Areas of Research

In the institute of Prof. Heiko J. Luhmann I investigate the cellular physiology of the immature cerebral cortex and the role of developmental disorders in the generation of neuronal pathologies. I'm also interested in the role of early network activity in the development and refinement of cortical structures and connections. In addition, I investigated the effects of extra- und intracellular pH on cellular properties and synaptic transmission. To adress this questions I mainly use electrophysiological, but also micro- fluorimetric, immunohistochemical and molecular methods.	Dr. Werner Kilb
Cellular physiology of the immature cortex
Morphology of different neuronal cell types in immature cortex. DIC-immage of a Cajal-Retzius cell (A), an undifferentiated (B) and a differentiated (C) pyramidal neuron and of a subplate neuron (D)(from: Luhmann et al. 2000).	Cortical neurons were generated in the ventricular zone and reach their final position by radially migrating along processes of radial glial cells. When the migrating neurons reach the superficial marginal zone, they detach from radial glial cells, stop their migration and start to differentiate. In addition to these precursors of projecting and interneurons, transient cell populations are present in the developing cerebral cortex. Cajal-Retzius cells are located in the marginal zone and play an important role in the termination of neuronal migration. Subplate neurons are located at the border between the corticals layers and white matter and play an important role in the formation of corticopetal and corticofugal connections.
Main focus of our investigations are the electrophysiological properties of those transient cell types. In particular, we are interested which neurotransmitter receptors are functionally expressed on these neurons and whether they are integrated in immature cortical networks. In addition, we examine how neuronal differentiation affects the functional properties of neurons.	Membrane responses of an immature pyramidal neuron (left) and a Cajal-Retzius cell (right)upon de- und hyperpolarizing current injections (from: Okabe et al. 2004).
Glycin induces a membrane depolarization in Cajal-Retzius cells, but inhibits the generation of action potentials (from: Kilb et al., 2003).	The central issue of my investigations are the mechanism and fuctional consequences on the increased intracellular Cl^- concentration in immature neurons. In contrast to the hyperpolarizing responses of GABA and glycine in mature neurons, both neurotransmitters evoke depolarizing responses in immature neurons. This membrane depolarization probably reflects a Cl^- efflux due to an elevated intracellular Cl^- concentration. Thus we investigate which transporters contribute to the active Cl^- accumulation and how this transport processes are regulated. In addition, we analyze whether this membrane depolarization mediates inhibition or excitation in the immature nervous system.
*Related publications:* Kilb, W., Hanganu, I.L., Okabe, A., Sava, B.A., Shimizu-Okabe, C., Fukuda, A., Luhmann, H.J. (2008) Glycine Receptors Mediate Excitation of Subplate Neurons in Neonatal Rat Cerebral Cortex. J. Neurophysiol Epub ahead of Print Shimizu-Okabe, C., Okabe, A., Kilb, W., Sato, K., Luhmann, H.J. & Fukuda, A. (2007) Changes in the expression of cation-Cl - cotransporters, NKCC1 and KCC2, during cortical malformation induced by neonatal freeze-lesion. Neuroscience Research 59: 288-295 Achilles K., Okabe A., Ikeda M., Shimizu-Okabe C., Yamada J., Fukuda A., Luhmann H.J. & Kilb W.(2007) Kinetic properties of Cl^- uptake mediated by Na⁺-dependent K⁺-2Cl^--cotransport in immature rat neocortical neurons J. Neurosci. 27:8616-8627 Yamada, J., Okabe, A., Toyoda, H., Kilb, W., Luhmann, H.J. & Fukuda, A. (2004) Cl- uptake promoting depolarizing GABA actions in immature rat neocortical neurones is mediated by NKCC1. J. Physiol. (Lond.) 557: 829-841. #105 Okabe, A., Kilb, W., Shimizu, C., Hanganu, I.L., Fukuda, A., Luhmann, H.J. (2004) Homogenous glycine receptor expression in cortical plate neurons and Cajal-Retzius cells of neonatal rat cerebral cortex. Neuroscience 123: 715-724. # 100 Shimizu-Okabe, C., Yokokura, M., Okabe, A., Ikeda, M., Sato, K., Kilb, W. Luhmann, H.J., Fukuda, A. (2002) Layer-specific expression of Cl- transporters and differential [Cl-]i in newborn rat cortex. NeuroReport 13: 2433-2437. #089 Hanganu, I.L., Kilb, W. & Luhmann, H.J. (2002) Functional synaptic projections onto subplate neurons in neonatal rat somatosensory cortex J. Neurosci. 22:7165-76. # 087 Kilb, W., Ikeda, M., Uchida, K., Okabe, A., Fukuda, A. & Luhmann, H.J. (2002) Depolarizing glycine responses in Cajal-Retzius cells of neonatal rat cerebral cortex Neuroscience 112: 299-307. # 085 Luhmann, H.J., Reiprich, R.A., Hanganu, IL., Kilb, W. (2000) Cellular physiology of the neonatal rat cerebral cortex: intrinsic membrane properties, sodium and calcium currents J Neurosci Res.62: 574-584. # 070
Pathophysiology of the immature cortex
In contrast to control neurons (A), an altered morphology and enhanced synaptic inputs were observed in neurones of a cortex in which neuronal migration was affected by the NMDA-antagonist MK-801 (B) (from: Reiprich et al. 2004).	A variety of neuronal disorders are caused by structural alterations in the cerebral cortex, which may originate from a disturbed neuronal development. To elucidate the mechanisms of these kind of deficits we focally induce migration disordes and investigate the resulting changes in functional and morphological properties of single cells and cortical structures.
