Activity-dependent Stimulation Research Articles

Delay-Dependent Response in Weakly Electric Fish under Closed-Loop Pulse Stimulation.

In this paper, we apply a real time activity-dependent protocol to study how freely swimming weakly electric fish produce and process the timing of their own electric signals. Specifically, we address this study in the elephant fish, Gnathonemus petersii, an animal that uses weak discharges to locate obstacles or food while navigating, as well as for electro-communication with conspecifics. To investigate how the inter pulse intervals vary in response to external stimuli, we compare the response to a simple closed-loop stimulation protocol and the signals generated without electrical stimulation. The activity-dependent stimulation protocol explores different stimulus delivery delays relative to the fish’s own electric discharges. We show that there is a critical time delay in this closed-loop interaction, as the largest changes in inter pulse intervals occur when the stimulation delay is below 100 ms. We also discuss the implications of these findings in the context of information processing in weakly electric fish.

Characterisation of neurons derived from a cortical human neural stem cell line CTX0E16.

IntroductionConditionally immortalised human neural progenitor cells (hNPCs) represent a robust source of native neural cells to investigate physiological mechanisms in both health and disease. However, in order to recognise the utility of such cells, it is critical to determine whether they retain characteristics of their tissue of origin and generate appropriate neural cell types upon differentiation. To this end, we have characterised the conditionally immortalised, cortically-derived, human NPC line, CTX0E16, investigating the molecular and cellular phenotype of differentiated neurons to determine whether they possess characteristics of cortical glutamatergic neurons.MethodsDifferentiated CTX0E16 cells were characterised by assessing expression of several neural fates markers, and examination of developing neuronal morphology. Expression of neurotransmitter receptors, signalling proteins and related proteins were assessed by q- and RT-PCR and complemented by Ca2+ imaging, electrophysiology and assessment of ERK signalling in response to neurotransmitter ligand application. Finally, differentiated neurons were assessed for their ability to form putative synapses and to respond to activity-dependent stimulation.ResultsDifferentiation of CTX0E16 hNPCs predominately resulted in the generation of neurons expressing markers of cortical and glutamatergic (excitatory) fate, and with a typical polarized neuronal morphology. Gene expression analysis confirmed an upregulation in the expression of cortical, glutamatergic and signalling proteins following differentiation. CTX0E16 neurons demonstrated Ca2+ and ERK1/2 responses following exogenous neurotransmitter application, and after 6 weeks displayed spontaneous Ca2+ transients and electrophysiological properties consistent with that of immature neurons. Differentiated CTX0E16 neurons also expressed a range of pre- and post-synaptic proteins that co-localized along distal dendrites, and moreover, displayed structural plasticity in response to modulation of neuronal activity.ConclusionsTaken together, these findings demonstrate that the CTX0E16 hNPC line is a robust source of cortical neurons, which display functional properties consistent with a glutamatergic phenotype. Thus CTX0E16 neurons can be used to study cortical cell function, and furthermore, as these neurons express a range of disease-associated genes, they represent an ideal platform with which to investigate neurodevelopmental mechanisms in native human cells in health and disease.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0136-8) contains supplementary material, which is available to authorized users.

Neuroplasticity subserving the operation of brain–machine interfaces

Neuroplasticity is key to the operation of brain machine interfaces (BMIs)—a direct communication pathway between the brain and a man-made computing device. Whereas exogenous BMIs that associate volitional control of brain activity with neurofeedback have been shown to induce long lasting plasticity, endogenous BMIs that use prolonged activity-dependent stimulation – and thus may curtail the time scale that governs natural sensorimotor integration loops – have been shown to induce short lasting plasticity. Here we summarize recent findings from studies using both categories of BMIs, and discuss the fundamental principles that may underlie their operation and the longevity of the plasticity they induce. We draw comparison to plasticity mechanisms known to mediate natural sensorimotor skill learning and discuss principles of homeostatic regulation that may constrain endogenous BMI effects in the adult mammalian brain. We propose that BMIs could be designed to facilitate structural and functional plasticity for the purpose of re-organization of target brain regions and directed augmentation of sensorimotor maps, and suggest possible avenues for future work to maximize their efficacy and viability in clinical applications.

Volitional muscle activity paired with transcranial magnetic stimulation increases corticospinal excitability.

