Joost le Feber

The Effect of Slow Electrical Stimuli to Achieve Learning in Cultured Networks of Rat Cortical Neurons

Learning, or more generally, plasticity may be studied using cultured networks of rat cortical neurons on multi electrode arrays. Several protocols have been proposed to affect connectivity in such networks. One of these protocols, proposed by Shahaf and Marom, aimed to train the input-output relationship of a selected connection in a network using slow electrical stimuli. Although the results were quite promising, the experiments appeared difficult to repeat and the training protocol did not serve as a basis for wider investigation yet. Here, we repeated their protocol, and compared our ‘learning curves’ to the original results. Although in some experiments the protocol did not seem to work, we found that on average, the protocol showed a significantly improved stimulus response indeed. Furthermore, the protocol always induced functional connectivity changes that were much larger than changes that occurred after a comparable period of random or no stimulation. Finally, our data shows that stimulation at a fixed electrode induces functional connectivity changes of similar magnitude as stimulation through randomly varied sites; both larger than spontaneous connectivity fluctuations. We concluded that slow electrical stimulation always induced functional connectivity changes, although uncontrolled. The magnitude of change increased when we applied the adaptive (closed-loop) training protocol. We hypothesize that networks develop an equilibrium between connectivity and activity. Induced connectivity changes depend on the combination of applied stimulus and initial connectivity. Plain stimuli may drive networks to the nearest equilibrium that accommodates this input, whereas adaptive stimulation may direct the space for exploration and force networks to a new balance, at a larger distance from the initial state.

Growth dynamics explain the development of spatiotemporal burst activity of young cultured neuronal networks in detail.

A typical property of isolated cultured neuronal networks of dissociated rat cortical cells is synchronized spiking, called bursting, starting about one week after plating, when the dissociated cells have sufficiently sent out their neurites and formed enough synaptic connections. This paper is the third in a series of three on simulation models of cultured networks. Our two previous studies [26], [27] have shown that random recurrent network activity models generate intra- and inter-bursting patterns similar to experimental data. The networks were noise or pacemaker-driven and had Izhikevich-neuronal elements with only short-term plastic (STP) synapses (so, no long-term potentiation, LTP, or depression, LTD, was included). However, elevated pre-phases (burst leaders) and after-phases of burst main shapes, that usually arise during the development of the network, were not yet simulated in sufficient detail. This lack of detail may be due to the fact that the random models completely missed network topology .and a growth model. Therefore, the present paper adds, for the first time, a growth model to the activity model, to give the network a time dependent topology and to explain burst shapes in more detail. Again, without LTP or LTD mechanisms. The integrated growth-activity model yielded realistic bursting patterns. The automatic adjustment of various mutually interdependent network parameters is one of the major advantages of our current approach. Spatio-temporal bursting activity was validated against experiment. Depending on network size, wave reverberation mechanisms were seen along the network boundaries, which may explain the generation of phases of elevated firing before and after the main phase of the burst shape.In summary, the results show that adding topology and growth explain burst shapes in great detail and suggest that young networks still lack/do not need LTP or LTD mechanisms.

Phase-dependent effects of stimuli locked to oscillatory activity in cultured cortical networks.

The archetypal activity pattern in cultures of dissociated neurons is spontaneous network-wide bursting. Bursts may interfere with controlled activation of synaptic plasticity, but can be suppressed by the application of stimuli at a sufficient rate. We sinusoidally modulated (4 Hz) the pulse rate of random background stimulation (RBS) and found that cultures were more active, burst less frequently, and expressed oscillatory activity. Next, we studied the effect of phase-locked tetani (four pulses, 200 s(-1)) on network activity. Tetani were applied to one electrode at the peak or trough of mRBS stimulation. We found that when tetani were applied at the peak of modulated RBS (mRBS), a significant potentiation of poststimulus histograms (PSTHs) occurred. Conversely, tetani applied at the trough resulted in a small but insignificant depression of PSTHs. In addition to PSTHs, electrode-specific firing rate profiles within spontaneous bursts before and after mRBS were analyzed. Here, significant changes in firing rate profiles were found only for stimulation at the peak of mRBS. Our study shows that rhythmic activity in culture is possible, and that the network responds differentially to strong stimuli depending on the phase at which they are delivered. This suggests that plasticity mechanisms may be differentially accessible in an oscillatory state.

