The malleable brain: plasticity of neural circuits and behavior: A review from students to students

One of the most intriguing features of the brain is its ability to be malleable, allowing it to adapt continually to changes in the environment. Specific neuronal activity patterns drive long-lasting increases or decreases in the strength of synaptic connections, referred to as long-term potentiation (LTP) and long-term depression (LTD) respectively. Such phenomena have been described in a variety of model organisms, which are used to study molecular, structural, and functional aspects of synaptic plasticity. This review originated from the first International Society for Neurochemistry (ISN) and Journal of Neurochemistry (JNC) Flagship School held in Alpbach, Austria (Sep 2016), and will use its curriculum and discussions as a framework to review some of the current knowledge in the field of synaptic plasticity. First, we describe the role of plasticity during development and the persistent changes of neural circuitry occurring when sensory input is altered during critical developmental stages. We then outline the signaling cascades resulting in the synthesis of new plasticity-related proteins, which ultimately enable sustained changes in synaptic strength. Going beyond the traditional understanding of synaptic plasticity conceptualized by LTP and LTD, we discuss system-wide modifications and recently unveiled homeostatic mechanisms, such as synaptic scaling. Finally, we describe the neural circuits and synaptic plasticity mechanisms driving associative memory and motor learning. Evidence summarized in this review provides a current view of synaptic plasticity in its various forms, offers new insights into the underlying mechanisms and behavioral relevance, and provides directions for future research in the field of synaptic plasticity.Fil: Schaefer, Natascha. University of Wuerzburg; AlemaniaFil: Rotermund, Carola. University of Tuebingen; AlemaniaFil: Blumrich, Eva Maria. Universitat Bremen; AlemaniaFil: Lourenco, Mychael V.. Universidade Federal do Rio de Janeiro; BrasilFil: Joshi, Pooja. Robert Debre Hospital; FranciaFil: Hegemann, Regina U.. University of Otago; Nueva ZelandaFil: Jamwal, Sumit. ISF College of Pharmacy; IndiaFil: Ali, Nilufar. Augusta University; Estados UnidosFil: García Romero, Ezra Michelet. Universidad Veracruzana; MéxicoFil: Sharma, Sorabh. Birla Institute of Technology and Science; IndiaFil: Ghosh, Shampa. Indian Council of Medical Research; IndiaFil: Sinha, Jitendra K.. Indian Council of Medical Research; IndiaFil: Loke, Hannah. Hudson Institute of Medical Research; AustraliaFil: Jain, Vishal. Defence Institute of Physiology and Allied Sciences; IndiaFil: Lepeta, Katarzyna. Polish Academy of Sciences; ArgentinaFil: Salamian, Ahmad. Polish Academy of Sciences; ArgentinaFil: Sharma, Mahima. Polish Academy of Sciences; ArgentinaFil: Golpich, Mojtaba. University Kebangsaan Malaysia Medical Centre; MalasiaFil: Nawrotek, Katarzyna. University Of Lodz; ArgentinaFil: Paid, Ramesh K.. Indian Institute of Chemical Biology; IndiaFil: Shahidzadeh, Sheila M.. Syracuse University; Estados UnidosFil: Piermartiri, Tetsade. Universidade Federal de Santa Catarina; BrasilFil: Amini, Elham. University Kebangsaan Malaysia Medical Centre; MalasiaFil: Pastor, Verónica. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia ; ArgentinaFil: Wilson, Yvette. University of Melbourne; AustraliaFil: Adeniyi, Philip A.. Afe Babalola University; NigeriaFil: Datusalia, Ashok K.. National Brain Research Centre; IndiaFil: Vafadari, Benham. Polish Academy of Sciences; ArgentinaFil: Saini, Vedangana. University of Nebraska; Estados UnidosFil: Suárez Pozos, Edna. Instituto Politécnico Nacional; MéxicoFil: Kushwah, Neetu. Defence Institute of Physiology and Allied Sciences; IndiaFil: Fontanet, Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia ; ArgentinaFil: Turner, Anthony J.. University of Leeds; Reino Unid

