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R. Grantyn Dept. Sensory and Developmental Physiology, Institute of Neurophysiology, Charité Berlin JOHANNES-MÜLLER-INSTITUT FÜR PHYSIOLOGIE Mainz GRK 2005 Development of synaptic inhibition in cortical structures: Focus on mechanisms regulating the E/I balance of synaptic input The group in Berlin Christian Henneberger Jochen Meier Bhumika Singh Our collaborators in Madrid Rubenstein & Merzenich* (2003): More excitable (more weekly inhibited) cortex is, by its nature, more poorly functionally differentiated; this type of cortex will lead to broad-ranging abnormalities in perception, memory and cognition, and motor control. Moreover ‘noisy‘ (hyperexcitable, poorly functionally differentiated) cortex is inherently unstable, and susceptible to epilepsy. A short list of diseases associated with a disturbed balance between E and I: Epilepsy Nonsyndromic mental retardation Autism Rett syndrom *Dept. Physiology and Otolaryngology, University of California at San Francisco Does inhibition balance excitation in the cerebral cortex? Typical compartmental model to study synaptic actions Synaptic conductance as Σgtotal Impact of synaptic inhibition From: Trevelyan, Watkinson (2005) Prog Biophys Mol Biol 87:109 Hippocampal cultures An I synaptic input cancels an E input in accordance with its time cource (τD 15-25 ms) and a space constant λ of 10 µm From: Liu (2004) Nat Neurosci 7:373 Cerebral cortical cultures The hypothesis of ‘homeostatic plasticity‘ of synaptic transmission in the cerbral cortex From: Turrigiano, Nelson (2004) Nat Rev Neurosci 5:97 Main hypothesis: The number/activity of E synapses always matches the number/activity of I synapses Co-scaling model E I Control E/I 2:1 Enhanced E input leads to enhanced I input E/I remains 2:1 Our main question: Which signals determine the ratio of E/I input on the level of single cortical neurons developing under definded growth conditions? Hippocampal cultures The E/I ratio of synaptic terminals received by a given neuron is determined on the basis of transfection-labeling and immunostaining EGFP EGFP E/I ratio: typically 2:1 IPSC τD=17.2 ms Probability of PSC occurrence The E/I ratio of synaptic input can be determined on the basis of the probability of occurence of an PSC with a certain decay kinetics E/I ratio: typically 3:1 EPSC 0.16 AMPA 0.08 GABA 0.00 0 1 log τD 2 Hippocampal neurons Is the relationship between E and I synapses stable throughout a population of neurons? From: Salama-Cohen, Arevalo, Grantyn, Rodríguez-Tébar (2005) submitted Cultured hippocampal neurons Is the relationship between E and I synapses stable over the length of a dendrite? From: Liu (2004) Nature Neurosci 7:373 Postsynaptic adjustment of E synapses to altered network activity Cerebral cortical cultures GluR-IR GFP-IR From: Wierenga, Ibata, Turrigiano (2005) J Neurosci 25:2895 Block of GABAARs stimulates the formation of inhibitory synapses in situ After 2.5 h in ACSF+BMI 50 µM After 2.5 hr in ACSF P1 superior colliculus slices Immediately fixed O hr From: Meier, Akyeli, Kirischuk, Grantyn (2003) Mol Cell Neurosci 23:600 The induction of new inhibitory synaptic terminals requires the activation of PKC ** *** 20 0 80 - superior colliculus - superficial layers only - P1 WT mouse - tangential slices - submerged at 25 oC - images at 100x 60 40 20 0 0h ACSF PKC block PDBu 40 ** * 100 VIAAT+ GAD65+ (%) 60 0h ACSF PKC block PDBu VIAAT / VF 80 ** *** ****** From: Meier, Akyeli, Kirischuk, Grantyn (2003) Mol Cell Neurosci 23:600 Elimination of the PKC phosphorylation site Ser343 impairs postsynaptic accumulation of GFP::γ2L GABAAR γ2L contains an additional sequence of 8 amino acids, among them the phosphorylation site Ser343 PDBu Meier & Grantyn (2004) J Physiol 559:355 As a rule of thumb: Conditions promoting postsynaptic GABAAR receptor accumulation also increase the number of inhibitory terminals Low GABAAR activity (comp. antag.) Low level of group I mGluR activity Block of Ca influx Activation of Ca-dep PKC activity Syp VIAAT Syp VIAAT GAD65 GABAAR High GABAAR activity (benzodiazepines) High level of group I mGluR activity Permanently elevated ic Ca Depletion of Ca-dep PKC activity Syp VIAAT GAD65 Block of repeatedly elevated local Ca transients promotes filopodial growth O hr From: Lohmann, Finski, Bonhoeffer (2005) Nat Neurosci 8:305 Does the number of newly formed synapses correlate with dendrite number or branching? O hr P<0.01 Hippocampal neurons Number of Syp+ terminals 30 20 10 0 0 10 20 30 Number of branch points