Miri Goldin

GABAergic Hub Neurons Orchestrate Synchrony in Developing Hippocampal Networks

Coordinating Neuronal Assemblies Theoretical models predict the existence of so-called hub neurons—highly connected cells that strongly influence the synchronization of spiking activity in a large group of neurons. However, experimental evidence for the existence of these neuronal hubs is lacking. Bonifazi et al. (p. 1419) used high-resolution, two-photon calcium imaging to measure spontaneous calcium fluctuations in hundreds of neurons simultaneously and determined the relative timing of these fluctuations. Examination of functional connectivity maps, based on temporal correlation measurements, revealed a subpopulation of GABAergic hub neurons displaying a remarkably widespread axonal arborization that orchestrated network synchrony in developing hippocampal networks. Spontaneous network synchronizations in developing hippocampus caused giant depolarizing potentials in individual neurons, and manipulating the spike activity in potential hub cells influenced network activity. A model for the topology of brain networks incorporates a morpho-functional description of neuronal hubs. Brain function operates through the coordinated activation of neuronal assemblies. Graph theory predicts that scale-free topologies, which include “hubs” (superconnected nodes), are an effective design to orchestrate synchronization. Whether hubs are present in neuronal assemblies and coordinate network activity remains unknown. Using network dynamics imaging, online reconstruction of functional connectivity, and targeted whole-cell recordings in rats and mice, we found that developing hippocampal networks follow a scale-free topology, and we demonstrated the existence of functional hubs. Perturbation of a single hub influenced the entire network dynamics. Morphophysiological analysis revealed that hub cells are a subpopulation of γ-aminobutyric acid–releasing (GABAergic) interneurons possessing widespread axonal arborizations. These findings establish a central role for GABAergic interneurons in shaping developing networks and help provide a conceptual framework for studying neuronal synchrony.

Protein kinase C and ERK involvement in dendritic spine plasticity in cultured rodent hippocampal neurons

The roles of protein kinase C and the MAP‐kinase extracellular receptor kinase in structural changes associated with long‐term potentiation of network activity were examined in cultured hippocampal neurons. A brief exposure to a conditioning medium that favours activation of the N‐methyl‐d‐aspartate receptor caused a rapid and specific increase in staining of neurons for the phosphorylated form of extracellular receptor kinase as well as of cyclic AMP response element binding protein. Exposure of the cultures to the conditioning medium was followed by a protein synthesis‐dependent formation of novel dendritic spines. An extracellular receptor kinase antagonist PD98059 blocked the phosphorylated form of extracellular receptor kinase response and the formation of novel spines. A selective protein kinase C agonist, phorbol 12‐myristate 13‐acetate, caused activation of extracellular receptor kinase and formation of novel spines. The protein kinase C antagonist GF109203x blocked the phosphorylated form of extracellular receptor kinase response and the subsequent spine formation by phorbol 12‐myristate 13‐acetate. Both the conditioning medium and phorbol 12‐myristate 13‐acetate caused a delayed increase in mean amplitude of miniature excitatory postsynaptic currents recorded in the hippocampal neurons. These results indicate that activation of extracellular receptor kinase mediates the effect of a conditioning protocol on the formation of dendritic spines. The formation of novel spines was associated with long‐term increase in network activity and functional synaptic connectivity among the cultured neurons.

Synaptic Kainate Receptors Tune Oriens-Lacunosum Moleculare Interneurons to Operate at Theta Frequency

GABAergic interneurons of the hippocampus play an important role in the generation of behaviorally relevant network oscillations. Among this heterogeneous neuronal population, somatostatin (SOM)-positive oriens-lacunosum moleculare (O-LM) interneurons are remarkable because they are tuned to operate at theta frequencies (6–10 Hz) in vitro and in vivo. Recent studies show that a high proportion of glutamatergic synapses that impinge on O-LM interneurons are mediated by kainate receptors (KA-Rs). In the present study, we thus tested the hypothesis that KA-Rs transmit afferent inputs in O-LM neurons during synaptic stimulation at theta frequency. We combined multibeam two-photon calcium imaging in hippocampal slices from SOM–enhanced green fluorescent protein (EGFP) mice, to record the activity of SOM cells as well as hundreds of neurons simultaneously, and targeted electrophysiological recordings and morphological analysis to describe the morphofunctional features of particular cells. We report that EGFP-positive O-LM neurons are the only subtype of interneuron that reliably follows synaptic stimulation of the alveus in the theta frequency range. Electrophysiological recordings revealed the crucial contribution of KA-Rs to the firing activity and to the glutamatergic response to theta stimuli in O-LM cells compared with other cell types. The reliable activation of O-LM cells in the theta frequency range did not simply result from the longer kinetics of KA-R-mediated postsynaptic events (EPSPKA) but presumably from a specific interaction between EPSPKA and their intrinsic active membrane properties. Such preferential processing of excitatory inputs via KA-Rs by distally projecting GABAergic microcircuits could provide a key role in theta band frequency oscillations.

