Presynaptic HCN channel activity is required for the expression of long-term potentiation at lateral amygdala to basal amygdala synapses.

Recently, we reported that auditory fear conditioning leads to the presynaptic potentiation at lateral amygdala to basal amygdala (LA-BA) synapses that shares the mechanism with high-frequency stimulation (HFS)-induced long-term potentiation (LTP) ex vivo. In the present study, we further examined the molecular mechanisms underlying the HFS-induced presynaptic LTP. We found that a presynaptic elevation of Ca2+ was required for the LTP induction. Interestingly, the blockade of presynaptic but not postsynaptic HCN channels with ZD7288 completely abolished LTP induction. While ZD7288 did not alter basal synaptic transmission, the blocker fully reversed previously established LTP, indicating that HCN channels are also required for the maintenance of LTP. Indeed, HCN3 and HCN4 channels were preferentially localized in the presynaptic boutons of LA afferents. Furthermore, an inhibition of either GABAB receptors or GIRK channels eliminated the inhibitory effect of HCN blockade on the LTP induction. Collectively, we suggest that activation of presynaptic HCN channels may counteract membrane hyperpolarization during tetanic stimulation, and thereby contributes to the presynaptic LTP at LA-BA synapses.

TRAF2 regulates the protein stability of HIPK2.

A nuclear serine/threonine kinase homeodomain-interacting protein kinase 2 (HIPK2) is a critical regulator of development and DNA damage response. HIPK2 can induce apoptosis under cellular stress conditions and thus its protein level is maintained low by constant proteasomal degradation. In the present study, we present evidence that TNF receptor-associated factor 2 (TRAF2) regulates the protein stability of HIPK2. Overexpression of TRAF2 decreased while its knockdown increased the HIPK2 protein level. The TRAF2-mediated decrease in HIPK2 protein expression was blocked by proteasomal inhibitor. In addition, TRAF2 decreased the protein half-life of HIPK2. We found that HIPK2 and TRAF2 co-immunoprecipitated. Interestingly, the co-immunoprecipitation was reduced while HIPK2 protein level increased following TNFα treatment, suggesting TNFα induced dissociation of TRAF2 from HIPK2 to accumulate HIPK2. Inhibition of HIPK2 partially suppressed TNFα-induced cell death, indicating that the accumulated HIPK2 may contribute to the TNFα-induced cell death. Our results suggest that TRAF2 can regulate proapoptotic function of HIPK2 by promoting proteasomal degradation.

Auditory fear conditioning facilitates neurotransmitter release at lateral amygdala to basal amygdala synapses.

The lateral amygdala (LA) is a main sensory input site from the cortical and thalamic regions. In turn, LA glutamatergic pyramidal neurons strongly project to the basal amygdala (BA). Although it is well known that auditory fear conditioning involves synaptic potentiation in the LA, it is not clear whether the LA-BA synaptic transmission is modified upon auditory fear conditioning. Here we found that high-frequency stimulation ex vivo resulted in long-term potentiation (LTP) with a concomitant enhancement of neurotransmitter release at LA-BA synapses. Auditory fear conditioning also led to the presynaptic facilitation at LA-BA synapses. Meanwhile, AMPA/NMDA current ratio was not changed upon fear conditioning, excluding the involvement of postsynaptic mechanism. Notably, fear conditioning occluded electrically induced ex vivo LTP in the LA-BA pathway, indicating that the conditioning and electrically induced LTP share common mechanisms. Our findings suggest that the presynaptic potentiation of LA-BA synapses may be involved in fear conditioning.

Regulation of habenular G-protein gamma 8 on learning and memory via modulation of the central acetylcholine system.