PRDM12 Is Required for Initiation of the Nociceptive Neuron Lineage during Neurogenesis

Summary: The sensation of pain is essential for the preservation of the functional integrity of the body. However, the key molecular regulators necessary for the initiation of the development of pain-sensing neurons have remained largely unknown. Here, we report that, in mice, inactivation of the transcriptional regulator PRDM12, which is essential for pain perception in humans, results in a complete absence of the nociceptive lineage, while proprioceptive and touch-sensitive neurons remain. Mechanistically, our data reveal that PRDM12 is required for initiation of neurogenesis and activation of a cascade of downstream pro-neuronal transcription factors, including NEUROD1, BRN3A, and ISL1, in the nociceptive lineage while it represses alternative fates other than nociceptors in progenitor cells. Our results thus demonstrate that PRDM12 is necessary for the generation of the entire lineage of pain-initiating neurons. : The sensation of pain, temperature, and itch by neurons of the nociceptive lineage is essential for animal survival. Bartesaghi et al. report that the transcriptional regulator PRDM12 is indispensable in neural crest cells (NCCs) for the initiation of the sensory neuronal differentiation program that generates the entire nociceptive lineage. Keywords: neurogenesis, pain, nociceptive neurons, Prdm12, neural crest cell

Sprouty4 Is an Endogenous Negative Modulator of TrkA Signaling and Neuronal Differentiation Induced by NGF

The Sprouty (Spry) family of proteins represents endogenous regulators of downstream signaling pathways induced by receptor tyrosine kinases (RTKs). Using real time PCR, we detect a significant increase in the expression of Spry4 mRNA in response to NGF, indicating that Spry4 could modulate intracellular signaling pathways and biological processes induced by NGF and its receptor TrkA. In this work, we demonstrate that overexpression of wild-type Spry4 causes a significant reduction in MAPK and Rac1 activation and neurite outgrowth induced by NGF. At molecular level, our findings indicate that ectopic expression of a mutated form of Spry4 (Y53A), in which a conserved tyrosine residue was replaced, fail to block both TrkA-mediated Erk/MAPK activation and neurite outgrowth induced by NGF, suggesting that an intact tyrosine 53 site is required for the inhibitory effect of Spry4 on NGF signaling. Downregulation of Spry4 using small interference RNA knockdown experiments potentiates PC12 cell differentiation and MAPK activation in response to NGF. Together, these findings establish a new physiological mechanism through which Spry4 regulates neurite outgrowth reducing not only the MAPK pathway but also restricting Rac1 activation in response to NGF

Etv4 and Etv5 transcription factors as mediators of neurotrophic factor signaling and development

