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.     

Latency-Related Development of Functional Connections in Cultured Cortical Networks

To study plasticity, we cultured cortical networks on multielectrode arrays, enabling simultaneous recording from multiple neurons. We used conditional firing probabilities to describe functional network connections by their strength and latency. These are abstract representations of neuronal pathways and may arise from direct pathways between two neurons or from a common input. Functional connections based on direct pathways should reflect synaptic properties. Therefore, we searched for long-term potentiation (this mechanism occurs in vivo when presynaptic action potentials precede postsynaptic ones with interspike intervals up to ∼20 ms) in vitro. To investigate if the strength of functional connections showed a similar latency-related development, we selected periods of monotonously increasing or decreasing strength. We observed increased incidence of short latencies (5-30 ms) during strengthening, whereas these rarely occurred during weakening. Furthermore, we saw an increased incidence of 40-65 ms latencies during weakening. Conversely, functional connections tended to strengthen in periods with short latency, whereas strengthening was significantly less than average during long latency. Our data suggest that functional connections contain information about synaptic connections, that conditional firing probability analysis is sensitive enough to detect it and that a substantial fraction of all functional connections is based on direct pathways.     

Effects of Lipids on ENaC Activity in Cultured Mouse Cortical Collecting Duct Cells

Direct effects on epithelial Na⁺ channels (ENaC) activity by lipids, e.g., arachidonic acid (AA), eicosatetraynoic acid (ETYA), linoleic acid (LA), stearic acid (SA), hydroxyeicosatetraenoic acid (HETE), 11,12–epoxyeicosatrienoic acid (EET), (PGF2), and (PGE2), in cultured mouse cortical collecting duct (M1) cells were clarified by using single-channel recordings in this study. In a cell-attached recording, a bath application of 10 μM AA significantly reduced the ENaC open probability (NPo), whereas 10 μM ETYA or 5 μM LA only induced a slight inhibition. The inside-out recording as a standard protocol was thereafter performed to examine effects of these lipids on ENaC activity. Within 10 min after the formation of the inside-out configuration, the NPo of ENaC in cultured mouse cortical collecting duct (M1) cells remained relatively constant. Application of ETYA or LA or SA exhibited a similar inhibition on the channel NPo when applied to the extracellular side, suggesting that fatty acids could exert a nonspecific inhibition on ENaC activity. 11,12-EET, a metabolite of AA via the cytochrome P450 epoxygenase pathway, significantly inhibited the ENaC NPo, whereas 20-HETE, a metabolite of AA via the hydroxylase pathway, only caused a small inhibition of the ENaC NPo, to a similar degree as that seen with ETYA and LA. However, both PGE2 and PGF2α significantly enhanced the ENaC NPo. These results suggest that fatty acids exert a nonspecific effect on ENaC activity due to the interaction between the channel proximity and the lipid. The opposite effects of 11,12-EET and prostaglandin (PG) implicate different mechanisms in regulation of ENaC activity by activation of epoxygenase and cyclooxygenase.     

Saccadic Reaction Times to Audiovisual Stimuli Show Effects of Oscillatory Phase Reset

Initiating an eye movement towards a suddenly appearing visual target is faster when an accessory auditory stimulus occurs in close spatiotemporal vicinity. Such facilitation of saccadic reaction time (SRT) is well-documented, but the exact neural mechanisms underlying the crossmodal effect remain to be elucidated. From EEG/MEG studies it has been hypothesized that coupled oscillatory activity in primary sensory cortices regulates multisensory processing. Specifically, it is assumed that the phase of an ongoing neural oscillation is shifted due to the occurrence of a sensory stimulus so that, across trials, phase values become highly consistent (phase reset). If one can identify the phase an oscillation is reset to, it is possible to predict when temporal windows of high and low excitability will occur. However, in behavioral experiments the pre-stimulus phase will be different on successive repetitions of the experimental trial, and average performance over many trials will show no signs of the modulation. Here we circumvent this problem by repeatedly presenting an auditory accessory stimulus followed by a visual target stimulus with a temporal delay varied in steps of 2 ms. Performing a discrete time series analysis on SRT as a function of the delay, we provide statistical evidence for the existence of distinct peak spectral components in the power spectrum. These frequencies, although varying across participants, fall within the beta and gamma range (20 to 40 Hz) of neural oscillatory activity observed in neurophysiological studies of multisensory integration. Some evidence for high-theta/alpha activity was found as well. Our results are consistent with the phase reset hypothesis and demonstrate that it is amenable to testing by purely psychophysical methods. Thus, any theory of multisensory processes that connects specific brain states with patterns of saccadic responses should be able to account for traces of oscillatory activity in observable behavior.     