Studies of activity-dependent stimulation in non-human primates suggest that pairing each instance of volitional muscle activity with immediate intracortical stimulation causes long-term-potentiation-like effects. This technique holds promise for clinical rehabilitation, yet few investigators have tested activity-dependent stimulation in human subjects. In addition, no one has studied activity-dependent stimulation on the cortical representation for two separate target muscles in human subjects. We hypothesized that 40 min of transcranial magnetic stimulation (TMS) triggered from ballistic muscle activity at a mean repetition rate of 1 Hz would cause greater increases in corticospinal excitability than TMS-cued muscle activity, and that these changes would be specific to the muscle of study. Ten healthy human subjects participated in 4 separate sessions in this crossover study: (1) visually cued volitional activation of the abductor pollicis brevis (APB) muscle triggering TMS (APB-Triggered TMS), (2) volitional activation of APB in response to TMS delivered from a recording of the prior APB-Triggered TMS session (TMS-Cued APB), (3) visually cued volitional activation of the extensor digitorum (ED) triggering TMS (ED-Triggered TMS), and (4) volitional activation of ED in response to TMS delivered from a recording of the prior ED-Triggered TMS session (TMS-Cued ED). Contrary to our hypothesis, we discovered evidence of increased corticospinal excitability for all conditions as measured by change in area of the motor evoked potential. We conclude that single TMS pulses paired either before or after muscle activity may increase corticospinal excitability and that further studies are needed to clarify the optimal time window for inducing neural plasticity with activity-dependent stimulation. These findings will inform the design of future activity-dependent stimulation protocols for clinical rehabilitation.

Applications of closed-loop approaches to therapeutic brain stimulation

Event Abstract Back to Event Applications of closed-loop approaches to therapeutic brain stimulation Randolph Nudo1* 1 University of Kansas Medical Center, Department of Rehabilitation Medicine, United States Artificial modulation of neural activity following acquired central nervous system injuries has received increasing attention over the past several years as therapeutic interventions. Examples include numerous non-invasive approaches, including transcranial magnetic stimulation or transcranial direct current stimulation to excite or suppress neuronal activity in the damaged or intact cortex after stroke. More invasive technologies, such as epidural stimulation, vagus nerve stimulation and deep brain stimulation, also have demonstrated promising results in neurological populations. Most of these neuromodulation therapies have employed continuous stimulation, and indications in research settings continue to expand for these neuromodulatory therapies. However, recently, various implementations of closed-loop approaches have begun to attract attention for altering neuronal function. These closed-loop modalities are often described as responsive neurostimulation, adaptive brain stimulation or activity-dependent stimulation (ADS), and typically involve invasive technologies. For example, subdural or epidural electrodes have been used to record electroencephalographic activity, triggering brain stimulation when epileptiform discharges are detected to suppress a seizure. In animal models, it has long been known that synaptic facilitation can be induced with particular patterns of open-loop stimulation (e.g., theta burst stimulation), or by spike-timing dependent stimulation. Recent studies using ADS have been used in our laboratory to reinforce synaptic activity in specific cortico-cortical pathways following cortical injury. Such closed-loop stimulation resulted in rapid recovery of sensorimotor skills in rats (Guggenmos et al. 2013, PNAS). We have recently found that cortico-cortical functional connectivity can be reinforced in either intact or injured brains, even under anesthesia, providing a convenient platform for testing hypotheses regarding spike-timing dependent plasticity in neuronal systems. These results suggest that cortico-cortical entrainment immediately after cortical injury can be evoked more readily than in healthy brains. However, cortico-cortical potentiation appears to differ depending upon pre-existing strength of anatomical connectivity between the areas. These results are placed in the context of diaschisis-like phenomena that result from focal injury. Conference: 2015 International Workshop on Clinical Brain-Machine Interfaces (CBMI2015), Tokyo, Japan, 13 Mar - 15 Mar, 2015. Presentation Type: Oral presentation / lecture Topic: Clinical Brain-Machine Interfaces Citation: Nudo R (2015). Applications of closed-loop approaches to therapeutic brain stimulation. Conference Abstract: 2015 International Workshop on Clinical Brain-Machine Interfaces (CBMI2015). doi: 10.3389/conf.fnhum.2015.218.00015 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 23 Apr 2015; Published Online: 29 Apr 2015. * Correspondence: PhD. Randolph Nudo, University of Kansas Medical Center, Department of Rehabilitation Medicine, Kansas City, KS, 66103, United States, randynudo@gmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Randolph Nudo Google Randolph Nudo Google Scholar Randolph Nudo PubMed Randolph Nudo Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Restoring motor function with bidirectional neural interfaces.