In addition some neurological diseases, like e.g. epilepsia, have higher incidence rates during early childhood. To understand the mechanisms and conditions that contribute to such developmental differences, we investigate epileptiform activity in an in-vitro model and examine whether the conditions leading to this activity and their interference with antiepileptic substances differs between adult and immature nervous systems.	The GABA_A Antagonist pircotoxin (PTX) induces ictal like epileptiform activity, although GABA mediates depolarizing membrane responses during this period.
*Related publications:* Kilb W., Sinning A. & Luhmann, H.J. (2007) Model-specific effects of bumetanide on epileptiform activity in the invitro intact hippocampus of the newborn mouse Neuropharmacology 53: 524-33 Kilb W, Dierkes PW, Sykova E, Vargova L, Luhmann HJ (2006) Hypoosmolar conditions reduce extracellular volume fraction and enhance epileptiform activity in the CA3 region of the immature rat hippocampus. J Neurosci Res. #128 Moser J, Kilb W, Werhahn KJ, Luhmann HJ (2006) Early developmental alterations of low-Mg2+ -induced epileptiform activity in the intact corticohippocampal formation of the newborn mouse in vitro. Brain Res 1077: 170-177. #126 Heck N, Kilb W, Reiprich P, Kubota H, Furukawa T, Fukuda A, Luhmann HJ (2006) GABA-A Receptors Regulate Neocortical Neuronal Migration In Vitro and In Vivo. Cereb Cortex. #125 Karl, C., Couillard-Despres, S., Prang, P., Munding, M., Kilb, W., Brigadski, T., Plötz, S., Mages, W., Luhmann, H.J., Winkler, J., Bogdahn, U. & Aigner, L. (2005) Neuronal precursor specific activity of a human doublecortin regulatory sequence. J. Neurochem., 92:264-282. #112 Reiprich, P., Kilb, W. & Luhmann, H.J. (2004) Neonatal NMDA receptor blockade disturbs neuronal migration in rat somatosensory cortex in vivo. Cerebral Cortex, in press. #106
Network activity in the immature cortex
The development of the cerebral cortex is guverned not only by internal genetic programs, but spontaeous synaptic activity substantially contributes to the functional maturation of this structure. To investigate the influence of early synaptic activity of the maturation of the cortex, we characterized spontaneous synaptic activity and neurotransmitter-triggered network oscillations in the immature cortex.	Field potential recording (A) und spectrogram (B) of a carbachol-induced oscillation in a whole-cortex preparation of immature brain (from: Kilb & Luhmann, 2003).
*Related publications:* Dupont E, Hanganu IL, Kilb W, Hirsch S, Luhmann HJ (2006) Rapid developmental switch in the mechanisms driving early cortical columnar networks. Nature 439: 79-83. #123 Kilb, W. & Luhmann, H.J. (2003) Carbachol-induced Network Oscillations in the Intact Cerebral Cortex of the Newborn Rat Cereb. Cortex 13: 409-421. # 091 Kilb, W. & Luhmann, H.J. (2001) Spontaneous GABAergic postsynaptic currents in Cajal-Retzius cells in neonatal rat cerebral cortex Eur. J. Neurosci. 13: 1387-1390. # 080 Hanganu, IL., Kilb, W. & Luhmann, H.J. (2001) Spontaneous synaptic activity of subplate neurons in neonatal rat somatosensory cortex Cerebral Cortex 11: 400-410. # 079
Effect of intra- and extracellular pH
Many physiological processes, like electrical activity or synaptic transmission, as well as pathophysiological insults, like ischemia or epileptic seizures, induce rather massive changes in extra- and intracellular pH. To elucidate if these pH changes interfere with CNS function, I investigated the effect of extra- and inctracellular pH-changes on cellular properties and synaptic transmission in the medicinal leech (Hirudo medicinalis).	The glutamatergic agonist kainate induced membrane depolarization, a Na⁺ influx and intracellular acidification (from: Kilb & Schlue, 1999).
*Related Publications:* Kilb W, Schlue WR (2001) Feedback control of intracellular pH by means of iontophoretic H+/OH- injection. Pflügers Arch 443: 54-60. # 073 Hochstrate P, Dierkes PW, Kilb W, Schlue WR (2001) Modulation of Ca2+ influx in leech Retzius neurons. I. Effect of extracellular pH. J Membr Biol 184: 13-25. # 072 Hochstrate P, Dierkes PW, Kilb W, Schlue WR (2001) Modulation of Ca2+ influx in leech Retzius neurons II. Effect of extracellular Ca2+. J Membr Biol 184: 27-33. # 071 Kilb W, Schlue WR (1999) Mechanism of the kainate-induced intracellular acidification in leech Retzius neurons. Brain Res 824: 168-182. # 055