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In systems consolidation, encoded memories are replayed by the hippocampus during slow-wave sleep (SWS), and permanently stored in the neocortex. Declarative memory consolidation is believed to benefit from the oscillatory rhythms and low cholinergic tone observed in this sleep stage, but underlying mechanisms remain unclear. To clarify the role of cholinergic modulation and synchronized activity in memory consolidation, we applied repeated electrical stimulation in mature cultures of dissociated rat cortical neurons with high or low cholinergic tone, mimicking the cue replay observed during systems consolidation under distinct cholinergic concentrations. In the absence of cholinergic input, these cultures display activity patterns hallmarked by network bursts, synchronized events reminiscent of the low frequency oscillations observed during SWS. They display stable activity and connectivity, which mutually interact and achieve an equilibrium. Electrical stimulation reforms the equilibrium to include the stimulus response, a phenomenon interpreted as memory trace formation. Without cholinergic input, activity was burst-dominated. First application of a stimulus induced significant connectivity changes, while subsequent repetition no longer affected connectivity. Presenting a second stimulus at a different electrode had the same effect, whereas returning to the initial stimuli did not induce further connectivity alterations, indicating that the second stimulus did not erase the ‘memory trace’ of the first. Distinctively, cultures with high cholinergic tone displayed reduced network excitability and dispersed firing, and electrical stimulation did not induce significant connectivity changes. We conclude that low cholinergic tone facilitates memory formation and consolidation, possibly through enhanced network excitability. Network bursts or SWS oscillations may merely reflect high network excitability.

The understanding of the neurophysiological processes that occur in the areas that surround the core of a brain infarct is crucial for the creation of new therapies and treatments to improve neuronal recovery. The present study aims to demonstrate that both rodent and human neuronal networks lose their activity under low oxygen conditions and that electrical stimulation can increase the probability of recovery. Hypoxia was induced in rodent and human neurons and the effects of electrical stimulation were assessed in the rat cultures. The results obtained show that neuronal activation, in the form of electrical stimulation, has the potential to maintain the networks at higher levels of activity and, therefore, to improve cell survival. This study will open the way for new treatment strategies based on brain-stimulation to enhance neuronal recovery and will be of large relevance for patients, families, and society.

The brain is the most complex organ of the body, and many pathological processes underlying various brain disorders are poorly understood. Limited accessibility hinders observation of such processes in the in vivo brain, and experimental freedom is often insufficient to enable informative manipulations. In vitro preparations (brain slices or cultures of dissociated neurons) offer much better accessibility and reduced complexity and have yielded valuable new insights into various brain disorders. Both types of preparations have their advantages and limitations with regard to lifespan, preservation of in vivo brain structure, composition of cell types, and the link to behavioral outcome is often unclear in in vitro models. While these limitations hamper general usage of in vitro preparations to study, e.g., brain development, in vitro preparations are very useful to study neuronal and synaptic functioning under pathologic conditions. This chapter addresses several brain disorders, focusing on neuronal and synaptic functioning, as well as network aspects. Recent progress in the fields of brain circulation disorders, excitability disorders, and memory disorders will be discussed, as well as limitations of current in vitro models.

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.

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.

The objective of this work was to study the relation between afferent bladder nerve activity and bladder mechanics and the mechanisms that initiate and terminate bladder contractions. Bladder nerve activity, pressure and volume were recorded during the micturition cycle in the rat. The highest correlation was found between afferent nerve activity and stress (pressure x volume). Afferent nerve activity depended linearly on stress within 6%, and both slope and offset were independent of the bladder-filling rate. The levels of afferent bladder nerve activity at the onset and cessation of efferent firing to the bladder were highly reproducible with coefficients of variation ofor=17%. We propose a model in which afferent activity is proportional to bladder wall stress, and bladder contraction is initiated when afferent activity exceeds a threshold due to an increasing pressure and volume. The contraction continues until afferent activity drops below a threshold again as a result of a decreasing volume.