Closed-loop brain-computer interfaces have bidirectional connections that allow activity-dependent stimulation of the brain, spinal cord, or muscles. Such bidirectional brain-computer interfaces (BBCI) have three major applications that can be used to restore lost motor function. First, the brain could learn to incorporate a long-term artificial recurrent connection into normal behavior, exploiting the brain's ability to adapt to consistent sensorimotor conditions. The obvious clinical application for restoring motor function is to use an artificial recurrent connection to bridge a lost biological connection. Second, activity-dependent stimulation can generate synaptic plasticity on the cellular level. The corresponding clinical application is to strengthen weakened neural connections, such as occur in stroke. A third application involves delivery of activity-dependent deep brain stimulation at subcortical reward sites, which can operantly reinforce the activity that generates the stimulation. The BBCI paradigm has numerous specific applications, depending on the source of the signals and the stimulated targets.

Strengthening of corticospinal connection through recurrent neural interface

Individuals with neural damage hope to regain their impaired functions. However, therapeutic rehabilitation is lacking. Nishimura et al. (2013) have documented that the strength of neural connections between the motor cortex and the spinal cord can be modified using an autonomous recurrent neural interface that delivers electrical stimuli in the spinal cord and this is triggered by action potentials of corticospinal cells during free behavior. This activity-dependent stimulation modifies the strength of the terminal connections of single corticomotoneuronal cells, which is consistent with the bidirectional spike-timing-dependent plasticity rule previously derived from in vitro experiments. The success of our protocol suggests neurorehabilitative treatments could be possible for patients with damaged pathways.

Coupling brain-machine interfaces with cortical stimulation for brain-state dependent stimulation: enhancing motor cortex excitability for neurorehabilitation

Motor recovery after stroke is an unsolved challenge despite intensive rehabilitation training programs. Brain stimulation techniques have been explored in addition to traditional rehabilitation training to increase the excitability of the stimulated motor cortex. This modulation of cortical excitability augments the response to afferent input during motor exercises, thereby enhancing skilled motor learning by long-term potentiation-like plasticity. Recent approaches examined brain stimulation applied concurrently with voluntary movements to induce more specific use-dependent neural plasticity during motor training for neurorehabilitation. Unfortunately, such approaches are not applicable for the many severely affected stroke patients lacking residual hand function. These patients require novel activity-dependent stimulation paradigms based on intrinsic brain activity. Here, we report on such brain state-dependent stimulation (BSDS) combined with haptic feedback provided by a robotic hand orthosis. Transcranial magnetic stimulation (TMS) of the motor cortex and haptic feedback to the hand were controlled by sensorimotor desynchronization during motor-imagery and applied within a brain-machine interface (BMI) environment in one healthy subject and one patient with severe hand paresis in the chronic phase after stroke. BSDS significantly increased the excitability of the stimulated motor cortex in both healthy and post-stroke conditions, an effect not observed in non-BSDS protocols. This feasibility study suggests that closing the loop between intrinsic brain state, cortical stimulation and haptic feedback provides a novel neurorehabilitation strategy for stroke patients lacking residual hand function, a proposal that warrants further investigation in a larger cohort of stroke patients.

Spike-timing-dependent plasticity in primate corticospinal connections induced during free behavior.

Motor learning and functional recovery from brain damage involve changes in the strength of synapticconnections between neurons. Relevant invivo evidence on the underlying cellular mechanisms remains limited and indirect. We found that the strength of neural connections between motor cortex and spinal cord in monkeys can be modified with an autonomous recurrent neural interface that delivers electrical stimuli in the spinal cord triggered by action potentials of corticospinal cells during free behavior. The activity-dependent stimulation modified the strength of the terminal connections of single corticomotoneuronal cells, consistent with a bidirectional spike-timing-dependent plasticity rule previously derived from invitro experiments. For some cells, the changes lasted for days after the end of conditioning, but most effects eventually reverted to preconditioning levels. These results provide direct evidence of corticospinal synaptic plasticity invivo at the level of single neurons induced by normal firing patterns during free behavior.