From: Singh, Henneberger, Meier, Rodríguez-Tébar, Grantyn (2005) submitted 40 Long ago, abnormal dendrite growth has been considered to be a likely cause of mental retardation (MR) O hr Dendritic spine abnormalities in Golgi-stained cerebral cortex of normal (a) and MR (b) infant at 10 month From: Purpura (1972) Science 186:1126 The more recent discovery that single gene defects on the X chromosome affect the expression of rho GTPase-regulating proteins and produce nonsyndromic MR (NS-MR) has revived the idea that some forms of MR are primarily caused by abnormal dendrite structure The OPHN1-defective young male displays MR, reduced visual acuity and congenital strabism; the mother had neither learning disabilities, nor visual or oculomotor deficits The cause: genomic deletion of exon 19 causing a frameshift at the site Xq12; OPHN1 is proposed to act as inhibitor of rhoA and might be necessary for the maintenance of spine motility, at least during some critical steps in the development of visuo-motor behavior From Bergmann, ...& Ramaekers (2003) Brain 126:1537 The XL-MR genes ARHGEF6 and OPHN1 are likely to be involved in the control of dendrite initiation an spine motility, respectively XLMR genes Rho family GTPases Effector Cytoskeleton Dendrite growth Predicted deficits in dendrite growth ARHGEF6 (αPIX) CDc42 ? Rac1 OPHN1 RhoA GTPase Pak3 Actin filament assembly Dendrite initiation ARHGEF6-/-: Reduced dendritic branching Actin filament extension Actomyosin contraction Dendrite elongation Spine motility ZZ-/-: reduced dendrite elongation OPHN1-/-: Increased threshold for spine plasticity Overexpressing or silencing NS-XLMR genes may allow one to manipulate dendrite initiation, dendrite elongation or spine motility, and to test for changes in synapse formation OPHN1::DsRedE VGluT VIAAT 15 µm From: Meier, Grantyn, unpubl. Overexpression of OPHN1 promotes both the formation of new primary dendrites and the formation of glutamatergic synapses From: Meier, Grantyn, unpubl. In search for genes able to alter dendrite outgrowth and E/I relationships we also considered the group of ‘proneural‘ genes O hr (Ngn3?) Ngn1/2 Neurogenin3 Mash1 From: Bertrand, Castro, Guellemot (2002) Nature Rev Neurosci 3:517 Overexpression of Ngn3 stimulates the outgrowth of new dendrites O EGFP hr From: Sallama-Cohen, Arevalo, Grantyn, Rodríguez-Tébar (2005) submitted MAP2 Overexpression of Ngn3 increases the E/I ratio by suppressing the formation of inhibitory synapses From: Salama-Cohen, Arevalo, Grantyn, Rodríguez-Tébar (2005) submitted Thus, OPHN1 and Ngn3 are potentially dangerous genes... Reverse scaling model Control E/I 2:1 Disinhibition of both OPHN1 and Ngn3 E/I 8:1 If short-term+local: Activity-dependent synapse reorganization (Hepp-type plasticity) If long-term+global: States of overexcitation (Brain pathology) What controls the expression/activity of these genes? Our experiments suggest a new major role of the neurotrophin NGF From: Salama-Cohen, Arevalo, Grantyn, Rodríguez-Tébar (2005) submitted Treatment of hippocampal neurons with nerve growth (NGF) factor strongly promotes inhibitory synaptogenesis (It also suppresses the outgrowth of new dendrites, not shown) From: Salama-Cohen, Arevalo, Grantyn, Rodríguez-Tébar (2005) submitted Tentative scheme on the role of NGF in the control of the E/I balance in hippocampal neurons Do all members of the neurotrophin family reduce E/I? The I-promoting effect of NGF is new and surprizing, considering that Brain-derived neurotrophic factor (BDNF) is well known for its epileptogenic action. BDNF promotes LTP of glutamatergic synapses and causes a postsynaptic depression of GABAergic inhibition... bdnf-/- P12 50 pA 50 ms P15 ** eIPSC amplitude (pA) 200 160 120 80 40 0 From: ** ** +/+ -/P12 +/+ -/- -/P15 +BDNF Henneberger, Jüttner, Rothe, Grantyn (2002) J Neurophysiol 88:595 Henneberger,...Grantyn (2005) Eur J Neurosci 21:431 How does the presence of BDNF in a postsynaptic neuron affect the formation/stabilization of E vs. I synapses? Control EGFP VGluT tBDNF VIAAT From: Singh, Henneberger, Meier, Rodríguez-Tébar, Grantyn (2005) submitted BDNF shifts the E/I balance towards excitation. A BDNF-expressing neuron attracts a larger number of E synapses than a neuron free of BDNF Control is bdnf-/- From: Singh, Henneberger, Meier, Rodríguez-Tébar, Grantyn (2005) submitted Thus, NGF and BDNF are antagonists with respect to the E/I ratios (and dendrite outgrowth, not shown) The reverse scaling model Control E/I 1.5:1-2:1 Exogenous NGF E/I~0.75 tBDNF, exogenous BDNF E/I ~5:1