Guanine nucleotide binding protein (G protein) gamma 8 (Gng8) is a subunit of G proteins and expressed in the medial habenula (MHb) and interpeduncular nucleus (IPN). Recent studies have demonstrated that Gng8 is involved in brain development; however, the roles of Gng8 on cognitive function have not yet been addressed. In the present study, we investigated the expression of Gng8 in the brain and found that Gng8 was predominantly expressed in the MHb-IPN circuit of the mouse brain. We generated Gng8 knockout (KO) mice by CRISPR/Cas9 system in order to assess the role of Gng8 on cognitive function. Gng8 KO mice exhibited deficiency in learning and memory in passive avoidance and Morris water maze tests. In addition, Gng8 KO mice significantly reduced long-term potentiation (LTP) in the hippocampus compared to that of wild-type (WT) mice. Furthermore, we observed that levels of acetylcholine (ACh) and choline acetyltransferase (ChAT) in the MHb and IPN of Gng8 KO mice were significantly decreased, compared to WT mice. The administration of nAChR α4ß2 agonist A85380 rescued memory impairment in the Gng8 KO mice, suggesting that Gng8 regulates cognitive function via modulation of cholinergic activity. Taken together, Gng8 is a potential therapeutic target for memory-related diseases and/or neurodevelopmental diseases.

PARP-1 regulates mouse embryonic neural stem cell proliferation by regulating PDGFRα expression.

PARP-1 is a multifunctional enzyme that regulates DNA repair, chromatin remodeling, inflammation and cell survival. Our previous study revealed that PARP-1 is required for maintaining normal level of neural stem cell proliferation. In the present study, we present evidence indicating that PARP-1 regulates neural stem cell proliferation by upregulating the expression of platelet-derived growth factor receptor α (PDGFRα). PARP-1 knockout neural stem cells exhibited striking downregulation of PDGFRα expression. We found that PARP-1 promotes the transcription of PDGFRα independently of its enzymatic activity. Overexpression of PDGFRα in the PARP-1 knockout neural stem cells reversed the proliferation defect of the knockout cells. Conversely, knockdown or blocking antibody of PDGFRα suppressed the proliferation of neural stem cells. In addition, blockade of PDGFRα increased cell death rate. Consistent with the downregulation of PDGFRα in the absence of PARP-1, PDGF-AA promoted proliferation of wild-type neural stem cells but not that of PARP-1 knockout cells. These results suggest that PARP-1 can control the neural stem cell proliferation by regulating the expression of PDGFRα.

Defective neurogenesis and schizophrenia-like behavior in PARP-1-deficient mice.

In the current study we present evidence suggesting that PARP-1 regulates neurogenesis and its deficiency may result in schizophrenia-like behavioral deficits in mice. PARP-1 knockout neural stem cells exhibited a marked upregulation of embryonic stem cell phosphatase that can suppress the proliferative signaling of PI3K-Akt and ERK. The suppressed activity of Akt and ERK in the absence of PARP-1 results in the elevation of FOXO1 activity and its downstream target genes p21 and p27, leading to the inhibition of neural stem cell proliferation. Moreover, expression of neurogenic factors and neuronal differentiation were decreased in the PARP-1 knockout neural stem cells whereas glial differentiation was increased. In accordance with the in vitro data, PARP-1 knockout mice exhibited reduced brain weight with enlarged ventricle as well as decreased adult neurogenesis in the hippocampus. Interestingly, PARP-1 knockout mice exhibited schizophrenia-like symptoms such as anxiety, depression, social interaction deficits, cognitive impairments, and prepulse inhibition deficits. Taken together, our results suggest that PARP-1 regulates neurogenesis during development and in adult and its absence may lead to the schizophrenia-like behavioral abnormality in mice.

Chronic social defeat stress increases burst firing of nucleus accumbens-projecting ventral subicular neurons in stress-susceptible mice.

The ventral subiculum (vSub) is the major output structure of the hippocampus and serves as a main limbic region in mediating the brain's response to stress. Previously, we reported that there are three subtypes of vSub neurons based on their firing patterns: regular-spiking (RS), weak-bursting (WB) and strong-bursting (SB) neurons and chronic social defeat stress (CSDS) increased SB neurons especially in the proximal vSub. Here, we found that neurons in the proximal vSub projected to the nucleus accumbens (NAc). CSDS significantly increased SB neurons but decreased RS neurons among the NAc-projecting vSub neuronal population. Interestingly, these changes were only apparent in mice susceptible to CSDS, but not in CSDS-resilient ones. Given that ventral hippocampal inputs to the NAc regulate susceptibility to CSDS, the bursting activity of NAc-projecting vSub neurons might be functionally relevant to behavioral susceptibility to CSDS.