Behavioral driving through on line monitoring and activity-dependent stimulation in weakly electric fish

This work has been supported by Brazilian agencies FAPESP, CNPq, CAPES and grants MICINN BFU2009-08473 and TIN 2010-19607, Spanish-Brazilian Cooperation PHB2007-0008 and 7a Convocatoria De PROYECTOS de COOPERACION INTERUNIVERSITARIAUAM-SANTANDER con America Latina

Assisted closed-loops for brain-computer interfaces

The dynamic clamp technology to implement artificial membrane or synaptic conductances is one of the most successful examples of closed-loop interactions with the nervous system for observation and control purposes (for a review see [1]). We recently proposed a generalization of the dynamic clamp concept to design goal-driven closed-loop interactions with biological systems beyond electrical stimulation and recording in the context of in vitro electrophysiology and animal ethology [2]. Following the same activity-dependent stimulation approach, we designed an assisted closed-loop (ACL) to optimize the efficiency of brain-computer interfaces (BCI) based on steady state visually evoked potentials (SSVEP) [3]. SSVEP based BCIs use multiple visual stimuli such as LEDs or regions on a screen flickering at different frequencies. A subject's brain generates electrical activity at the same fundamental frequency as the visual stimulus on which the subject has focused his/her attention. The ACL consists in the delivery of online information with regard to the control over the given BCI goal both to the human subject and to the system, resulting in an online adaptation of BCI stimuli properties, in this first proof of concept study the combination of the employed flicker frequencies (Figure ​(Figure1).1). The optimization process is realized by first performing a real-time closed-loop search for the best set of stimulation frequencies to achieve the given BCI goal, which informs the system about the most effective flicker frequencies to produce SSVEPs in a given subject. Secondly, a continuous auditory feedback informs the subject about the actual distance to a predefined detection threshold. Figure 1 The assisted closed-loop (ACL) provides online information to the system about the most effective flicker frequencies and to the human subject about the actual distance to the pre-defined SSVEP detection threshold by a continuous auditory feedback (speaker ... Our results show that this shared online information leads to an enhanced BCI efficiency, as compared to classic BCI protocols, by helping them to reach the BCI goal in their interaction. The analysis also shows that the ACL protocol takes into account interindividual variabilities. In particular, baseline resting state EEG measures seem to predict subjects' BCI performances, which indicates that the ACL based BCIs might be expanded to innovative new diagnostic/therapeutic tools for clinical contexts and as new paradigms for basic research. Taking all together, these results illustrate that assisted closed-loop protocols can reveal dynamics otherwise hidden under traditional stimulation techniques, provide control of regular and pathological states, induce learning processes, bridge between distinct levels of analysis and lead to automation of experiments. The proposed approach might have a large impact for applied uses, such as computer control and biomedical or prosthetic uses, but also in novel paradigms for neuroscientific and biomedical research.

New modalities of brain stimulation for stroke rehabilitation

Stroke is a leading cause of disability, and the number of stroke survivors continues to rise. Traditional neurorehabilitation strategies aimed at restoring function to weakened limbs provide only modest benefit. New brain stimulation techniques designed to augment traditional neurorehabilitation hold promise for reducing the burden of stroke-related disability. Investigators discovered that repetitive transcranial magnetic stimulation (rTMS), transcranial direct current stimulation (tDCS), and epidural cortical stimulation (ECS) can enhance neural plasticity in the motor cortex post-stroke. Improved outcomes may be obtained with activity-dependent stimulation, in which brain stimulation is contingent on neural or muscular activity during normal behavior. We review the evidence for improved motor function in stroke patients treated with rTMS, tDCS, and ECS and discuss the mediating physiological mechanisms. We compare these techniques to activity-dependent stimulation, discuss the advantages of this newer strategy for stroke rehabilitation, and suggest future applications for activity-dependent brain stimulation.

Generalization of the Dynamic Clamp Concept in Neurophysiology and Behavior

The idea of closed-loop interaction in in vitro and in vivo electrophysiology has been successfully implemented in the dynamic clamp concept strongly impacting the research of membrane and synaptic properties of neurons. In this paper we show that this concept can be easily generalized to build other kinds of closed-loop protocols beyond (or in addition to) electrical stimulation and recording in neurophysiology and behavioral studies for neuroethology. In particular, we illustrate three different examples of goal-driven real-time closed-loop interactions with drug microinjectors, mechanical devices and video event driven stimulation. Modern activity-dependent stimulation protocols can be used to reveal dynamics (otherwise hidden under traditional stimulation techniques), achieve control of natural and pathological states, induce learning, bridge between disparate levels of analysis and for a further automation of experiments. We argue that closed-loop interaction calls for novel real time analysis, prediction and control tools and a new perspective for designing stimulus-response experiments, which can have a large impact in neuroscience research.

