Scientific experience : Activity patterns and synaptic plasticity in the developing and adult cortical networks

             Cortical networks, including the hippocampal circuit, often express spontaneous, physiological  patterns of activity which strongly influence neuronal synchonisation. The relative timing of neuronal discharges is a critical factor for the induction of activity-dependent synaptic plasticity, that may support cognitive functions such as learning and memory in the adult, circuit maturation during the early stages of development, or even cell death during pathological synchronisations. GABAergic inhibition mediated by various categories of interneurons with both divergent and dense axonal projections plays a central role in the control of neuronal synchronisation and global excitability of the network. Using various electrophysiological (sharp electrodes, patch clamp, extracellular unit and multi-unit recordings using simple of more elaborate multi-site recording probes) and imaging (confocal Ca2+ imaging) techniques in rodent preparations (slices, intact neonatal septo-hippocampal complex in vitro, anesthetized and freely moving animals), we investigate the expression, mechanisms of generation and consequences of spontaneous patterns of activity expressed by cortical structures in various behavioral situations in the adult and neonatal rat, with a particular emphasis on the interactions between GABAergic and glutamatergic neuronal circuits.  

1) Activity patterns in the immature rat hippocampal network in vitro (PhD : development, slices; with Vadim Tseeb and Rustem Khazipov (PhD supervisor) in Ben-Ari's lab)

             During my PhD, I have used a combination of imaging (confocal microscopy with the fluorescent Ca2+ sensors Fluo3 and Fluo3-AM) and electrophysiological (patch clamp in whole cell and cell attached configurations) techniques and observed that during the first postnatal week (P0-5), rat hippocampal neurons displayed synchronous intracellular Ca2+ oscillations mediated by synaptically generated Giant Depolarising Potentials (GDPs). In fact, we have shown that interactions between immature neurons had very different properties than in adult: 1) GABA, which is the principal inhibitory transmitter of the adult brain, has excitatory effects in neonatal neurons due to a reverse Cl- gradient, and facilitates the activation of NMDA receptors at a time when excitatory transmission via AMPA receptors is quiescent; 2) this GABA-NMDA synergy provides simultaneous presynaptic firing, postsynaptic Ca increase and activation of NMDA receptors during Giant Depolarising Potentials (GDPs) that are spontaneously expressed by the immature hippocampal network and could therefore modulate the formation of the neuronal network by activity dependent synaptic plasticity (hebbian model).

 Publications :

- Leinekugel X., Tseeb V., Ben-Ari Y. & Bregestovski Y. (1995): Synaptic GABAA activation induces Ca2+ rise in pyramidal cells and interneurons from rat neonatal hippocampal slices. Journal of Physiology (London) 487, p. 319-329 Leinekugel-et-al-J-Physiol-1995.pdf

- Leinekugel X., Medina I., Khalilov I., Ben-Ari Y. & Khazipov R. (1997): Ca2+ oscillations mediated by the synergistic excitatory actions of GABAA and NMDA receptors in the neonatal hippocampus, Neuron 18, p. 243-255 Leinekugel-et-al-neuron-1997.pdf

- Khazipov R., Leinekugel X., Khalilov I., Jean-Luc Gaļarsa & Ben-Ari Y. (1997): Synchronization of GABAergic interneuronal network in CA3 region of neonatal hippocampus. Journal of Physiology (London) 498.3, p. 763-772 Khazipov-et-al-J-Physiol-1997.pdf

  

2) Activity patterns in intact immature cortical networks, in vitro and in vivo (postdoc : development, intact preparations; with Ilgam Khalilov, Rustem Khazipov and Gyorgy Buzsaki in Ben-Ari's lab, with Rustem Khazipov, Anton Sirota and Gyorgy Buzsaki in Buzsaki-lab)

             In order to study the generation and propagation of GDPs in intact networks, we developped in Dr. Ben-Ari's laboratory a new preparation that allows to record neuronal activity from intact cortical structures in vitro. Using multiple simultaneous electrophysiological recordings on this preparation (single or double patch clamp combined to 4 or 5 extracellular electrodes), I found that spontaneous Giant Depolarizing Potentials (GDPs) are present and propagate within the intact septo-hippocampal complex (both hippocampi and septum functionally connected) in vitro. In fact, the septal pole of hippocampus acts as a pace maker for the generation of GDPs that subsequently propagate towards both septal poles and medial septum. Therefore, the maturation of these two networks that form a functional system in the adult brain may be modulated by a common pattern of activity.

