Characterization of Voltage-Gated Ca2+ Conductances in Layer 5 Neocortical Pyramidal Neurons from Rats

Neuronal voltage-gated Ca2+ channels are involved in electrical signalling and in converting these signals into cytoplasmic calcium changes. One important function of voltage-gated Ca2+ channels is generating regenerative dendritic Ca2+ spikes. However, the Ca2+ dependent mechanisms used to create these spikes are only partially understood. To start investigating this mechanism, we set out to kinetically and pharmacologically identify the sub-types of somatic voltage-gated Ca2+ channels in pyramidal neurons from layer 5 of rat somatosensory cortex, using the nucleated configuration of the patch-clamp technique. The activation kinetics of the total Ba2+ current revealed conductance activation only at medium and high voltages suggesting that T-type calcium channels were not present in the patches. Steady-state inactivation protocols in combination with pharmacology revealed the expression of R-type channels. Furthermore, pharmacological experiments identified 5 voltage-gated Ca2+ channel sub-types – L-, N-, R- and P/Q-type. Finally, the activation of the Ca2+ conductances was examined using physiologically derived voltage-clamp protocols including a calcium spike protocol and a mock back-propagating action potential (mBPAP) protocol. These experiments enable us to suggest the possible contribution of the five Ca2+ channel sub-types to Ca2+ current flow during activation under physiological conditions.

[1]  D. Linden,et al.  Regulation of the rebound depolarization and spontaneous firing patterns of deep nuclear neurons in slices of rat cerebellum. , 1999, Journal of neurophysiology.

[2]  G. Dayanithi,et al.  Calcium channel subtypes responsible for voltage-gated intracellular calcium elevations in embryonic rat motoneurons , 1998, Neuroscience.

[3]  H. Reuter.  Calcium channel modulation by neurotransmitters, enzymes and drugs , 1983, Nature.

[4]  P. Mermelstein,et al.  Properties of Q-Type Calcium Channels in Neostriatal and Cortical Neurons are Correlated with β Subunit Expression , 1999, The Journal of Neuroscience.

[5]  D. DiFrancesco,et al.  Action of the hyperpolarization-activated current (Ih) blocker ZD 7288 in hippocampal CA1 neurons , 1997, Pflügers Archiv.

[6]  Huxley Af,et al.  A quantitative description of membrane current and its application to conduction and excitation in nerve. 1952. , 1990 .

[7]  T. Snutch,et al.  Decay of prepulse facilitation of N type calcium channels during G protein inhibition is consistent with binding of a single Gbeta subunit. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[8]  P. J. Sjöström,et al.  Dendritic excitability and synaptic plasticity. , 2008, Physiological reviews.

[9]  R C Foehring,et al.  Characterization of pharmacologically identified voltage-gated calcium channel currents in acutely isolated rat neocortical neurons. II. Postnatal development. , 1995, Journal of neurophysiology.

[10]  S. Ikeda.  Double‐pulse calcium channel current facilitation in adult rat sympathetic neurones. , 1991, The Journal of physiology.

[11]  C. Hidalgo,et al.  Calcium microdomains and gene expression in neurons and skeletal muscle cells. , 2006, Cell calcium.

[12]  P. Jonas,et al.  Differential Gating and Recruitment of P/Q-, N-, and R-Type Ca2+ Channels in Hippocampal Mossy Fiber Boutons , 2007, The Journal of Neuroscience.

[13]  G. Wang,et al.  An R-Type Ca2+ Current in Neurohypophysial Terminals Preferentially Regulates Oxytocin Secretion , 1999, The Journal of Neuroscience.

[14]  B. Gähwiler,et al.  L-Type Ca2+ channels mediate the slow Ca2+-dependent afterhyperpolarization current in rat CA3 pyramidal cells in vitro. , 1998, Journal of neurophysiology.

[15]  R. Tsien,et al.  Contrasting biophysical and pharmacological properties of T-type and R-type calcium channels , 1997, Neuropharmacology.

[16]  P. Kostyuk,et al.  Developmental changes in the expression of low‐voltage‐activated Ca2+ channels in rat visual cortical neurones , 1998, The Journal of physiology.

[17]  E. Perez-Reyes,et al.  State-Dependent Inactivation of the α1g T-Type Calcium Channel , 1999, The Journal of general physiology.

[18]  D. Johnston,et al.  Characterization of single voltage‐gated Na+ and Ca2+ channels in apical dendrites of rat CA1 pyramidal neurons. , 1995, The Journal of physiology.

[19]  D. H. Cox,et al.  Pharmacological discrimination of N-type from L-type calcium current and its selective modulation by transmitters , 1992, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[20]  N. Spruston.  Pyramidal neurons: dendritic structure and synaptic integration , 2008, Nature Reviews Neuroscience.

[21]  Shigeo Watanabe,et al.  Synaptically Activated Ca2+ Waves in Layer 2/3 and Layer 5 Rat Neocortical Pyramidal Neurons , 2003, The Journal of physiology.

[22]  H. Markram,et al.  Dendritic calcium transients evoked by single back‐propagating action potentials in rat neocortical pyramidal neurons. , 1995, The Journal of physiology.

[23]  E Neher,et al.  Sodium and calcium channels in bovine chromaffin cells , 1982, The Journal of physiology.

[24]  B Sakmann,et al.  R-type Ca2+ currents evoke transmitter release at a rat central synapse. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[25]  Angus M. Brown,et al.  Voltage dependence and activation kinetics of pharmacologically defined components of the high-threshold calcium current in rat neocortical neurons. , 1993, Journal of neurophysiology.

