LDT cells. Immunohistochemical processing confirmed that calcium imaging recordings were obtained from the LDT nucleus as determined by presence of cholinergic neurons. Our results contribute to the body of evidence suggesting that neurobiological changes are induced if gestation is accompanied by nicotine exposure. We conclude that in light of the role played by the LDT in motivated behaviour, the cellular changes in the LDT induced by exposures to nicotine prenatally, when combined with alterations in other reward-related regions, could contribute to the increased susceptibility to smoking observed in the offspring.

Functional consequences of age-related morphologic changes to pyramidal neurons of the rhesus monkey prefrontal cortex.

Layer 3 (L3) pyramidal neurons in the lateral prefrontal cortex (LPFC) of rhesus monkeys exhibit dendritic regression, spine loss and increased action potential (AP) firing rates during normal aging. The relationship between these structural and functional alterations, if any, is unknown. To address this issue, morphological and electrophysiological properties of L3 LPFC pyramidal neurons from young and aged rhesus monkeys were characterized using in vitro whole-cell patch-clamp recordings and high-resolution digital reconstruction of neurons. Consistent with our previous studies, aged neurons exhibited significantly reduced dendritic arbor length and spine density, as well as increased input resistance and firing rates. Computational models using the digital reconstructions with Hodgkin-Huxley and AMPA channels allowed us to assess relationships between demonstrated age-related changes and to predict physiological changes that have not yet been tested empirically. For example, the models predict that in both backpropagating APs and excitatory postsynaptic currents (EPSCs), attenuation is lower in aged versus young neurons. Importantly, when identical densities of passive parameters and voltage- and calcium-gated conductances were used in young and aged model neurons, neither input resistance nor firing rates differed between the two age groups. Tuning passive parameters for each model predicted significantly higher membrane resistance (R m ) in aged versus young neurons. This R m increase alone did not account for increased firing rates in aged models, but coupling these R m values with subtle differences in morphology and membrane capacitance did. The predicted differences in passive parameters (or parameters with similar effects) are mathematically plausible, but must be tested empirically.

Nicotine and ethanol cooperate to enhance ventral tegmental area AMPA receptor function via α6-containing nicotinic receptors.

Nicotine + ethanol co-exposure results in additive and/or synergistic effects in the ventral tegmental area (VTA) to nucleus accumbens (NAc) dopamine (DA) pathway, but the mechanisms supporting this are unclear. We tested the hypothesis that nAChRs containing α6 subunits (α6* nAChRs) are involved in the response to nicotine + ethanol co-exposure. Exposing VTA slices from C57BL/6 WT animals to drinking-relevant concentrations of ethanol causes a marked enhancement of α-amino-3-hydroxy-5-methyl-isoxazolepropionic acid (AMPA) receptor (AMPAR) function in VTA neurons. This effect was sensitive to α-conotoxin MII (an α6ß2* nAChR antagonist), suggesting that α6* nAChR function is required. In mice expressing hypersensitive α6* nAChRs (α6L9S mice), we found that lower concentrations (relative to C57BL/6 WT) of ethanol were sufficient to enhance AMPAR function in VTA neurons. Exposure of live C57BL/6 WT mice to ethanol also produced AMPAR functional enhancement in VTA neurons, and studies in α6L9S mice strongly suggest a role for α6* nAChRs in this response. We then asked whether nicotine and ethanol cooperate to enhance VTA AMPAR function. We identified low concentrations of nicotine and ethanol that were capable of strongly enhancing VTA AMPAR function when co-applied to slices, but that did not enhance AMPAR function when applied alone. This effect was sensitive to both varenicline (an α4ß2* and α6ß2* nAChR partial agonist) and α-conotoxin MII. Finally, nicotine + ethanol co-exposure also enhanced AMPAR function in VTA neurons from α6L9S mice. Together, these data identify α6* nAChRs as important players in the response to nicotine + ethanol co-exposure in VTA neurons.

Stress induces pain transition by potentiation of AMPA receptor phosphorylation.

Chronic postsurgical pain is a serious issue in clinical practice. After surgery, patients experience ongoing pain or become sensitive to incident, normally nonpainful stimulation. The intensity and duration of postsurgical pain vary. However, it is unclear how the transition from acute to chronic pain occurs. Here we showed that social defeat stress enhanced plantar incision-induced AMPA receptor GluA1 phosphorylation at the Ser831 site in the spinal cord and greatly prolonged plantar incision-induced pain. Interestingly, targeted mutation of the GluA1 phosphorylation site Ser831 significantly inhibited stress-induced prolongation of incisional pain. In addition, stress hormones enhanced GluA1 phosphorylation and AMPA receptor-mediated electrical activity in the spinal cord. Subthreshold stimulation induced spinal long-term potentiation in GluA1 phosphomimetic mutant mice, but not in wild-type mice. Therefore, spinal AMPA receptor phosphorylation contributes to the mechanisms underlying stress-induced pain transition.