             Moreover, during my postdoc in Pr. Buzsaki laboratory (Rutgers University, Newark, NJ, USA), I used multi-extracellular and patch clamp recordings from freely behaving and anesthetized 3-6 day-old pups, which allowed us to describe recurrent collective bursts of action potentials, reminiscent of both adult sharp waves and immature in vitro giant depolarizing potentials. Since these synchronous events provide synchronized pre- and postsynaptic firing at the CA3-CA1 synapse, they may contribute to the maturation and maintenance of cortical circuits in the newborn rat by Hebbian modulation of developing synapses.

             After my return to France with a permanent research position, I have continued collaboration with Pr. Buzsaki and R. Khazipov (INMED, Marseille, France) for the study of neuronal activity in immature cortical structures. Recording spontaneous movements together with cortical multi-unit and intracellular activity in neonatal rat somato-sensory S1 cortex, we observed that sensory feedback signals generated by endogenous spinal cord bursts cooperate with immature cortical network properties for the expression of a spontaneous spindle pattern of activity, potentially implicated in the establishment and maintenance of body maps in S1 cortex. 

Publications (* : equally contributing authors) :

- Khalilov I., Esclapez M., Medina I., Aggoun D., Lamsa K., Leinekugel X., Khazipov R. & Ben-Ari Y. (1997): A Novel In Vitro Preparation: The Intact Hippocampal Formation. Neuron 19, p. 743-749. Khalilov-et-al-neuron-1997.pdf

- Leinekugel X, Khalilov I., Ben-Ari Y. & Khazipov R. (1998): Giant Depolarising Potentials: the septal pole of hippocampus paces the activity of the intact neonatal septo-hippocampal complex in vitro. Journal of Neuroscience 18 (16), p. 6349-6357. Leinekugel-et-al-JN-1998.pdf

- Leinekugel X.*, Khazipov R.*, Cannon R., Hirase H., Ben-Ari Y. & Buzsaki G.* (2002) : Correlated bursts of activity in the neonatal hippocampus in vivo. Science 296 (5575), p. 2049-2052. Leinekugel-et-al-science-2002.pdf

- Khazipov R.*, Sirota A.*, Leinekugel X.*, Holmes G., Ben-Ari Y. & Buzsaki G. (2004) : Early Motor Activity Drives Spindle-Bursts in the Developing Somatosensory Cortex, Nature 432 (7018), p.758-761. Khazipov-et-al-nature-2004.pdf 

For review, you can have a look at Leinekugel-J-Physiol-Paris-2003.pdf

Ou pour une revue en franēais, Leinekugel-Neurologies-2006.pdf

 

3) Activity patterns and synaptic plasticity in the adult rat (postdoc : adult hippocampus in vivo, with Hajime Hirase, Andras Czurco, Jozsef Csicsvari, Darrell Henze, Charles King, Ken Harris, Lisa Marshall and Editt Papp in Buzsaki-lab)

            Information encoding and storage in the mammalian central nervous structures seem to result from tight interactions between the basic integrative properties of individual cells and network driven background oscillations (theta, gamma or Sharp Waves) observed in the behaving animal. Long term modification of synaptic weights within a circuit is largely dependent on the discharge type (single action potentials or bursts) and their synchrony. During my postdoc in Pr. Buzsaki laboratory (Rutgers University, Newark, NJ, USA), I have investigated what determines the spiking patterns of individual cells during different behavioural states of the adult hippocampal network and the relationships between network patterns and synaptic plasticity.

 Although the hebbian model of synaptic plasticity is the main reference as basic cellular mechanism of learning, the physiological conditions implicated in synaptic plasticity are still unclear. Using combined intracellular and extracellular recordings in anesthetised adult rats, we have observed that experimentally pairing the intracellularly recorded cell with the spontaneous network activity (SPWs), recorded in extracellular, produced long-term enhancement of the synaptic drive to this cell. Therefore, spontaneously occuring SPWs represent a relevant pattern for long term synaptic plasticity induction in the adult rat hippocampus.

 I also performed large scale extracellular unit recordings in freely moving animals using "tetrodes", that allow to identify the spiking activity of 5 to 10 individual neurons (per tetrode) simultaneously, together with the field potential collective activity. Using 8 tetrodes simultaneously during various types of behaviour (exploration of familiar or new environments, wheel-running, sleep...), we made the following observations :

            First, we could provide experimental support to the hypothesis that the firing rate of hippocampal cells during sleep is determined by an interaction between novelty-induced experience and network regulating mechanisms that maintain homeostatic population activity. Therefore, any activity-dependent change in the matrix of synaptic weights would be compensated by opposite plasticity to maintain constant global activity within the network.