[26]  R. Tsien,et al.  Molecular diversity of voltage-dependent Ca2+ channels. , 1991, Trends in pharmacological sciences.

[27]  R. Tsien,et al.  Multiple types of neuronal calcium channels and their selective modulation , 1988, Trends in Neurosciences.

[28]  B. Sakmann,et al.  Patch-clamp recordings from the soma and dendrites of neurons in brain slices using infrared video microscopy , 1993, Pflügers Archiv.

[29]  A. Hodgkin,et al.  A quantitative description of membrane current and its application to conduction and excitation in nerve , 1952, The Journal of physiology.

[30]  G. Dayanithi,et al.  Interaction of SNX482 with Domains III and IV Inhibits Activation Gating of α1E (CaV2.3) Calcium Channels , 2001 .

[31]  William A. Horne,et al.  The naming of voltage-gated calcium channels , 1994, Neuron.

[32]  B. Sakmann,et al.  Voltage‐gated K+ channels in layer 5 neocortical pyramidal neurones from young rats: subtypes and gradients , 2000, The Journal of physiology.

[33]  B. Sakmann,et al.  Action potential initiation and propagation in rat neocortical pyramidal neurons , 1997, The Journal of physiology.

[34]  N. Akaike,et al.  Calcium currents in pyramidal neurons acutely dissociated from the rat frontal cortex: a study by the nystatin perforated patch technique , 1993, Brain Research.

[35]  J. Bargas,et al.  Cellular and molecular characterization of Ca2+ currents in acutely isolated, adult rat neostriatal neurons , 1994, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[36]  G. Stuart,et al.  Action Potential Initiation and Propagation in Layer 5 Pyramidal Neurons of the Rat Prefrontal Cortex: Absence of Dopamine Modulation , 2003, The Journal of Neuroscience.

[37]  B. Sakmann,et al.  Active propagation of somatic action potentials into neocortical pyramidal cell dendrites , 1994, Nature.

[38]  J. Roder,et al.  Neuronal calcium sensor 1 and phosphatidylinositol 4-OH kinase β interact in neuronal cells and are translocated to membranes during nucleotide-evoked exocytosis , 2002, Journal of Cell Science.

[39]  R.,et al.  Low-Threshold Calcium Currents in Central Nervous System Neurons , 2003 .

[40]  B Sakmann,et al.  Spatial profile of dendritic calcium transients evoked by action potentials in rat neocortical pyramidal neurones. , 1995, The Journal of physiology.

[41]  Otto D. Creutzfeldt,et al.  Generality of the functional structure of the neocortex , 1977, Naturwissenschaften.

[42]  K Takahashi,et al.  Kinetic properties of T-type Ca2+ currents in isolated rat hippocampal CA1 pyramidal neurons. , 1991, Journal of neurophysiology.

[43]  Alon Korngreen,et al.  Dendritic voltage‐gated K+ conductance gradient in pyramidal neurones of neocortical layer 5B from rats , 2007, The Journal of physiology.

[44]  R. Tsien,et al.  Distinctive pharmacology and kinetics of cloned neuronal Ca2+ channels and their possible counterparts in mammalian CNS neurons , 1993, Neuropharmacology.

[45]  G. Dayanithi,et al.  Interaction of SNX482 with domains III and IV inhibits activation gating of alpha(1E) (Ca(V)2.3) calcium channels. , 2001, Biophysical journal.

[46]  P. Ascher,et al.  Activation and desensitization of N‐methyl‐D‐aspartate receptors in nucleated outside‐out patches from mouse neurones. , 1992, The Journal of physiology.

[47]  J. Zhu,et al.  Maturation of layer 5 neocortical pyramidal neurons: amplifying salient layer 1 and layer 4 inputs by Ca2+ action potentials in adult rat tuft dendrites , 2000, The Journal of physiology.

[48]  A. Ricci,et al.  Biophysical and Pharmacological Characterization of Voltage‐Gated Calcium Currents in Turtle Auditory Hair Cells , 2003, The Journal of physiology.

[49]  L. Jan,et al.  The distribution and targeting of neuronal voltage-gated ion channels , 2006, Nature Reviews Neuroscience.

[50]  E. Neher.  Correction for liquid junction potentials in patch clamp experiments. , 1992, Methods in enzymology.

[51]  J. Ting,et al.  Neurotransmitter : Release , 2008 .

[52]  B Sakmann,et al.  Calcium Channel Types with Distinct Presynaptic Localization Couple Differentially to Transmitter Release in Single Calyx-Type Synapses , 1999, The Journal of Neuroscience.

[53]  B. Sakmann,et al.  Dendritic mechanisms underlying the coupling of the dendritic with the axonal action potential initiation zone of adult rat layer 5 pyramidal neurons , 2001, The Journal of physiology.

[54]  R. J. Miller,et al.  Multiple calcium channels and neuronal function. , 1987, Science.

[55]  R. Foehring,et al.  Characterization of pharmacologically identified voltage-gated calcium channel currents in acutely isolated rat neocortical neurons. I. Adult neurons. , 1995, Journal of neurophysiology.

[56]  B. Sakmann,et al.  Calcium electrogenesis in distal apical dendrites of layer 5 pyramidal cells at a critical frequency of back-propagating action potentials. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[57]  Stuart L. Johnson,et al.  Biophysical properties of CaV1.3 calcium channels in gerbil inner hair cells , 2008, The Journal of physiology.

[58]  N. Akaike,et al.  Calcium antagonist effects on low-threshold (T-type) calcium current in rat isolated hippocampal CA1 pyramidal neurons. , 1991, The Journal of pharmacology and experimental therapeutics.