Real-time control of stepper motors for mechano-sensory stimulation

Mechanical stimulation is widely used to study sensory encoding in the nervous system of living organisms. The stimulation of mechano-receptor neurons is achieved through a large variety of devices that generate movement or vibration. In many situations, a hard real-time (RT) control of the device (in the millisecond time scale) is needed to produce realistic mechanical stimuli. The real-time control can be required to achieve the desired precision in the device or to implement activity-dependent stimulation protocols that imply the detection of physiological events to drive the stimulus in real time. In this paper we show that real-time software technology can be used to control stepper motors for mechano-receptor stimulation, and to implement artificial closed-loops to address the sensory-motor transformation. We illustrate this using as an example the control of a stepper motor to precisely move gravimetric organs in in vitro preparations.

Activity‐dependent control of bulk endocytosis by protein dephosphorylation in central nerve terminals

Bulk endocytosis is the process by which nerve terminals retrieve large amounts of synaptic vesicle membrane during periods of strong stimulation intensity. The process is rapidly activated and is most probably calcium dependent in a similar manner to synaptic vesicle exocytosis. This article briefly summarizes the current knowledge of bulk endocytosis with respect to its activation, kinetics and molecular mechanism. It also presents recent data from our laboratory showing that the dephosphorylation of a group of endocytosis proteins called the dephosphins by the Ca(2+)-dependent protein phosphatase calcineurin is key to the activity-dependent stimulation of the process. Possible downstream effectors of calcineurin are discussed such as the large GTPase dynamin I and its phosphorylation-dependent interaction partner syndapin I.

Glutamate exocytosis from astrocytes controls synaptic strength

The release of transmitters from glia influences synaptic functions. The modalities and physiological functions of glial release are poorly understood. Here we show that glutamate exocytosis from astrocytes of the rat hippocampal dentate molecular layer enhances synaptic strength at excitatory synapses between perforant path afferents and granule cells. The effect is mediated by ifenprodil-sensitive NMDA ionotropic glutamate receptors and involves an increase of transmitter release at the synapse. Correspondingly, we identify NMDA receptor 2B subunits on the extrasynaptic portion of excitatory nerve terminals. The receptor distribution is spatially related to glutamate-containing synaptic-like microvesicles in the apposed astrocytic processes. This glial regulatory pathway is endogenously activated by neuronal activity-dependent stimulation of purinergic P2Y1 receptors on the astrocytes. Thus, we provide the first combined functional and ultrastructural evidence for a physiological control of synaptic activity via exocytosis of glutamate from astrocytes.

CO, an Immuno- and Neuromodulator

Two groups report signaling mediated by carbon monoxide (CO), a gas much better known for its ability to displace oxygen in hemoglobin, which can result in death. Morse et al. suggest that CO produced by stress-stimulated heme oxygenase-1 (HO-1) may inhibit cytokine production, resulting in a protective effect in a mouse model of sepsis. Exposure of mice to CO before injection with lipopolysaccharide (LPS) decreased morbidity and decreased circulating cytokine levels [interleukin-6 (IL-6) and IL-1β]. Analysis of CO-exposed cultured macrophages suggested that the decrease in cytokine production was due to decreased transcription. Analysis of reporter gene expression and electrophoretic mobility shift assays indicated that CO disrupted activity of the AP-1 transcription factor complex, specifically the complex containing c-Fos. Finally, CO appeared to inhibit Jun-N-terminal kinase signaling, because the Jnk–/– mice were not rescued from injected LPS-induced death by CO, nor were inflammatory cytokine levels triggered by nonlethal doses of LPS decreased by treatment with CO. Boehning et al. show that in neurons CO production is stimulated by activity-dependent phosphorylation of HO-2. Using in vitro phosphorylation assays, phosphopeptide mapping, and site-directed mutagenesis, the authors determined that a single serine in HO-2 was phosphorylated by casein kinase 2 (CK2). In hippocampal cultures and cultured olfactory neurons, HO-2 activity and phosphorylation were stimulated by phorbol esters and odorant stimulation, respectively. Pharmacologic analysis suggested that the upstream pathways leading to CK stimulation of HO-2 were different in the two neuronal cultures. The final system where CO-mediated neurotransmission was analyzed was dissected internal anal sphincter (IAS) preparations, in which relaxation is known to be mediated by nonadrenergic noncholinergic transmission involving both nitric oxide and CO. Treatment with CK2 inhibitors reduced IAS relaxation in response to electric field stimulation, and the effectiveness of the tested compounds correlated with their potency as CK2 inhibitors. Thus, neuronal activity-dependent stimulation of CK2 appears to be a common mechanism by which the synthesis of CO can be achieved in neurons.D. Morse, S. E. Pischke, Z. Zhou, R. J. Davis, R. A. Flavell, T. Loop, S. L. Otterbein, L. E. Otterbein, A. M. K. Choi, Suppression of inflammatory cytokine production by carbon monoxide involves the JNK pathway and AP-1. J. Biol. Chem. 278, 36993-36998 (2003). [Abstract] [Full Text]D. Boehning, C. Moon, S. Sharma, K. J. Hunt, L. D. Hester, G. V. Ronnett, D. Shugar, S. H. Snyder, Carbon monoxide neurotransmission activated by CK2 phosphorylation of heme oxygenase-2. Neuron 40, 129-137 (2003). [Online Journal]