            Second, we found that temporal correlation among neurons depends on the distance between the recorded neurons as well as on the state of the network. Therefore, even though the hippocampus does not code information in a simple topographic format, nearby hippocampal neurons may discharge together due to the state-dependent participation of different sets of afferents during various tasks. This would affect the spatial clustering of neuronal discharges in the hippocampal CA1 region and be responsible for local clustering of similarly responding neurons.

            Third, we found that burst initiation probability and burst length from hippocampal pyramidal cells were inversely correlated to the last inter-spike interval. Therefore, bursts length and probability both increased during silent periods. While crucial involvement of burst firing has been hypothesized in the induction of synaptic plasticity, the behavioral, network, and cellular conditions under which a burst will be initiated are not yet clear. Our results suggest that bursts may function as "conditional synchrony detectors", signaling strong afferent synchrony after neuronal silence, and that single spikes triggered by a weak input may suppress the later induction of a burst by a strong input.

            Fourth, analysis of the firing phase of hippocampal cells in respect to theta oscillations revealed that the spike phase precession phenomenon is not a hallmark of spatial behavior, as suggested previously, but rather a manifestation of a more fundamental principle governing the timing of pyramidal cell discharge, that involves the interplay between the magnitude of dendritic excitation and rhythmic inhibition of the somatic region.

 Publications (* equally contributing authors) :

- King C., Henze D., Leinekugel X. & Buzsaki G. (1999) : Hebbian modification of a hippocampal population pattern. Journal of Physiology (London) 521(Pt 1), p. 159-167. King-et-al-J-Physiol-1999.pdf

- Papp E., Leinekugel X., Henze D.A., Lee J. & Buzsaki G. (2001) : The apical shaft of CA1 pyramidal cells is under GABAergic interneuronal control. Neuroscience102(4), p. 715-721. Papp-et-al-neurosci-2001.pdf

- Szabo I., Czurko A., Csicsvari J., Hirase H., Leinekugel X. & Buzsaki G. (2001) : The application of printed circuit board technology for fabrication of multi-channel micro-drives. Journal of Neuroscience Methods 105 (1), p. 105-110. Szabo-et-al-JN-meth-2001.pdf

- Hirase H., Leinekugel X., Csicsvari J., Czurko A. & Buzsaki G. (2001) : Behavior-dependent states of the hippocampal network affect functional clustering of neurons. Journal of Neuroscience 21 (10), RC 145. Hirase-et-al-JN-2001.pdf

- Hirase H.*, Leinekugel X.*, Czurko A.*, Csicsvari J. & Buzsaki G. (2001) : Firing rates of hippocampal neurons are preserved during subsequent sleep episodes and modified by novel awake experience. Proc Natl Acad Sci U S A 98 (16), p. 9386-9390. Hirase-et-al-PNAS-2001.pdf

- Harris K.D.*, Hirase H.*, Leinekugel X.*, Henze D.A. & Buzsaki G. (2001) : Temporal interaction between single spikes and complex spike bursts in hippocampal pyramidal cells. Neuron 32 (1), p. 141-149. Harris-et-al-neuron-2001.pdf

- Marshall L., Henze D.A., Hirase H., Leinekugel X., Dragoi G. & Buzsaki G. (2002) : Hippocampal pyramidal cell-interneuron spike transmission is frequency dependent and responsible for place modulation of interneuron discharge. Journal of Neuroscience 22 (2), RC197. Marshall-et-al-JN-2002.pdf

- Harris K.D., Henze D.A., Hirase H., Leinekugel X., Dragoi G., Czurko A. & Buzsaki G. (2002) : Spike train dynamics predicts theta-related phase precession in hippocampal pyramidal cells. Nature 417 (6890), p. 738-741. Harris-et-al-nature-2002.pdf

 

4) Physiopathology of activity patterns (Principal Investigator : freely moving, head restrained and anesthetized rats and transgenic mice, with Michele Pignatelli, Colin Molter, Eva Pastalkova, Hajime Hirase, Gyorgy Buzsaki, Yoon Cho, Joseph O'Neill, Thibault Maviel, Cyril Dejean, Jialing Liu, Bruno Bontempi, in CNIC/IMN, Bordeaux)

            In normal animals, brain states alternate between two major segregated patterns: an active state associated with high cholinergic activity, cortical desynchronization and hippocampal theta rhythm, and a slow-wave state (SWS) associated with low cholinergic activity, neocortical slow oscillations and hippocampal sharp-wave ripples. Establishing my own research group in Bordeaux, I have investigated the expression of brain activity patterns in normal animals and in pathological models of human diseases (Huntington's, Alzheimer's, epiliepsy, stroke) as well as their contribution to information encoding.