El preciso desarrollo de las conexiones neuronales es esencial para el correcto funcionamiento del sistema nervioso, y la falta de precisión en este proceso está asociada al desarrollo de diferentes patologías. La formación de los circuitos neuronales depende de la interacción de factores celulares intrínsecos y extrínsecos, que regulan la transmisión de la información en el sistema nervioso. Entre estos, los factores neurotróficos (FN) cumplen roles esenciales en el mantenimiento y sobrevida, crecimiento dendrítico y axonal, sinaptogénesis y plasticidad sináptica de distintas poblaciones neuronales del sistema nervioso central y periférico. Para ello estos factores inducen la expresión de programas transcripcionales específicos implicados en el desarrollo neuronal. Durante los últimos años, las evidencias indican que diversos aspectos del desarrollo neuronal están dirigidos por la expresión de combinaciones específicas de factores transcripcionales. Es por ello que uno de los desafíos de la neurobiología del desarrollo es entender como múltiples señales son integradas por las neuronas en programas transcripcionales que generan patrones específicos de conectividad. El objetivo general de este proyecto es identificar nuevos elementos de los programas transcripcionales y vías de señalización disparadas por los factores neurotróficos, para controlar la conectividad de distintas poblaciones neuronales del sistema nervioso central y periférico. Con el objeto de identificar genes involucrados en la diferenciación neuronal hemos realizado ensayos de expresión génica diferencial en un modelo celular análogo a neuroblastos en proliferación, que en presencia del factor de crecimiento nervioso (NGF, nerve growth factor) detienen su división celular y adquieren un fenotipo neuronal. Este ensayo permitió identificar dos genes que codifican para dos factores de transcripción denominados: Etv4 (también conocido como E1AF o Pea3) y Etv5 (también conocido como Erm) que son inducidos por NGF. En la primera sección de este trabajo demostramos que estos dos miembros de la familia Pea3 son expresados en neuronas sensoriales que responden a NGF durante el período de inervación cutánea y son inducidos local y distalmente por esta neurotrofina. Ensayos de pérdida y ganancia de función para Etv4 o Etv5, indicaron que estos factores son esenciales en el crecimiento neurítico de las neuronas sensoriales inducidas por NGF sugiriendo que estos factores cumplen un rol fisiológico durante el período de inervación periférica inducida por esta neurotrofina. En este trabajo también mostramos que Etv4 y Etv5 son expresados en neuronas hipocampales de las áreas CA1, CA3 y el giro dentado. Describimos que estos factores son inducidos en neuronas hipocampales cultivadas en respuesta al factor neurotrófico derivado del cerebro (BDNF, brain derived neurotrofic factor) y utilizando ensayos de inmunoprecipitación in vivo, mostramos que Etv4 y Etv5 interactúan en el hipocampo. Análisis in vitro de pérdida y ganancia de función indicaron que estos factores median los efectos de crecimiento y ramificación del árbol dendrítico disparados por la neurotrofina BDNF en neuronas hipocampales. El estudio de animales deficientes para Etv4 y Etv5 evidenció un importante rol de estos factores transcripcionales en el desarrollo de la conectividad neuronal hipocampal. En resúmen, nuestros resultados demuestran que Etv4 y Etv5 son moleculas esenciales del programa transcripcional disparado por neurotrofinas que llevan al correcto establecimiento de las conexiones neuronales.Construction of the neural networks depends largely on the precision with which neuronal circuits are established during development. The accuracy of this process is fundamental for normal nervous system function and its aberrant connectivity leads to nervous system disorders. The accuracy of this process depends on the combined actions of extrinsic and intrinsic factors. Between them, neurotrophic factors play key roles in the maintenance and survival of different neuronal populations, dendritic and axonal sprouting, synaptogenesis and synaptic plasticity in the peripheral and central nervous system. To this end, the soluble factors induce the expression of specific transcriptional programs involved in neuronal development. In recent years, the evidence indicates that several aspects of neural development are driven by the expression of specific combinations of transcription factors. Identifying the transcriptional programs and signalling pathways triggered by extracellular cues that control neuronal circuit formation will be of great importance in order to be able to decipher and understand the functioning of mature nervous system. Our general aim focus on the identification of transcriptional programs and signaling pathways triggered by extracellular cues, such as neurotrophic factors, to control the connectivity between specific populations of central and peripheral neurons. In order to identify genes involved in neuronal differentiation and proliferation of neuronal precursors we performed differential gene expression assays in a cellular model analogous to proliferating neuroblasts which in the presence of nerve growth factor (NGF) stops cell division and acquire a neuronal phenotype. This assay allowed us to identify, among others, genes encoding two transcription factors named Etv4 (also known as E1AF, E1A enhancer binding protein) and Etv5 (also known as Erm, ETS related molecule). In this thesis we studied the role of Etv4 and Etv5 in the development of different neuronal type, in central or peripheral nervous system. In the first part of this work we demonstrate that this two members of the Pea3 family are expressed in sensory neurons positive for the NGF receptor, TrkA, during the period of cutaneous innervation and are induced by this neurotrophin. Lost and gain of function assays for Etv4 and Etv5 indicated that these factors are essential for the neurite growth of sensory neurons induced by NGF, suggesting that these factors play a physiological role during the period of peripheral innervation induced by this neurotrophin. In this work we also present data that demonstrate that Etv4 and Etv5 are expressed by CA1, CA3 hippocampal neurons and cells from the dentate gyrus. We describe that these transcriptional factors are induced in hippocampal neuron cultures in response to brain derived neurotrophc factor (BDNF) and immunoprecipitation assays from hippocampus showed that Etv4 and Etv5 interacts in vivo. Moreover, in vitro, lost and gain of function assays indicated that these factors mediate dendrite branching and outgrowth triggered by the BDNF in hippocampal neurons. Animals deficient in Etv4 evidenced a crucial role of it in the development of hippocampal connectivity. In summary, our results demonstrate that Etv4 and Etv5 are essential molecules of the transcriptional program triggered by neurotrophins that leads to the correct establishment of the neuronal connections.Fil:Fontanet, Paula A.. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Sprouty4 restricts Erk/MAPK activation in response to NGF.