Ezrin Mediates Neuritogenesis via Down-Regulation of RhoA Activity in Cultured Cortical Neurons

Neuronal morphogenesis is implicated in neuronal function and development with rearrangement of cytoskeletal organization. Ezrin, a member of Ezrin/Radixin/Moesin (ERM) proteins links between membrane proteins and actin cytoskeleton, and contributes to maintenance of cellular function and morphology. In cultured hippocampal neurons, suppression of both radixin and moesin showed deficits in growth cone morphology and neurite extensions. Down-regulation of ezrin using siRNA caused impairment of netrin-1-induced axon outgrowth in cultured cortical neurons. However, roles of ezrin in the neuronal morphogenesis of the cultured neurons have been poorly understood. In this report, we performed detailed studies on the roles of ezrin in the cultured cortical neurons prepared from the ezrin knockdown (Vil2^kd/kd) mice embryo that showed a very small amount of ezrin expression compared with the wild-type (Vil2^+/+) neurons. Ezrin was mainly expressed in cell body in the cultured cortical neurons. We demonstrated that the cultured cortical neurons prepared from the Vil2^kd/kd mice embryo exhibited impairment of neuritogenesis. Moreover, we observed increased RhoA activity and phosphorylation of myosin light chain 2 (MLC2), as a downstream effector of RhoA in the Vil2^kd/kd neurons. In addition, inhibition of Rho kinase and myosin II rescued the impairment of neuritogenesis in the Vil2^kd/kd neurons. These data altogether suggest a novel role of ezrin in the neuritogenesis of the cultured cortical neurons through down-regulation of RhoA activity.     

Antioxidative and Proapoptotic Effects of Riluzole on Cultured Cortical Neurons

Abstract : Riluzole is used clinically in patients with amyotrophic lateral sclerosis. As oxidative stress, in addition to excitotoxicity, may be a major mechanism of motoneuron degeneration in patients with amyotrophic lateral sclerosis, we examined whether riluzole protects against nonexcitotoxic oxidative injury. Probably reflecting its weak antiexcitotoxic effects, riluzole (1-30 μ M ) attenuated submaximal neuronal death induced by 24-h exposure to 30 μ M kainate or NMDA, but not that by 100 μ M NMDA, in cortical cultures. Riluzole also attenuated nonexcitotoxic oxidative injury induced by exposure to FeCl₃ in the presence of MK-801 and CNQX. Consistent with its antioxidative effects, riluzole reduced Fe³⁺-induced lipid peroxidation, and inhibited cytosolic phospholipase A₂. By contrast, riluzole did not attenuate neuronal apoptosis induced by staurosporine. Rather unexpectedly, 24-48-h exposure to 100-300 μ M riluzole induced neuronal death accompanied by nuclear and DNA fragmentations, which was attenuated by caspase inhibitor carbobenzyloxy-Val-Ala-Asp-fluoromethyl ketone but not by protein synthesis inhibitor cycloheximide. The present study demonstrates that riluzole has direct antioxidative actions, perhaps in part by inhibiting phospholipase A₂. However, in the same neurons, riluzole paradoxically induces neuronal apoptosis in a caspase-sensitive manner. Considering current clinical use of riluzole, further studies are warranted to investigate its potential cytolethal effects.     

