Hugh P. C. Robinson

Human cerebral cortex development from pluripotent stem cells to functional excitatory synapses

Efforts to study the development and function of the human cerebral cortex in health and disease have been limited by the availability of model systems. Extrapolating from our understanding of rodent cortical development, we have developed a robust, multistep process for human cortical development from pluripotent stem cells: directed differentiation of human embryonic stem (ES) and induced pluripotent stem (iPS) cells to cortical stem and progenitor cells, followed by an extended period of cortical neurogenesis, neuronal terminal differentiation to acquire mature electrophysiological properties, and functional excitatory synaptic network formation. We found that induction of cortical neuroepithelial stem cells from human ES cells and human iPS cells was dependent on retinoid signaling. Furthermore, human ES cell and iPS cell differentiation to cerebral cortex recapitulated in vivo development to generate all classes of cortical projection neurons in a fixed temporal order. This system enables functional studies of human cerebral cortex development and the generation of individual-specific cortical networks ex vivo for disease modeling and therapeutic purposes.

Simultaneous Induction of Pathway-Specific Potentiation and Depression in Networks of Cortical Neurons

Activity-dependent modification of synaptic efficacy is widely recognized as a cellular basis of learning, memory, and developmental plasticity. Little is known, however, of the consequences of such modification on network activity. Using electrode arrays, we examined how a single, localized tetanic stimulus affects the firing of up to 72 neurons recorded simultaneously in cultured networks of cortical neurons, in response to activation through 64 different test stimulus pathways. The same tetanus produced potentiated transmission in some stimulus pathways and depressed transmission in others. Unexpectedly, responses were homogeneous: for any one stimulus pathway, neuronal responses were either all enhanced or all depressed. Cross-correlation of responses with the responses elicited through the tetanized site revealed that both enhanced and depressed responses followed a common principle: activity that was closely correlated before tetanus with spikes elicited through the tetanized pathway was enhanced, whereas activity outside a 40-ms time window of correlation to tetanic pathway spikes was depressed. Response homogeneity could result from pathway-specific recurrently excitatory circuits, whose gain is increased or decreased by the tetanus, according to its cross-correlation with the tetanized pathway response. The results show how spatial responses following localized tetanic stimuli, although complex, can be accounted for by a simple rule for activity-dependent modification.

The mechanisms of generation and propagation of synchronized bursting in developing networks of cortical neurons

The characteristics and mechanisms of synchronized firing in developing networks of cultured cortical neurons were studied using multisite recording through planar electrode arrays (PEAs). With maturation of the network (from 3 to 40 d after plating), the frequency and propagation velocity of bursts increased markedly (approximately from 0.01 to 0.5 Hz and from 5 to 100 mm/sec, respectively), and the sensitivity to extracellular magnesium concentration (0–10 mM) decreased. The source of spontaneous bursts, estimated from the relative delay of onset of activity between electrodes, varied randomly with each burst. Physical separation of synchronously bursting networks into several parts using an ultraviolet laser, divided synchronous bursting into different frequencies and phases in each part. Focal stimulation through the PEA was effective at multiple sites in eliciting bursts, which propagated over the network from the site of stimulation. Stimulated bursts exhibited both an absolute refractory period and a relative refractory period, in which partially propagating bursts could be elicited. Periodic electrical stimulation (at 1 to 30 sec intervals) produced slower propagation velocities and smaller numbers of spikes per burst at shorter stimulation intervals. These results suggest that the generation and propagation of spontaneous synchronous bursts in cultured cortical neurons is governed by the level of spontaneous presynaptic firing, by the degree of connectivity of the network, and by a distributed balance between excitation and recovery processes.

Spontaneous periodic synchronized bursting during formation of mature patterns of connections in cortical cultures.

Long-term recording of spontaneous activity in cultured cortical neuronal networks was carried out using substrates containing multi-electrode arrays. Spontaneous uncorrelated firing appeared within the first 3 days and transformed progressively into synchronized bursting within a week. By 30 days from the establishment of the culture, the network exhibited a complicated non-periodic, synchronized activity pattern which showed no changes for more than 2 months and thus represented the mature state of the network. Pharmacological inhibition of activity only during the period when regular synchronized bursting was observed was capable of producing a different mature activity pattern from the control. These results suggest that periodic synchronized bursting plays a critical role in the development of synaptic connections.

Injection of digitally synthesized synaptic conductance transients to measure the integrative properties of neurons.

A novel technique was developed for injecting a time-varying conductance into a neuron, to allow quantitative measurement of the processing of synaptic inputs. In current-clamp recording mode, the membrane potential was sampled continuously and used to calculate and update the level of injected current within 60 microseconds, using a real-time computer, so as to mimic the electrical effect of a given conductance transient. Cellular responses to synthetic conductance transients modelled on the fast (non-N-methyl-D-aspartate) phase of the glutamatergic postsynaptic potential were measured in cultured rat hippocampal neurons.