<p>A) Erk2/MAPK activation (Phospho-Erk2/MAPK) was evaluated by transient transfection of HA-tagged Erk2 plasmid with a control or a Myc-tagged Sprouty4 vector into PC12 cells. After 36 h cells were serum-starved and stimulated with or without NGF for 10 min. The level of Erk2 activation (P-Erk2) was evaluated by HA-immunoprecipitation followed by immunoblotting with a specific antibody that recognizes the phosphorylated forms of ERK/MAPK. Reprobing of the same blot with anti-HA and anti-Myc antibodies is shown. B) Histogram shows quantification of Erk2/MAPK activation. Results are presented as averages ± SD from three independent experiments. *p<0.05 (Student's t test). C) Erk/MAPK activation (P-MAPK) in cell lysates of parental and PC12-Spry4 (Clon S2) cells treated with NGF (50 ng/ml) and detected by immunoblot (IB). Reprobing of the same blot with anti–tubulin and anti-Myc antibodies is shown. D) Akt activation (P-Akt) in cell lysates of parental and stable transfected PC12-Spry4 (Clon S2) cells treated with NGF (50 ng/ml) and detected by IB. Reprobing of the same blot with anti–tubulin and anti-Myc antibodies is shown. E) Ligand-independent Erk/MAPK activation (P-MAPK) was evaluated by transient transfection of HA-tagged Ras-V12 plasmid (constitutively active Ras) together with a control or a Myc-Sprouty4 vector into PC12 cells. After 36 h, cells were serum-starved and the levels of Erk/MAPK activation were evaluated by immunoblot with a specific antibody that recognizes the phosphorylated forms of ERK/MAPK. Reprobing of the same blot with anti-HA, anti-Myc and anti–tubulin antibodies is shown. Fold change relative to the level of constitutive active HA-Ras V12 is indicated.</p

Sprouty4 Y53A mutant loses its ability to block both Erk/MAPK pathway and neurite outgrowth of PC12 cells in response to NGF.