Objective There is limited understanding of the nature of dynamic neuronal processes leading to either secondary irreversible damage or recovery in the ischemic penumbra. We use cultured neuronal networks to study neuronal dynamics during partial hypoxia, focusing on changes of synaptic transmission and membrane integrity. Methods Twenty-two cultured networks of rat cortical neurons grown over multi electrode arrays were exposed to hypoxia of various depth and duration (pO 2 of 160 mmHg lowered to 90 or 30 mmHg, during 6–48 h). Synaptic functioning was assessed before, during, and after hypoxia by the amount of spontaneous network activity and network responses to electrical stimulation. Action potential shapes and direct (non-synaptic) stimulus responses were used as measures of neuronal integrity. Results During pO 2 =30 mmHg, synaptic transmission failed within an hour. Isolated synaptic failure was reversible after 6–12 h of hypoxia, but partly irreversible after 24 h. Irreversible membrane failure occurred between 24 and 48 h, but later with pO 2 = 90 mmHg. Conclusion The cascade of neuronal pathophysiological events in the ischemic penumbra includes reversible synaptic failure, irreversible synaptic failure, and membrane failure, respectively. The timescale of this cascade depends on remaining perfusion levels.

Extremely synchronized firing patterns such as those observed in brain diseases like epilepsy may result from excessive network excitability. Although network excitability is closely related to (excitatory) connectivity, a direct measure for network excitability remains unavailable. Several methods currently exist for estimating network connectivity, most of which are related to cross-correlation. An example is the conditional firing probability (CFP) analysis which calculates the pairwise probability (CFPi,j) that electrode j records an action potential at time t = τ, given that electrode i recorded a spike at t = 0. However, electrode i often records multiple spikes within the analysis interval, and CFP values are biased by the on-going dynamic state of the network. Here we show that in a linear approximation this bias may be removed by deconvoluting CFPi,j with the autocorrelation of i (i.e. CFPi,i), to obtain the single pulse response (SPRi,j)-the average response at electrode j to a single spike at electrode i. Thus, in a linear system SPRs would be independent of the dynamic network state. Nonlinear components of synaptic transmission, such as facilitation and short term depression, will however still affect SPRs. Therefore SPRs provide a clean measure of network excitability. We used carbachol and ghrelin to moderately activate cultured cortical networks to affect their dynamic state. Both neuromodulators transformed the bursting firing patterns of the isolated networks into more dispersed firing. We show that the influence of the dynamic state on SPRs is much smaller than the effect on CFPs, but not zero. The remaining difference reflects the alteration in network excitability. We conclude that SPRs are less contaminated by the dynamic network state and that mild excitation may decrease network excitability, possibly through short term synaptic depression.

Background: While ghrelin was initially related to appetite stimulation and growth hormone secretion, it also has a neuroprotective effect in neurodegenerative diseases and regulates cognitive function. The cellular basis of those processes is related to synaptic efficacy and plasticity. Previous studies have shown that ghrelin not only stimulates synapse formation in cultured cortical neurons and hippocampal slices, but also alters some of the electrophysiological properties of neurons in the hypothalamus, amygdala and other subcortical areas. However, direct evidence for ghrelin’s ability to modulate the activity in cortical neurons is not available yet. In this study, we investigated the effect of acylated ghrelin on the development of the activity level and activity patterns in cortical neurons, in relation to its effect on synaptogenesis. Additionally, we quantitatively evaluated the expression of the receptor for acylated ghrelin – growth hormone secretagogue receptor-1a (GHSR-1a) during development. Results: We performed electrophysiology and immunohistochemistry on dissociated cortical cultures from neonates, treated chronically with acylated ghrelin. On average 76 ± 4.6% of the cortical neurons expressed GHSR-1a. Synapse density was found to be much higher in ghrelin treated cultures than in controls across all age groups (1, 2 or 3 weeks). In all cultures (control and ghrelin treated), network activity gradually increased until it reached a maximum after approximately 3 weeks, followed by a slight decrease towards a plateau. During early developmental stages (1–2 weeks), the activity was much higher in ghrelin treated cultures and consequently, they reached the plateau value almost a week earlier than controls. Conclusions: Acylated ghrelin leads to earlier network formation and activation in cultured cortical neuronal networks, the latter being a possibly consequence of accelerated synaptogenesis.