Regulation of rat brain vesicular monoamine transporter by chronic treatment with ovarian hormones

Ovarian steroids play an important role in neuroregulation and in the pathophysiology of various neuropsychiatric disorders. Most of the studies focused on the impact of gonadal steroids on post-synaptic receptors and plasma membrane transporters. In the present study, we evaluated the effect of chronic treatment with ovarian steroids on the expression of rat brain vesicular monoamine transporter (VMAT2). Ovariectomized rats were treated for 21 days with estradiol, progesterone or both. VMAT2 gene expression was assessed on the protein level by high affinity [3H]dihydrotetrabenazine ([3H]TBZOH) binding using autoradiography and on the mRNA level by in situ hybridization. Progesterone administration led to a decrease in [3H]TBZOH binding in the middle striatum and in the nucleus accumbens and to a parallel decrease in VMAT2 mRNA level in the substantia nigra pars compacta and dorsal raphe nuclei. Chronic estradiol treatment reduced VMAT2 mRNA level in the dorsal raphe and [3H]TBZOH binding in middle part of the striatum and nucleus accumbens but did not affect VMAT2 mRNA level in the substantia nigra pars compacta. Simultaneous administration of both ovarian steroids did not modulate VMAT2 mRNA in the substantia nigra pars compacta as well as [3H]TBZOH binding in the striatum or the nucleus accumbens but reduced VMAT2 mRNA level in the dorsal raphe. It appears that ovarian steroids may play a crucial role in the regulation of VMAT2 gene expression in the dopamine and serotonin systems. This modulatory activity may be relevant to synaptic and neuronal plasticity as well as to the molecular and cellular pathophysiology of gender-specific neuropsychiatric disorders.

In vitro large-scale experimental and theoretical studies for the realization of bi-directional brain-prostheses.

Brain-machine interfaces (BMI) were born to control “actions from thoughts” in order to recover motor capability of patients with impaired functional connectivity between the central and peripheral nervous system. The final goal of our studies is the development of a new proof-of-concept BMI—a neuromorphic chip for brain repair—to reproduce the functional organization of a damaged part of the central nervous system. To reach this ambitious goal, we implemented a multidisciplinary “bottom-up” approach in which in vitro networks are the paradigm for the development of an in silico model to be incorporated into a neuromorphic device. In this paper we present the overall strategy and focus on the different building blocks of our studies: (i) the experimental characterization and modeling of “finite size networks” which represent the smallest and most general self-organized circuits capable of generating spontaneous collective dynamics; (ii) the induction of lesions in neuronal networks and the whole brain preparation with special attention on the impact on the functional organization of the circuits; (iii) the first production of a neuromorphic chip able to implement a real-time model of neuronal networks. A dynamical characterization of the finite size circuits with single cell resolution is provided. A neural network model based on Izhikevich neurons was able to replicate the experimental observations. Changes in the dynamics of the neuronal circuits induced by optical and ischemic lesions are presented respectively for in vitro neuronal networks and for a whole brain preparation. Finally the implementation of a neuromorphic chip reproducing the network dynamics in quasi-real time (10 ns precision) is presented.

Electron microscopic 3D-reconstruction of dendritic spines in cultured hippocampal neurons undergoing synaptic plasticity

Dendritic spines are assumed to constitute the locus of neuronal plasticity, and considerable effort has been focused on attempts to demonstrate that new memories are associated with the formation of new spines. However, few studies that have documented the appearance of spines after exposure to plasticity‐producing paradigms could demonstrate that a new spine is touched by a bona fida presynaptic terminal. Thus, the functional significance of plastic dendritic spine changes is not clearly understood. We have used quantitative time lapse confocal imaging of cultured hippocampal neurons before and after their exposure to a conditioning medium which activates synaptic NMDA receptors. Following the experiment the cultures were prepared for 3D electron microscopic reconstruction of visually identified dendritic spines. We found that a majority of new, 1‐ to 2‐h‐old spines was touched by presynaptic terminals. Furthermore, when spines disappeared, the parent dendrites were sometime touched by a presynaptic bouton at the site where the previously identified spine had been located. We conclude that new spines are most likely to be functional and that pruned spines can be transformed into shaft synapses and thus maintain their functionality within the neuronal network.

The role of the neuro-astro-vascular unit in the etiology of ataxia telangiectasia.