Simultaneous measurement of intracellular calcium and electrical activity from patterned neural networks in culture

Multisite extracellular electrical activity and intracellular calcium were recorded simultaneously. Electrical signals were measured using microelectrode array substrates. A novel cell positioning technique was combined with a method for controlling neurite outgrowth, which allowed cell-electrode contacts to be established easily, thus facilitating the electrical recording. Intracellular calcium was measured optically using the indicator fluo-3. Under low-magnesium conditions, cultured rat cortical neurons showed periodic transients of fluo-3 fluorescence, which were synchronized with the periodic bursting observed electrically. The intervals between bursts could be determined by electrical stimulation through the substrate electrodes. The results suggest that functional synaptic connections are formed in the culture system.<<ETX>>

A system for MEA-based multisite stimulation

The capability for multisite stimulation is one of the biggest potential advantages of microelectrode arrays (MEAs). There remain, however, several technical problems which have hindered the development of a practical stimulation system. An important design goal is to allow programmable multisite stimulation, which produces minimal interference with simultaneous extracellular and patch or whole cell clamp recording. Here, we describe a multisite stimulation and recording system with novel interface circuit modules, in which preamplifiers and transistor transistor logic-driven solid-state switching devices are integrated. This integration permits PC-controlled remote switching of each substrate electrode. This allows not only flexible selection of stimulation sites, but also rapid switching of the selected sites between stimulation and recording, within 1.2 ms. This allowed almost continuous monitoring of extracellular signals at all the substrate-embedded electrodes, including those used for stimulation. In addition, the vibration-free solid-state switching made it possible to record whole-cell synaptic currents in one neuron, evoked from multiple sites in the network. We have used this system to visualize spatial propagation patterns of evoked responses in cultured networks of cortical neurons. This MEA-based stimulation system is a useful tool for studying neuronal signal processing in biological neuronal networks, as well as the process of synaptic integration within single neurons.

Strengthening of synchronized activity by tetanic stimulation in cortical cultures: application of planar electrode arrays

Rat cortical neurons were cultured on planar electrode arrays with 64 embedded electrodes. Whole-cell recording from single neurons and multisite extracellular recording were carried out simultaneously in the cultured cortical networks, and the effects of focal tetanic stimulation of the culture were studied. Both the number of action potentials and the propagation velocity of stimulated bursts were increased after tetanic stimulation. These changes were associated with a marked increase in the number of late components in the synaptic current, but with little or no increase in the early peak synaptic current. The effects of tetanic stimulation were consistent with a widespread increase in the reliability of monosynaptic transmission.

Abnormal Ca2+ homeostasis before cell death revealed by whole cell recording of ischemic CA1 hippocampal neurons

Slices were made from the hippocampus of gerbils following transient ischemia achieved by clamping the carotid arteries for 5 min, and changes in the electrophysiology of CA1 pyramidal neurons were studied by whole cell patch-clamp recording as well as conventional intracellular recording. The great majority of CA1 neurons in slices made 2.5-3 days after ischemia showed reduced resting potentials and were easily depolarized by prolonged low-frequency stimulation or by tetanic stimulation of the Schaffer collateral/commissural input. This stimulus-induced depolarization was accelerated by intracellular injection of D-myo-inositol 1,4,5-triphosphate, which depolarized membrane potentials towards 0 mV without synaptic input stimulation. Intracellular application of BAPTA, a Ca2+ chelator, effectively blocked the stimulus-induced depolarization. When recording from ischemic neurons with patch pipettes containing both D-myo-inositol 1,4,5-triphosphate and BAPTA, excitatory postsynaptic currents were transiently potentiated by stimulation, but the membrane potential did not show stimulus-induced depolarization and remained steady for long periods. These results lend support to the view that the intracellular Ca2+ regulation system is severely disturbed following ischemia, and that input fiber stimulation leads to abnormal Ca2+ accumulation in ischemic neurons resulted in neuronal death. The reduction of free Ca2+ inside the ischemic neuron by BAPTA apparently saves neurons which are otherwise destined to delayed neuronal death.

Disturbance of Membrane Function Preceding Ischemic Delayed Neuronal Death in the Gerbil Hippocampus

Slice preparations were made from the hippocampus of gerbils after 5 min of ischemia by carotid artery occlusion and the membrane properties of pyramidal neurons were examined. A majority of CA1 neurons lost the capacity for long-term potentiation following tetanic stimulation of the input fibers. CA3 pyramidal neurons, in contrast, preserved responses similar to those in the normal gerbil. Following ischemia, CA1 pyramidal neurons showed increased spontaneous firing that was highly voltage dependent and was blocked by intracellular injection of the Ca2+ chelator, EGTA. Thirty-five percent of CA1 neurons showed an abnormal slow oscillation of the membrane potential after 24 h following ischemia. Intracellular injection of GTPγS or IP3 produced facilitation of the oscillations followed by irreversible depolarization. Our results indicate that ischemia-damaged CA1 neurons suffer from abnormal Ca2+ homeostasis, involving IP3-induced liberation of Ca2+ from internal stores.