Chronic social defeat stress-induced enhancement of T-type calcium channels increases burst-firing neurons in the ventral subiculum.

The ventral subiculum (vSub), a representative output structure of the hippocampus, serves as a main limbic region in mediating the brain's response to stress. There are three subtypes of subicular pyramidal neurons based on their firing patterns: regular-spiking (RS), weak-bursting (WB) and strong-bursting (SB) neurons, located differently along proximal-distal axis. Here, we found that chronic social defeat stress (CSDS) in mice increased the population of SB neurons but decreased RS neurons in the proximal vSub. Specific blockers of T-type calcium channels inhibited the burst firings with a concomitant reduction of afterdepolarization, suggesting that T-type calcium channels underlie the burst-spiking activity. Consistently, CSDS increased both T-type calcium currents and expression of Cav3.1 proteins, a subtype of T-type calcium channels, in the proximal vSub. Therefore, we conclude that CSDS-induced enhancement of Cav3.1 expression increased bursting neuronal population in the vSub, which may contribute to stress-related behaviors.

Differential regulation of neuronal excitability by nicotine and substance P in subdivisions of the medial habenula.

The medial habenula (MHb) plays an important role in nicotine-related behaviors, such as aversion and withdrawal. The MHb is composed of distinct subregions with unique neurotransmitter expression and neuronal connectivity. Here, we showed that nicotine and substance P (SP) differentially regulate neuronal excitability in subdivisions of the MHb (ventrolateral division, MHbVL; dorsal division; MHbD and superior division: MHbS). Nicotine remarkably increased spontaneous neuronal firing in the MHbVL and MHbD, but not in the MHbS, which was consistent with different magnitudes of whole-cell inward currents evoked by nicotine in each subdivision. Meanwhile, SP enhanced neuronal excitability in the MHbVL and MHbS. Although the MHbD is composed of SP-expressing neurons, they did not respond to SP. Neurons in the MHbVL increased their firing in response to bath-applied nicotine, which was attenuated by neurokinin receptor antagonists. Furthermore, nicotine addiction and withdrawal attenuated and augmented excitatory SP effects in the MHbVL, respectively. On the whole, we suggest that MHb-involving nicotine-related behaviors might be associated with SP signaling in MHb subdivisions.

p300-mediated acetylation increased the protein stability of HIPK2 and enhanced its tumor suppressor function.

Homeodomain-interacting protein kinase 2 (HIPK2) is a nuclear serine/threonine kinase that functions in development and tumor suppression. One of the prominent features of this kinase is that it is tightly regulated by proteasomal degradation. In the present study, we present evidence suggesting that the protein stability of HIPK2 can be regulated by p300-mediated acetylation. p300 increased the protein level of HIPK2 via its acetyltransferase activity. p300 increased the acetylation of HIPK2 while decreased polyubiquitination and its proteasomal degradation. We also observed that DNA damage induced acetylation of HIPK2 along with an increase in the protein amount, which was inhibited by p300 RNAi. Importantly, p300 promoted p53 activation and the HIPK2-mediated suppression of cell proliferation, suggesting acetylation-induced HIPK2 stabilization contributed to the enhanced activation of HIPK2. Overexpression of p300 promoted the HIPK2-mediated suppression of tumor growth in mouse xenograft model as well. Taken together, our data suggest that p300-mediated acetylation of HIPK2 increases the protein stability of HIPK2 and enhances its tumor suppressor function.

Optogenetic activation of septal GABAergic afferents entrains neuronal firing in the medial habenula.