The growing recognition that brain pathologies do not affect neurons only but rather are, to a large extent, pathologies of glial cells as well as of the vasculature opens to new perspectives in our understanding of genetic disorders of the CNS. To validate the role of the neuron-glial-vascular unit in the etiology of genome instability disorders, we report about cell death and morphological aspects of neuroglia networks and the associated vasculature in a mouse model of Ataxia Telangiectasia (A-T), a human genetic disorder that induces severe motor impairment. We found that A-T-mutated protein deficiency was consistent with aberrant astrocytic morphology and alterations of the vasculature, often accompanied by reactive gliosis. Interestingly similar findings could also be reported in the case of other genetic disorders. These observations bolster the notion that astrocyte-specific pathologies, hampered vascularization and astrocyte-endothelium interactions in the CNS could play a crucial role in the etiology of genome instability brain disorders and could underlie neurodegeneration.

The emergence of dynamical instantaneous memory in the spontaneous activity of spatially confined neuronal assemblies in vitro

Understanding the dynamics between communicating cell assemblies is essential for deciphering the neural code and identifying the mechanism underlying memory formation. In this work, in order to unveil possible emergent intrinsic memory phenomena in the communication between cell assemblies, we study the spontaneous dynamics of in vitro spatially confined inter-connected neuronal circuits grown on multi-electrode arrays. The spontaneous dynamics of the global network was characterized by the coupling of the activity independently generated by each circuit. The asymptotic functional connectivity of the network reflected its modular organization. Instantaneous functional connectivity maps on ten seconds epochs, revealed more complex dynamical states with the simultaneous activation of distinct circuits. When looking at the similarity of the generated network events, we observed that spontaneous network events occurring at temporal distances below two dozens of seconds had an average higher similarity compared to randomly played network events. Such a memory phenomenon was not observed in networks where spontaneous events were less frequent and in networks topologically organized as open lines. These results support the hypothesis that dynamical instantaneous memory, characterized by drifting network dynamics with decaying degree of similarity, is an intrinsic property of neuronal networks.

Astrocytes restore connectivity and synchronization in dysfunctional cerebellar networks

Significance Within the long-standing debate of whether glial cells contribute to neuronal circuits, we provide evidence of the impact of astrocytes on the physiopathology and the structural–functional organization of cerebellar neuronal circuits derived from Atm-deficient mice. In vitro and adult mouse characterizations show how disrupted astrocyte morphology is associated with an increased presence of synaptic markers in Atm−/− compared with wild-type circuits, also related to reduce autophagy. Our study is corroborated by an in vitro demonstration of how replenishment of neuronal circuits with healthy astrocytes can restore Atm−/− neuronal circuits’ dynamics. Evidence suggests that astrocytes play key roles in structural and functional organization of neuronal circuits. To understand how astrocytes influence the physiopathology of cerebellar circuits, we cultured cells from cerebella of mice that lack the ATM gene. Mutations in ATM are causative of the human cerebellar degenerative disease ataxia-telangiectasia. Cerebellar cultures grown from Atm−/− mice had disrupted network synchronization, atrophied astrocytic arborizations, reduced autophagy levels, and higher numbers of synapses per neuron than wild-type cultures. Chimeric circuitries composed of wild-type astrocytes and Atm−/− neurons were indistinguishable from wild-type cultures. Adult cerebellar characterizations confirmed disrupted astrocyte morphology, increased GABAergic synaptic markers, and reduced autophagy in Atm−/− compared with wild-type mice. These results indicate that astrocytes can impact neuronal circuits at levels ranging from synaptic expression to global dynamics.

Design, Surface Treatment, Cellular Plating, and Culturing of Modular Neuronal Networks Composed of Functionally Inter-connected Circuits

The brain operates through the coordinated activation and the dynamic communication of neuronal assemblies. A major open question is how a vast repertoire of dynamical motifs, which underlie most diverse brain functions, can emerge out of a fixed topological and modular organization of brain circuits. Compared to in vivo studies of neuronal circuits which present intrinsic experimental difficulties, in vitro preparations offer a much larger possibility to manipulate and probe the structural, dynamical and chemical properties of experimental neuronal systems. This work describes an in vitro experimental methodology which allows growing of modular networks composed by spatially distinct, functionally interconnected neuronal assemblies. The protocol allows controlling the two-dimensional (2D) architecture of the neuronal network at different levels of topological complexity. A desired network patterning can be achieved both on regular cover slips and substrate embedded micro electrode arrays. Micromachined structures are embossed on a silicon wafer and used to create biocompatible polymeric stencils, which incorporate the negative features of the desired network architecture. The stencils are placed on the culturing substrates during the surface coating procedure with a molecular layer for promoting cellular adhesion. After removal of the stencils, neurons are plated and they spontaneously redirected to the coated areas. By decreasing the inter-compartment distance, it is possible to obtain either isolated or interconnected neuronal circuits. To promote cell survival, cells are co-cultured with a supporting neuronal network which is located at the periphery of the culture dish. Electrophysiological and optical recordings of the activity of modular networks obtained respectively by using substrate embedded micro electrode arrays and calcium imaging are presented. While each module shows spontaneous global synchronizations, the occurrence of inter-module synchronization is regulated by the density of connection among the circuits.