            It is widely assumed that theta power reflects the expression of sensory-motor integration underlying decisional and voluntary motor processes, so that a main function of theta would be to prepare and control relevant motor behaviour. Using large scale neuronal population recording with single cell resolution in the freely behaving rat, we observed the presence of robust rhythmic fluctuations of hippocampal theta power on a time scale of ~1.3s, expressed in a whole variety of behaviours such as REM sleep, open-field exploration, wheel and maze running. Taking into account the phase of spikes relative to this Slow Modulation of Theta Power (TPSM) provided an up to 2-fold increase in single spike information content. While previous reports have mainly considered theta power modulation as fluctuations of brain state or attention level, our results provide the first demonstration that theta power modulation might be used as a carrier for spatial and behavioural information encoding. Our observation that changes in theta power occur in a cyclic manner during a whole variety of behaviours is also intriguing because it might suggest that the rat's behaviour is controlled or updated on a predefined time schedule. Moreover, the phenomenon we report of encoding of information within a second order brain rhythm (i.e. using the rhythmic modulation of EEG power in a given frequency band) is the first of the kind.

            Attention was recently driven towards the potential contribution of sleep disorders to the various deficits affecting patients and animal models of Huntington's disease (HD). By performing electrophysiological recordings from the hippocampus and neocortex of freely moving, head restrained and anesthetized R6/1 mice, we report evidence for a global dysregulation of brain activity in HD, including systematic background interictal activity and altered brain states segregation. More specifically, we observed that the Slow-Wave state was invaded and disorganized by atropine-sensitive "ectopic" theta oscillations that entrained interictal spikes and phase-locked hippocampal sharp-wave ripples. These results point to major cholinergic-system dependent alterations of neuronal activity during rest in Huntington's disease, a crucial period for off-line processing and memory consolidation. This is to our knowledge the first time that the segregation of brain states, one of the most robust and conserved property of brain functioning, is proven to be altered in a pathology. Moreover, this is the first electrophysiological investigation at the system's level of neuronal activity related to Huntington's disease. The potential involvement of cholinergic systems seems to be both important for the basic understanding and potential therapeutic approaches to this pathology.

Publications :

- M. Pignatelli, A. Beyeler & X. Leinekugel (2012) : Neural circuits underlying the generation of theta oscillations. J. Physiol. Paris 106 (3-4):81-92. Pignatelli-et-al-J-Physiol-Paris-2012.pdf

- C. Molter, J. O'Neill, Y. Yamaguchi, H. Hirase & X. Leinekugel (2012) : Rhythmic modulation of theta oscillations supports encoding of spatial and behavioural information in the rat hippocampus. Neuron, 2012.06.036 (in press). Molter-et-al-Neuron-2012.pdf

- M. Pignatelli, F. Lebreton, Y. H. Cho & X. Leinekugel (2012) : "Ectopic" theta oscillations and interictal activity during slow-wave state in the R6/1 mouse model of Huntington's disease. Neurobiol. Dis. 48 (3), p. 409-417. Pignatelli-et-al-NBD-2012.pdf

 

 

5) synaptic interactions in the CA3 hippocampal circuit (PI : adult hippocampus in vitro and in vivo, with Anna Beyeler, Aude Retailleau, Hajime Hirase, Janos Szabadics, Colin Molter, in CNIC/IMN, Bordeaux)

            Feedback inhibition is considered to be of primary importance in the CA3 hippocampal circuit to support network oscillations as well as to avoid hyper-synchronisation (which ultimate form is seizures) due to recurrent excitation by pyramidal cells local collaterals. However, basic parameters regarding the integration of excitation and perisomatic inhibition in this circuit have not been described during spontaneous activity. Taking advantage of an original approach which consists in the extracellular detection of perisomatic inhibitory events from their target population (extracellular inhibitory post-synaptic potentials, eIPSPs), in combination with intracellular and extracellular multi-unit activity recordings, we could quantify the probabilities that CA3 pyramidal neurons recruit each other and recruit perisomatic inhibition. In addition to the powerful time-locked inhibition of CA3 spontaneous activity expected from perisomatic inhibition, analysis of the temporal dynamics of spike discharges relative to eIPSPs suggests that the CA3 recurrent excitatory/inhibitory loop operates on a 10 ms time scale, within which pyramidal cells recruit each other through recurrent collaterals and trigger powerful feedback inhibition. Such quantified parameters of neuronal interactions in the hippocampal network may serve as a basis for future characterisation of pathological conditions potentially affecting the interactions between excitation and inhibition in this circuit.