Orexin-A and Orexin-B during the postnatal development of the rat brain

Orexin-A and orexin-B are hypothalamic neuropeptides isolated from a small group of neurons in the hypothalamus, which project their axons to all major parts of the central nervous system. Despite the extensive information about orexin expression and function at different parts of the nervous system in adults, data about the development and maturation of the orexin system in the brain are a bit contradictory and insufficient. A previous study has found expression of orexins in the hypothalamus after postnatal day 15 only, while others report orexins detection at embryonic stages of brain formation. In the present study, we investigated the distribution of orexin-A and orexin-B neuronal cell bodies and fibers in the brain at three different postnatal stages: 1-week-, 2-week-old and adult rats. By means of immunohistochemical techniques, we demonstrated that a small subset of cells in the lateral hypothalamus, and the perifornical and periventricular areas were orexin-A and orexin-B positive not only in 2-week-old and adult rats but also in 1-week-old animals. In addition, orexin-A and orexin-B expressing neuronal varicosities were found in many other brain regions. These results suggest that orexin-A and orexin-B play an important role in the early postnatal brain development. The widespread distribution of orexinergic projections through all these stages may imply an involvement of the two neurotransmitters in a large variety of physiological and behavioral processes also including higher brain functions like learning and memory.

Ghrelin stimulates synaptic formation in cultured cortical networks in a dose-dependent manner

Ghrelin was initially related to appetite stimulation and growth hormone secretion. However, it also has a neuroprotective effect in neurodegenerative diseases and regulates cognitive function. The cellular basis of these processes is related to synaptic efficacy and plasticity. Previous studies indicated that ghrelin has an excitatory effect on neuronal activity, and stimulates synaptic plasticity in vivo. Plasticity in the adult brain occurs in many different ways, including changes in synapse morphology and number. Therefore, we used in vitro neuronal cultures to investigate how ghrelin affects synaptogenesis. We used dissociated cortical cultures of newborn rats, chronically treated with different doses of ghrelin (0.5, 1, 1.5 and 2μM). After one-, two-, three- or four weeks cultures were immunostained for the presynaptic marker synaptophysin. In parallel, additional groups of non-treated cultures were immunostained for detection of ghrelin receptor (GHSR1). During development, GHSR1was increasingly expressed in all type of neurons, as well as the synaptophysin. Synaptic density depended on ghrelin concentration, and was much higher than in controls in all age groups. In conclusion, ghrelin leads to earlier network formation in dissociated cortical networks and an increase in number of synapses. The effect is probably mediated by GHSR1. These findings suggest that ghrelin may provide a novel therapeutic strategy for the treatment of disorders related to synaptic impairment.

Functionally different α-synuclein inclusions yield insight into Parkinson's disease pathology

The formation of α-synuclein (α-S) amyloid aggregates, called Lewy bodies (LBs), is a hallmark of Parkinson’s disease (PD). The function of LBs in the disease process is however still unclear; they have been associated with both neuroprotection and toxicity. To obtain insight into this contradiction, we induced the formation of α-S inclusions, using three different induction methods in SH-SY5Y cells and rat-derived primary neuronal cells. Using confocal and STED microscopy we observed induction-dependent differences in α-S inclusion morphology, location and function. The aggregation of α-S in functionally different compartments correlates with the toxicity of the induction method measured in viability assays. The most cytotoxic treatment largely correlates with the formation of proteasome-associated, juxta-nuclear inclusions. With less toxic methods cytosolic deposits that are not associated with the proteasome are more prevalent. The distribution of α-S over at least two different types of inclusions is not limited to cell models, but is also observed in primary neuronal cells and in human mesencephalon. The existence of functionally different LBs, in vivo and in vitro, gives important insights in the impact of Lewy Body formation on neuronal functioning and may thereby provide a platform for discovering therapeutics.