The medial habenula (MHb) plays an important role in nicotine-related behaviors such as nicotine aversion and withdrawal. The MHb receives GABAergic input from the medial septum/diagonal band of Broca (MS/DB), yet the synaptic mechanism that regulates MHb activity is unclear. GABA (γ -aminobutyric acid) is a major inhibitory neurotransmitter activating both GABAA receptors and GABAB receptors. Depending on intracellular chloride concentration, however, GABAA receptors also function in an excitatory manner. In the absence of various synaptic inputs, we found that MHb neurons displayed spontaneous tonic firing at a rate of about ~4.4 Hz. Optogenetic stimulation of MS/DB inputs to the MHb evoked GABAA receptor-mediated synaptic currents, which produced stimulus-locked neuronal firing. Subsequent delayed yet lasting activation of GABAB receptors attenuated the intrinsic tonic firing. Consequently, septal GABAergic input alone orchestrates both excitatory GABAA and inhibitory GABAB receptors, thereby entraining the firing of MHb neurons.

PARP1 regulates the protein stability and proapoptotic function of HIPK2.

Homeodomain-interacting protein kinase 2 (HIPK2) is a nuclear serine/threonine kinase that functions in DNA damage response and development. In the present study, we propose that the protein stability and proapoptotic function of HIPK2 are regulated by poly(ADP-ribose) polymerase 1 (PARP1). We present evidence indicating that PARP1 promotes the proteasomal degradation of HIPK2. The tryptophan-glycine-arginine (WGR) domain of PARP1 was necessary and sufficient for the promotion of HIPK2 degradation independently of the PARP1 enzymatic activity. The WGR domain mediated the interaction between HIPK2 and C-terminus of HSP70-interacting protein (CHIP) via HSP70. We found that CHIP can function as a ubiquitin ligase for HIPK2. The interaction between PAPR1 and HIPK2 was weakened following DNA damage. Importantly, PARP1 reduced the HIPK2-mediated p53 phosphorylation, proapoptotic transcriptional activity and cell death. These results suggest that PARP1 can modulate the tumor-suppressing function of HIPK2 by regulating the protein stability of HIPK2.

The role of inositol 1,4,5-trisphosphate 3-kinase A in regulating emotional behavior and amygdala function.

Inositol 1,4,5-trisphosphate 3-kinase A (IP3K-A) is a molecule enriched in the brain and neurons that regulates intracellular calcium levels via signaling through the inositol trisphosphate receptor. In the present study, we found that IP3K-A expression is highly enriched in the central nucleus of the amygdala (CeA), which plays a pivotal role in the processing and expression of emotional phenotypes in mammals. Genetic abrogation of IP3K-A altered amygdala gene expression, particularly in genes involved in key intracellular signaling pathways and genes mediating fear- and anxiety-related behaviors. In agreement with the changes in amygdala gene expression profiles, IP3K-A knockout (KO) mice displayed more robust responses to aversive stimuli and spent less time in the open arms of the elevated plus maze, indicating high levels of innate fear and anxiety. In addition to behavioral phenotypes, decreased excitatory and inhibitory postsynaptic current and reduced c-Fos immunoreactivity in the CeA of IP3K-A KO mice suggest that IP3K-A has a profound influence on the basal activities of fear- and anxiety-mediating amygdala circuitry. In conclusion, our findings collectively demonstrate that IP3K-A plays an important role in regulating affective states by modulating metabotropic receptor signaling pathways and neural activity in the amygdala.

Promotion of Cortical Neurogenesis from the Neural Stem Cells in the Adult Mouse Subcallosal Zone.