<p>A) Schematic representation of the domain structures of mammalian Sprouty family members. The figure shows an amino acid alignment of a conserved motif located at the N-terminal half of the molecule. There, a conserved tyrosine residue (Y) is indicated in red. The conserved C-terminal cysteine-rich domain is also shown. B) Erk/MAPK activation (P-MAPK) in cell lysates of parental and PC12-Myc-Spry4 Y53A cells treated with NGF (50 ng/ml) and detected by IB with a specific antibody that recognizes the phosphorylated forms of ERK/MAPK. Reprobing of the same blot with anti–tubulin and anti-Myc antibodies is shown. The experiment was repeated two times with similar results. C) Photomicrographs show PC12 cells transfected with control or Myc-tagged Sprouty4 Y53A mutant together with a GFP expression vector. After 72 h of NGF treatment, the cells were fixed and stained with anti-Myc. Arrows indicate neuronal cell bodies and arrowheads denote neurite tips. Scale bar: 10 m. D) The histogram shows the quantification of the relative number of GFP positive PC12 cells bearing neurites longer than 1.5 cell body diameters after 72 h of treatment with NGF. The results are presented as averages SD of a representative experiment performed in triplicate. *p<0.05 (ANOVA followed by Student Newman Keuls). The experiment was repeated three times with similar results.</p

Sprouty4 inhibits neurite outgrowth of dorsal root ganglion neurons (DRG) in response to NGF.

<p>A) Dissociated DRG neurons transfected with GFP in the absence (Control) or in the presence of an excess of Myc-tagged Sprouty4 (Spry4) construct were cultured with NGF (50 ng/ml). After 36 h in culture, neurons were fixed and stained with anti–tubulin antibodies. Scale bar represents 20 m. Arrows indicate neuronal cell bodies and arrowheads denote neurite tips. B) Left panel, histogram showing the inhibition of neurite outgrowth in DRG neurons by exogenous expression of Sprouty4. The results are averages SEM of a representative experiment measured in six wells per experimental group, *, p<0.05 (Student's t test). The experiment was repeated three times with similar results. Right panel, histogram showing the survival of DRG neurons by exogenous expression of Sprouty4. Neuronal survival was evaluated using the nuclear staining DAPI. GFP-positive neurons containing fragmented or condensed nuclear staining were scored as apoptotic cells. The results are averages SEM of a representative experiment performed in triplicate. C) Histogram shows the distribution of neurons carrying neurites in different length categories after transfection with GFP in the absence (Control) or in the presence of Myc-tagged Sprouty4. A total of 43 control- and 40 Sprouty4-transfected neurons from a representative assay were evaluated. Note the noticeable shift to the left of the distribution of neurons that received the Sprouty4 construct.</p

Knockdown of Sprouty4 potentiates MAPK activation and PC12 cell differentiation in response to NGF.

<p>A) <i>Spry4</i> mRNA levels were analyzed by real-time PCR in PC12 cells transfected with scrambled (control, Ctrl) or <i>Spry4</i> shRNA constructs. Transfected cells were enriched by puromycin treatment in order to increase the population of cells expressing scrambled or <i>Spry4</i> shRNA constructs. Quantitative analysis is shown as averages SD of triplicate determinations. The levels of Spry4 mRNA were normalized using the expression of the housekeeping gene <i>Tbp</i>. *p<0.001 (Student's <i>t</i> test). B) Spry4 protein levels were analyzed by IB in Cos cells transfected with <i>Spry4</i> shRNA or a control vector together with Myc-Spry4. Actin is shown as loading control. C) Spry4 knockdown on Erk2/MAPK activation was analyzed in PC12 cells treated with NGF (50 ng/ml) for 10 min. Erk2/MAPK activation was evaluated by transient transfection of HA-tagged Erk2 plasmid with a control or <i>Spry4</i> shRNA construct into PC12 cells. Erk2 activation (P-Erk2) was evaluated by HA-immunoprecipitation (IP) followed by IB with a specific antibody that recognizes the phosphorylated forms of ERK/MAPK. Reprobing of the same blot with anti-HA antibody is shown. Numbers below the lanes are normalized to the levels of HA-Erk2. D) Morphological differentiation of PC12 cells transfected with scramble (Ctrl) or <i>Spry4</i> shRNA cloned in the retroviral vector pGFP-V-RS and treated with NGF (50 ng/ml) for 24 h. E) The histogram shows the quantification of the relative number of GFP positive neurite-bearing cells longer than 1 or 2 cell diameters in the different experimental conditions. The results are shown as averages SD of a representative experiment performed in quadruplicates. *p<0.001 (Student's <i>t</i> test).</p

NGF signaling induces Sprouty4 in neuronal cells.