Progression of Neuronal Damage in an In Vitro Model of the Ischemic Penumbra

Improvement of neuronal recovery in the ischemic penumbra around a brain infarct has a large potential to advance clinical recovery of patients with acute ischemic stroke. However, pathophysiological mechanisms leading to either recovery or secondary damage in the penumbra are not completely understood. We studied neuronal dynamics in a model system of the penumbra consisting of networks of cultured cortical neurons exposed to controlled levels and durations of hypoxia. Short periods of hypoxia (pO2≈20mmHg) reduced spontaneous activity, due to impeded synaptic function. After ≈6 hours, activity and connectivity partially recovered, even during continuing hypoxia. If the oxygen supply was restored within 12 hours, changes in network connectivity were completely reversible. For longer periods of hypoxia (12–30 h), activity levels initially increased, but eventually decreased and connectivity changes became partially irreversible. After ≈30 hours, all functional connections disappeared and no activity remained. Since this complete silence seemed unrelated to hypoxic depths, but always followed an extended period of low activity, we speculate that irreversible damage (at least partly) results from insufficient neuronal activation. This opens avenues for therapies to improve recovery by neuronal activation.

Changes within bursts during learning in dissociated neural networks

We have studied the effect of imprinting a new stimulus-response (SR) relationship into a neuronal network cultured on a multi electrode array (MEA). We have used the Conditional Repetitive Stimulation (CRS) algorithm introduced by Shahaf et al in 2004. In this algorithm focal electrical stimulation is delivered at a low rate (<1 Hz) and is withdrawn when a desired response is observed. We confirmed that CRS could train the network to strengthen an initially weak SR relationship. With the acquisition of a new SR relationship, we studied its effect on network activity. Specifically, spontaneously occurring network bursts measured before, during and after training were analyzed. The total firing rate within bursts was estimated with a temporal resolution of milliseconds (burst profiles). We have shown earlier that these profiles change shape on a time base of several hours during spontaneous development. We show that the rate of change of the profiles during training (i.e. CRS) was higher than when no stimulation was applied.

Do external stimuli, applied to train cultured cortical networks, disturb the balance between activity and connectivity?

Learning, or more generally, plasticity may be studied using cultured neuronal networks on multi electrode arrays. Many protocols have been proposed to change connectivity in such networks. So far, only one of these protocols, proposed by Shahaf and Marom, aimed to change the input-output relationship of a selected connection in the network. Although the results were quite promising, the experiments appeared difficult to repeat and the protocol did not serve as a basis for wider investigation yet. Here, we repeated their protocol, and compared our ‘learning curves’ to the original results. Although in some experiments the protocol did not seem to work, we found that on average, the protocol showed a significant learning effect indeed. We frequently found learning curves that initially declined as in the original results, but then increased again before finally settling at a low level.

Repeated stimulation of cultured networks of rat cortical neurons induces parallel memory traces

During systems consolidation, memories are spontaneously replayed favoring information transfer from hippocampus to neocortex. However, at present no empirically supported mechanism to accomplish a transfer of memory from hippocampal to extra-hippocampal sites has been offered. We used cultured neuronal networks on multielectrode arrays and small-scale computational models to study the effect of memory replay on the formation of memory traces. We show that input-deprived networks develop an activity⇔connectivity balance where dominant activity patterns support current connectivity. Electrical stimulation at one electrode disturbs this balance and induces connectivity changes. Intrinsic forces in recurrent networks lead to a new equilibrium with activity patterns that include the stimulus response. The new connectivity is no longer disrupted by this stimulus, indicating that networks memorize it. A different stimulus again induces connectivity changes upon first application but not subsequently, demonstrating the formation of a second memory trace. Returning to the first stimulus does not affect connectivity, indicating parallel storage of both traces. A computer model robustly reproduced experimental results, suggesting that spike-timing-dependent plasticity and short time depression suffice to store parallel memory traces, even in networks without particular circuitry constraints.