Neurogenesis occurs spontaneously in the subventricular zone (SVZ) of the lateral ventricle in adult rodent brain, but it has long been debated whether there is sufficient adult neurogenesis in human SVZ. Subcallosal zone (SCZ), a posterior continuum of SVZ closely associated with posterior regions of cortical white matter, has also been reported to contain adult neural stem cells (aNSCs) in both rodents and humans. However, little is known whether SCZ-derived aNSC (SCZ-aNSCs) can produce cortical neurons following brain injury. We found that SCZ-aNSCs exhibited limited neuronal differentiation potential in culture and after transplantation in mice. Neuroblasts derived from SCZ initially migrated toward injured cortex regions following brain injury, but later exhibited apoptosis. Overexpression of anti-apoptotic bcl-xL in the SCZ by retroviral infection rescued neuroblasts from cell death in the injured cortex, but neuronal maturation was still limited, resulting in atrophy. In combination with Bcl-xL, infusion of brain-derived neurotropic factor rescued atrophy, and importantly, a subset of such SCZ-aNSCs differentiated and attained morphological and physiological characteristics of mature, excitatory neurons. These results suggest that the combination of anti-apoptotic and neurotrophic factors might enable the use of aNSCs derived from the SCZ in cortical neurogenesis for neural replacement therapy.

Learning-induced synaptic potentiation in implanted neural precursor cell-derived neurons.

Neuronal loss caused by neurodegenerative diseases, traumatic brain injury and stroke results in cognitive dysfunctioning. Implantation of neural stem/precursor cells (NPCs) can improve the brain function by replacing lost neurons. Proper synaptic integration following neuronal differentiation of implanted cells is believed to be a prerequisite for the functional recovery. In the present study, we characterized the functional properties of immortalized neural progenitor HiB5 cells implanted into the rat hippocampus with chemically induced lesion. The implanted HiB5 cells migrated toward CA1 pyramidal layer and differentiated into vGluT1-positive glutamatergic neurons with morphological and electrophysiological properties of endogenous CA1 pyramidal cells. Functional synaptic integration of HiB5 cell-derived neurons was also evidenced by immunohistochemical and electrophysiological data. Lesion-caused memory deficit was significantly recovered after the implantation when assessed by inhibitory avoidance (IA) learning. Remarkably, IA learning preferentially produced long-term potentiation (LTP) at the synapses onto HiB5 cell-derived neurons, which occluded paring protocol-induced LTP ex vivo. We conclude that the implanted HiB5 cell-derived neurons actively participate in learning process through LTP formation, thereby counteracting lesion-mediated memory impairment.

Tricyclic Antidepressants Amitriptyline and Desipramine Induced Neurotoxicity Associated with Parkinson's Disease.

Recent studies report that a history of antidepressant use is strongly correlated with the occurrence of Parkinson's disease (PD). However, it remains unclear whether antidepressant use can be a causative factor for PD. In the present study, we examined whether tricyclic antidepressants amitriptyline and desipramine can induce dopaminergic cell damage, both in vitro and in vivo. We found that amitriptyline and desipramine induced mitochondria-mediated neurotoxicity and oxidative stress in SH-SY5Y cells. When injected into mice on a subchronic schedule, amitriptyline induced movement deficits in the pole test, which is known to detect nigrostriatal dysfunction. In addition, the number of tyrosine hydroxylase-positive neurons in the substantia nigra pars compacta was reduced in amitriptyline-injected mice. Our results suggest that amitriptyline and desipramine may induce PD-associated neurotoxicity.

Group I mGluR-dependent depotentiation in the lateral amygdala does not require the removal of calcium-permeable AMPA receptors.

There is conflicting evidence regarding whether calcium-permeable receptors are removed during group I mGluR-mediated synaptic depression. In support of this hypothesis, AMPAR rectification, a correlative index of the synaptic expression of GluA2-lacking calcium-permeable AMPARs (CP-AMPARs), is known to decrease after the induction of several types of group I mGluR-mediated long-term depression (LTD), suggesting that a significant proportion of synaptic CP-AMPARs is removed during synaptic depression. We have previously demonstrated that fear conditioning-induced synaptic potentiation in the lateral amygdala is reversed by group 1 mGluR-mediated depotentiation. Here, we examined whether CP-AMPARs are removed by mGluR1-mediated depotentiation of fear conditioning-induced synaptic potentiation. The synaptic expression of CP-AMPARs was negligible before, increased significantly 12 h after, and returned to baseline 48 h after fear conditioning, as evidenced by the changes in the sensitivity of lateral amygdala synaptic responses to NASPM. Importantly, the sensitivity to NASPM was not altered after induction of depotentiation. Our findings, together with previous results, suggest that the removal of CP-AMPARs is not required for the depotentiation of fear conditioning-induced synaptic potentiation at lateral amygdala synapses.