<p>A) Left panel, quantitative analysis of S<i>prouty4</i> mRNA expression by real-time PCR in PC12 cells treated with NGF (50 ng/ml) during the indicated times. The levels of <i>Sprouty4 (Spry4)</i> mRNA were normalized using the expression of the housekeeping gene <i>Tbp</i> (TATA binding protein). Shown are averages ± SD of triplicate determinations. *p<0.001 versus control (Ctrl) group (one-way ANOVA followed by Dunnett's test). Right panel, expression of <i>Spry1</i> and <i>Spry2</i> mRNAs examined by RT-PCR (35 cycles) in DRG and PC12 cells treated with NGF (50 ng/ml) for different time-points. Expression of the housekeeping gene <i>Tbp</i> was used as loading control. The experiment was repeated two times with similar results. B) Western blot analysis of Sprouty4 expression in PC12 cells treated with NGF (50 ng/ml). Reprobing control was done with antibodies against -tubulin. Fold change relative to -tubulin is indicated. C) Left panel, expression of <i>Spry</i>4 mRNA examined by semiquantitative RT-PCR (27 cycles) in PC12 cells treated with the specific MEK inhibitor PD98059 (50 M) and stimulated with NGF (50 ng/ml) as indicated. Expression of the housekeeping gene <i>Tbp</i> was used as loading control. Right panel shows a control experiment performed in parallel to verify the inhibitory activity of PD98059 on MAPK pathway. PD98059 activity was controlled measuring MAPK activation by immunoblotting of PC12 cells stimulated for 10 min with NGF (50 ng/ml). Reprobing control was done with antibodies against -tubulin. D) Colocalization of Sprouty4 and TrkA in DRG dissociated neurons obtained from E14.5 rat embryos detected by immunofluorescence. Scale bars: 10 m.E) Quantitative analysis of S<i>pry1,</i> S<i>pry2</i> and S<i>pry4</i> mRNA expression by real-time PCR in DRG neurons, treated with NGF (50 ng/ml) during the indicated times. The <i>Sproutys</i> mRNA levels were normalized using the expression of the housekeeping gene <i>Tbp</i>. Shown are averages ± SD of triplicate determinations. *p<0.01 versus control (Ctrl) group (one-way ANOVA followed by Dunnett's test).</p

High Plasticity of New Granule Cells in the Aging Hippocampus

Summary: During aging, the brain undergoes changes that impair cognitive capacity and circuit plasticity, including a marked decrease in production of adult-born hippocampal neurons. It is unclear whether development and integration of those new neurons are also affected by age. Here, we show that adult-born granule cells (GCs) in aging mice are scarce and exhibit slow development, but they display a remarkable potential for structural plasticity. Retrovirally labeled 3-week-old GCs in middle-aged mice were small, underdeveloped, and disconnected. Neuronal development and integration were accelerated by voluntary exercise or environmental enrichment. Similar effects were observed via knockdown of Lrig1, an endogenous negative modulator of neurotrophin receptors. Consistently, blocking neurotrophin signaling by Lrig1 overexpression abolished the positive effects of exercise. These results demonstrate an unparalleled degree of plasticity in the aging brain mediated by neurotrophins, whereby new GCs remain immature until becoming rapidly recruited to the network by activity. : Trinchero et al. show that development of new granule cells born in the adult hippocampus is strongly influenced by age. In the aging hippocampus, new neurons remain immature for prolonged intervals, yet voluntary exercise triggers their rapid growth and functional synaptogenesis. This extensive structural remodeling is mediated by neurotrophins. Keywords: adult neurogenesis, dentate gyrus, functional integration, neurotrophins, synaptogenesis, exercis