温度对神经元网络活动的影响

利用多通道微电极阵列上培养的海马神经元网络,通过分析28～41℃范围内不同温度时网络自发放电频率．幅度和相邻峰电位时间间隔(interspike interval,ISI)的变化,探讨了温度对网络活动的影响。发现温度升高过程中放电频率、幅度呈不同程度增大的趋势,峰电位数目的变化程度随其间隔的增大呈减小趋势。结果表明体外培养的胎鼠海马神经元的网络活动具有温度敏感性。     

Protective Effects of Humanin on Okadaic Acid-Induced Neurotoxicities in Cultured Cortical Neurons

Neurofibrillary tangles are pathological hallmarks of Alzheimer’s disease (AD), which are mostly composed of hyperphosphorylated tau and directly correlate with dementia in AD patients. Okadaic acid (OA), a toxin extracted from marine life, can specifically inhibit protein phosphatases (PPs), including PP1 and Protein phosphatase 2A (PP2A), resulting in tau hyperphosphorylation. Humanin (HN), a peptide of 24 amino acids, was initially reported to protect neurons from AD-related cell toxicities. The present study was designed to test if HN could attenuate OA-induced neurotoxicities, including neural insults, apoptosis, autophagy, and tau hyperphosphorylation. We found that administration of OA for 24 h induced neuronal insults, including lactate dehydrogenase released, decreased of cell viability and numbers of living cells, neuronal apoptosis, cells autophagy and tau protein hyperphosphorylation. Pretreatment of cells with HN produced significant protective effects against OA-induced neural insults, apoptosis, autophagy and tau hyperphosphorylation. We also found that OA treatment inhibited PP2A activity and HN pretreatment significantly attenuated the inhibitory effects of OA. This study demonstrated for the first time that HN protected cortical neurons against OA-induced neurotoxicities, including neuronal insults, apoptosis, autophagy, and tau hyperphosphorylation. The mechanisms underlying the protections of HN may involve restoration of PP2A activity.     

Encoding of Time-varying Stimuli in Populations of Cultured Neurons

We wondered whether random populations of dissociated cultured cortical neurons, despite of their lack of structure and/or regional specialization, are capable of modulating their neural activity as the effect of a time-varying stimulation – a simulated ‘sensory’ afference. More specifically, we used localized low-frequency, non-periodic trains of stimuli to simulate sensory afferences, and asked how much information about the original trains of stimuli could be extracted from the neural activity recorded at the different sites. Furthermore, motivated by the results of studies performed both in vivo and in vitro on different preparations, which suggested that isolated spikes and bursts may play different roles in coding time-varying signals, we explored the amount of such ‘sensory’ information that could be associated to these different firing modes. Finally, we asked whether and how such ‘sensory’ information is transferred from the sites of stimulation (i.e., the ‘sensory’ areas), to the other regions of the neural populations. To do this we applied stimulus reconstruction techniques and information theoretic concepts that are typically used to investigate neural coding in sensory systems. Our main results are that (1) slow variations of the rate of stimulation are coded into isolated spikes and in the time of occurrence of bursts (but not in the bursts’ temporal structure); (2) increasing the rate of stimulation has the effect of increasing the proportion of isolated spikes in the average evoked response and their importance in coding for the stimuli; and, (3) the ability to recover the time course of the pattern of stimulation is strongly related to the degree of functional connectivity between stimulation and recording sites. These observations parallel similar findings in intact nervous systems regarding the complementary roles of bursts and tonic spikes in encoding sensory information. Our results also have interesting implications in the field of neuro-robotic interfaces. In fact, the ability of populations of neurons to code information is a prerequisite for obtaining hybrid systems, in which neuronal populations are used to control external devices.     

Thoughts of Death Modulate Psychophysical and Cortical Responses to Threatening Stimuli

Existential social psychology studies show that awareness of one''s eventual death profoundly influences human cognition and behaviour by inducing defensive reactions against end-of-life related anxiety. Much less is known about the impact of reminders of mortality on brain activity. Therefore we explored whether reminders of mortality influence subjective ratings of intensity and threat of auditory and painful thermal stimuli and the associated electroencephalographic activity. Moreover, we explored whether personality and demographics modulate psychophysical and neural changes related to mortality salience (MS). Following MS induction, a specific increase in ratings of intensity and threat was found for both nociceptive and auditory stimuli. While MS did not have any specific effect on nociceptive and auditory evoked potentials, larger amplitude of theta oscillatory activity related to thermal nociceptive activity was found after thoughts of death were induced. MS thus exerted a top-down modulation on theta electroencephalographic oscillatory amplitude, specifically for brain activity triggered by painful thermal stimuli. This effect was higher in participants reporting higher threat perception, suggesting that inducing a death-related mind-set may have an influence on body-defence related somatosensory representations.     