The mechanisms behind memory have been studied mainly in artificial neural networks. Several mechanisms have been proposed, but it remains unclear yet if and how these findings can be translated to biological networks. Here we unravel part of the mechanism by showing that cultured neuronal networks develop an activity connectivity balance. External inputs disturb this balance and induce connectivity changes. The new connectivity is no longer disrupted by reapplication of the input, indicating that a network memorizes the input, analog to attractor memory networks as demonstrated in Hopfield network models. A different input again induces connectivity changes upon first application but not after repeated stimulation. Returning to the first input no longer affects connectivity, showing that memory traces are stored in parallel. A simple computer model robustly reproduces the experimental results and shows that spike timing dependent plasticity suffices to store memory traces of different inputs in parallel in neuronal 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.

We propose and validated a design for a highly selective “endcap” regenerative neural interface towards a neuroprosthesis. In vitro studies using rat cortical neurons determine if a branching microchannel structure can counter fasciculated growth and cause neurites to separate from one another, allowing for greater selective contact. Initial studies find that narrower branching microchannels achieve improved neurite separation. Electrical stimulation of neurites within microchannels is possible, as is recording of neurite action potentials with the microchannels acting as electrical signal amplifiers.

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. One of these protocols, proposed by Shahaf and Marom, aimed to change the input-output relationship of a selected connection in a 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. Furthermore, the protocol always induced connectivity changes that were much larger than changes that occurred after a comparable period of random stimulation. Finally, our data suggest that network connectivity changes are induced more easily using stimulation at a fixed, than using randomly changing electrodes.

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.

In this study we measured urethral pressure changes in response to efferent pudendal nerve stimulation in rats. All other neural pathways to the urethra were transected, and the urethra was continuously perfused. We found fast twitch-like contractions, superimposed on a slow relaxation. The amplitude of the twitches was independent of the stimulation frequency below 26 Hz, whereas the relaxation depended highly on this frequency. The twitches were caused by striated urethral muscles, and the relaxation was caused by smooth muscles. Both were mediated by acetylcholine. We calculated the effective urethral relaxation as the absolute relaxation multiplied by the time fraction between the twitches. Maximum effective relaxation occurred at 8–10 Hz, exactly the frequency of spontaneous oscillations during bladder voiding in rats. Although the oscillatory sphincter contractions in rats during voiding may be needed in other mechanisms for efficient voiding, our data suggest that they may be a side effect of the actual purpose: urethral relaxation.

In this study, the mechanism involved in the initiation of voiding was investigated. Bladder pressure and bladder and urethral nerve activity were recorded in the anesthetized rat. Bladder nerve activity was resolved into afferent and efferent activity by means of a theoretical model. The beginning of an active bladder contraction was defined as the onset of bladder efferent firing at a certain time ( t 0). From t 0 onward, bladder efferent activity increased linearly during δ t seconds (rise time) to a maximum. The pressure at t 0 was 1.0 ± 0.4 kPa, the afferent nerve activity at t 0 was 2.0 ± 0.6 μV (53 ± 15% of maximum total nerve activity), and δ t was 11 ± 13 s. Between contractions the afferent activity at t 0 was never exceeded. Urethral afferent nerve activity started at bladder pressures of 2.1 ± 1.1 kPa. Therefore, we concluded that urethral afferent nerve activity does not play a role in the initiation of bladder contractions; voiding contractions presumably are initiated by bladder afferent nerve activity exceeding a certain threshold.

In male urethan-anesthetized rats, activity was measured in nerves that run over the proximal urethra. The urethral nerve response to stepwise urethral perfusion could be described by a four-parameter model (fit error max), which was proportional to the instantaneous pressure. The duration of this first episode (δ t) was inversely proportional to the perfusion rate. After infusion of a fixed volume, the urethra opened and the NA decreased with a time constant ϕ−1 (∼1.8 s) to an elevated level (NAlevel). NAlevel was linearly related to the steady-state pressure. Accordingly, sensors in the urethra are sensitive to pressure rather than to the perfusion rate. The parameters NAmax, NAlevel, and δ t showed very good reproducibility (SD ∼19% of mean). The measured activity was most likely afferent and conducted to the major pelvic ganglion.

DBS parameters are set by trail and error. Moreover DBS produces nonselective stimulation of an unknown group of neuronal elements over an unknown volume of tissue by this high frequency stimulation. By culturing rat subthalamic neurons (STN) on multi-electrode arrays (MEA) flat unorganized neuronal cultures are produced.