Regional differences in acute corticosterone-induced dendritic remodeling in the rat brain and their behavioral consequences.

BACKGROUND: Glucocorticoid released by stressful stimuli elicits various stress responses. Acute treatment with a single dose of corticosterone (CORT; predominant glucocorticoid of rats) alone has previously been shown to trigger anxiety behavior and robust dendritic hypertrophy of neurons in the basolateral amygdala (BLA). Neurons in the medial prefrontal cortex (mPFC) are also known to be highly sensitive to stress and regulate anxiety-like behaviors. Nevertheless, we know less about acute CORT-induced structural changes of other brain regions and their behavioral outcomes. In addition, the temporal profile of acute CORT effects remains to be examined. The current study investigates time course changes of dendritic architectures in the stress vulnerable brain areas, the BLA and mPFC, and their behavioral consequences after acute treatment with a single dose of CORT. RESULTS: Acute CORT treatment produced delayed onset of dendritic remodeling in the opposite direction in the BLA and mPFC with different time courses. Acute CORT induced dendritic hypertrophy of BLA spiny neurons, which was paralleled by heightened anxiety, both peaked 12 days after the treatment. Meanwhile, CORT-induced dendritic atrophy of mPFC pyramidal neurons peaked on day 6, concomitantly with impaired working memory. Both changed dendritic morphologies and altered behavioral outcomes were fully recovered. CONCLUSION: Our results suggest that stress-induced heightened anxiety appears to be a functional consequence of dendritic remodeling of BLA neurons but not that of mPFC. Instead, stress-induced dendritic atrophy of mPFC neurons is relevant to working memory deficit. Therefore, structural changes in the BLA and the mPFC might be specifically associated with distinct behavioral symptoms observed in stress-related mental disorders. Remarkably, stress-induced dendritic remodeling in the BLA as well as mPFC is readily reversible. The related behavioral outcomes also follow the similar time course in a reversible manner. Therefore, further studies on the cellular mechanism for the plasticity of dendrites architecture might provide new insight into the etiological factors for stress-related mental illness such as posttraumatic stress disorder (PTSD).

Heptachlor induced nigral dopaminergic neuronal loss and Parkinsonism-like movement deficits in mice.

Epidemiological studies have suggested an association between pesticide exposure and Parkinson's disease. In this study, we examined the neurotoxicity of an organochlorine pesticide, heptachlor, in vitro and in vivo. In cultured SH-SY5Y cells, heptachlor induced mitochondria-mediated apoptosis. When injected into mice intraperitoneally on a subchronic schedule, heptachlor induced selective loss of dopaminergic neurons in the substantia nigra pars compacta. In addition, the heptachlor injection induced gliosis of microglia and astrocytes selectively in the ventral midbrain area. When the general locomotor activities were monitored by open field test, the heptachlor injection did not induce any gross motor dysfunction. However, the compound induced Parkinsonism-like movement deficits when assessed by a gait and a pole test. These results suggest that heptachlor can induce Parkinson's disease-related neurotoxicities in vivo.

SIRT1 negatively regulates the protein stability of HIPK2.

In the present study, we investigated whether a histone deacetylase sirtuin 1 (SIRT1) can regulate the protein stability of homeodomain-interacting protein kinase 2 (HIPK2). We observed the evidence of molecular interaction between SIRT1 and HIPK2. Interestingly, overexpression or pharmacological activation of SIRT1 promoted ubiquitination and the proteasomal degradation of HIPK2 whereas inhibition of SIRT1 activity increased the protein level of HIPK2. Furthermore, a SIRT1 activator decreased the level of HIPK2 acetylation whereas an inhibitor increased the acetylation level. These results suggest that SIRT1 may deacetylate and promote the ubiquitination and subsequent proteasomal degradation of HIPK2.