Ethanol''s Effects on Cortical Adenylate Cyclase Activity

The effects of ethanol on beta-adrenergic receptor-coupled adenylate cyclase (AC) of mouse cerebral cortex were examined. The addition of ethanol (20-500 mM) to incubation mixtures containing cortical membranes demonstrated that ethanol could increase AC activity and potentiate the stimulatory effects of guanylyl-imidodiphosphate [Gpp(NH)p] on AC activity. Ethanol increased the rate of activation of AC by guanine nucleotides and concomitantly decreased the EC50 for magnesium required to achieve maximal stimulation of cortical AC. The EC50 values for Gpp(NH)p and isoproterenol stimulation of AC activity were also altered by ethanol. Ethanol was capable of stimulating AC extracted by use of digitonin. The AC activity in the digitonin extract was no longer sensitive to the addition of Gpp(NH)p or NaF, but was still stimulated by ethanol. We propose multiple sites of action for ethanol in stimulating cortical AC activity. These sites include actions at the beta-adrenergic receptor, at the G/F coupling proteins, and at the catalytic unit of cortical AC. Comparison of ethanol's actions on cortical beta receptor coupled AC activity with prior reported actions of ethanol on striatal dopamine (DA)-sensitive AC indicated differential sensitivities of these two AC systems to ethanol. These differences may be determined by specific coupling characteristics of the striatal and cortical AC systems or by differences in the plasma membranes in which striatal and cortical AC systems are located.     

Task-Dependent Changes in Cross-Level Coupling between Single Neurons and Oscillatory Activity in Multiscale Networks

Understanding the principles governing the dynamic coordination of functional brain networks remains an important unmet goal within neuroscience. How do distributed ensembles of neurons transiently coordinate their activity across a variety of spatial and temporal scales? While a complete mechanistic account of this process remains elusive, evidence suggests that neuronal oscillations may play a key role in this process, with different rhythms influencing both local computation and long-range communication. To investigate this question, we recorded multiple single unit and local field potential (LFP) activity from microelectrode arrays implanted bilaterally in macaque motor areas. Monkeys performed a delayed center-out reach task either manually using their natural arm (Manual Control, MC) or under direct neural control through a brain-machine interface (Brain Control, BC). In accord with prior work, we found that the spiking activity of individual neurons is coupled to multiple aspects of the ongoing motor beta rhythm (10–45 Hz) during both MC and BC, with neurons exhibiting a diversity of coupling preferences. However, here we show that for identified single neurons, this beta-to-rate mapping can change in a reversible and task-dependent way. For example, as beta power increases, a given neuron may increase spiking during MC but decrease spiking during BC, or exhibit a reversible shift in the preferred phase of firing. The within-task stability of coupling, combined with the reversible cross-task changes in coupling, suggest that task-dependent changes in the beta-to-rate mapping play a role in the transient functional reorganization of neural ensembles. We characterize the range of task-dependent changes in the mapping from beta amplitude, phase, and inter-hemispheric phase differences to the spike rates of an ensemble of simultaneously-recorded neurons, and discuss the potential implications that dynamic remapping from oscillatory activity to spike rate and timing may hold for models of computation and communication in distributed functional brain networks.     

Robustness of Oscillatory Behavior in Correlated Networks

Understanding network robustness against failures of network units is useful for preventing large-scale breakdowns and damages in real-world networked systems. The tolerance of networked systems whose functions are maintained by collective dynamical behavior of the network units has recently been analyzed in the framework called dynamical robustness of complex networks. The effect of network structure on the dynamical robustness has been examined with various types of network topology, but the role of network assortativity, or degree–degree correlations, is still unclear. Here we study the dynamical robustness of correlated (assortative and disassortative) networks consisting of diffusively coupled oscillators. Numerical analyses for the correlated networks with Poisson and power-law degree distributions show that network assortativity enhances the dynamical robustness of the oscillator networks but the impact of network disassortativity depends on the detailed network connectivity. Furthermore, we theoretically analyze the dynamical robustness of correlated bimodal networks with two-peak degree distributions and show the positive impact of the network assortativity.     

Excessive Glutamate Stimulation Impairs ACE2 Activity Through ADAM17-Mediated Shedding in Cultured Cortical Neurons

The excitotoxicity of glutamate plays an important role in the progression of various neurological disorders via participating in inflammation and neuronal damage. In this study, we identified the role of excessive glutamate stimulation in the modulation of angiotensin-converting enzyme type 2 (ACE2), a critical component in the compensatory axis of the renin–angiotensin system (RAS). In primary cultured cortical neurons, high concentration of glutamate (100 µM) significantly reduced the enzymatic activity of ACE2. The elevated activity of ADAM17, a member of the ‘A Disintegrin And Metalloprotease’ (ADAM) family, was found to contribute to this glutamate-induced ACE2 down-regulation. The decrease of ACE2 activity could be prevented by pre-treatment with antagonists targeting ionotropic glutamate receptors. In addition, the glutamate-induced decrease in ACE2 activity was significantly attenuated when the neurons were co-treated with MitoTEMPOL or blockers that target oxidative stress-mediated signaling pathway. In summary, our study reveals a strong relationship between excessive glutamate stimulation and ADAM17-mediated impairment in ACE2 activity, suggesting a possible cross-talk between glutamate-induced excitotoxicity and dysregulated RAS.     

Cortical Influences on Neurons of the Lateral Reticular Nucleus Responding to Noxious Stimuli

The effect of frontoparietal sensorimotor (FPSM) cortex stimulation on both the spontaneous and the noxious evoked activity of neurons in the lateral reticular nucleus (LRN) was tested in barbiturate-anesthetized rats. Ninety-three LRN neurons that responded to a noxious heat stimulus (HS) were recorded (72% antidromically fired from the cerebellum). Of these, 66 neurons altered their spontaneous firing rates in response to cortical stimulation. Two patterns of responses were found: either an excitation followed by a suppression of spontaneous activity (52 neurons), or a pure suppression of spontaneous activity lasting 50-400 msec (14 neurons). In 46 of these neurons, it was found that cortical stimulation reduced HS-evoked activity to near the baseline level. Furthermore, it was found that when applied after a prolonged cortical stimulation, the HS was ineffective. It is concluded that FPSM cortex can influence nociceptive information in LRN neurons that respond to its stimulation, possibly interfering with the mechanisms underlying stimulation-produced analgesia (SPA). In this context, it is proposed that the cortex can modulate the activity of LRN neurons that activate, through local loops, a descending antinociceptive system and also a separate projection system to the cerebellum.     

Lactacystin Stimulates Stellation of Cultured Rat Cortical Astrocytes

Proteasome inhibition has been observed in many neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease. Here, the effect of proteasome inhibition on the morphology of cultured rat cortical astrocytes was investigated. Increasing evidence suggests that the function of astrocytes is related closely to its morphology. Lactacystin, a specific inhibitor of the 20S proteasome, can induce astrocytes stellation in a dose dependent manner and reorganize the cytoskeleton of astrocytes. Furthermore, decreased levels of expression of Rho A, total Akt, and Phospho-Akt were found in the process of astrocytes stellation and lysophosphatidic acid, an activator of Rho A, can largely reverse the astrocytes stellation caused by lactacystin. This suggests that proteasome inhibition in astrocytes could stabilize signals of morphological changes that might be processed through Rho and Akt signaling cascade. Our results suggest that proteasome inhibition might function as a factor regulating astrocytes morphology in some pathophysiological conditions. Qing-Guo Ren and Ying Yu contributed equally to this work.