CO, an Immuno- and Neuromodulator

Two groups report signaling mediated by carbon monoxide (CO), a gas much better known for its ability to displace oxygen in hemoglobin, which can result in death. Morse et al. suggest that CO produced by stress-stimulated heme oxygenase-1 (HO-1) may inhibit cytokine production, resulting in a protective effect in a mouse model of sepsis. Exposure of mice to CO before injection with lipopolysaccharide (LPS) decreased morbidity and decreased circulating cytokine levels [interleukin-6 (IL-6) and IL-1β]. Analysis of CO-exposed cultured macrophages suggested that the decrease in cytokine production was due to decreased transcription. Analysis of reporter gene expression and electrophoretic mobility shift assays indicated that CO disrupted activity of the AP-1 transcription factor complex, specifically the complex containing c-Fos. Finally, CO appeared to inhibit Jun-N-terminal kinase signaling, because the Jnk –/– mice were not rescued from injected LPS-induced death by CO, nor were inflammatory cytokine levels triggered by nonlethal doses of LPS decreased by treatment with CO. Boehning et al. show that in neurons CO production is stimulated by activity-dependent phosphorylation of HO-2. Using in vitro phosphorylation assays, phosphopeptide mapping, and site-directed mutagenesis, the authors determined that a single serine in HO-2 was phosphorylated by casein kinase 2 (CK2). In hippocampal cultures and cultured olfactory neurons, HO-2 activity and phosphorylation were stimulated by phorbol esters and odorant stimulation, respectively. Pharmacologic analysis suggested that the upstream pathways leading to CK stimulation of HO-2 were different in the two neuronal cultures. The final system where CO-mediated neurotransmission was analyzed was dissected internal anal sphincter (IAS) preparations, in which relaxation is known to be mediated by nonadrenergic noncholinergic transmission involving both nitric oxide and CO. Treatment with CK2 inhibitors reduced IAS relaxation in response to electric field stimulation, and the effectiveness of the tested compounds correlated with their potency as CK2 inhibitors. Thus, neuronal activity-dependent stimulation of CK2 appears to be a common mechanism by which the synthesis of CO can be achieved in neurons. D. Morse, S. E. Pischke, Z. Zhou, R. J. Davis, R. A. Flavell, T. Loop, S. L. Otterbein, L. E. Otterbein, A. M. K. Choi, Suppression of inflammatory cytokine production by carbon monoxide involves the JNK pathway and AP-1. J. Biol. Chem. 278 , 36993-36998 (2003). [Abstract] [Full Text] D. Boehning, C. Moon, S. Sharma, K. J. Hunt, L. D. Hester, G. V. Ronnett, D. Shugar, S. H. Snyder, Carbon monoxide neurotransmission activated by CK2 phosphorylation of heme oxygenase-2. Neuron 40 , 129-137 (2003). [Online Journal]

Experience-Dependent Modifications in MAP2 Phosphorylation in Rat Olfactory Bulb

Microtubule-associated protein 2 (MAP2) is a neuron-specific cytoskeletal protein, enriched in dendrites and cell bodies, that helps determine dendritic shape. MAP2 regulates microtubule stability in a phosphorylation-dependent manner. The present study used immunocytochemistry with phosphoepitope-specific and phosphorylation state-independent antibodies to examine experience-dependent changes in MAP2 expression during postnatal development of the olfactory bulb. Our results demonstrate that immunoreactivity reflecting total MAP2 expression reaches a maximal level by postnatal day 20 (P20). The degree of staining for phosphoindependent forms of MAP2 is relatively unaffected by blocking odorant passage to one half the nasal epithelium via unilateral naris closure, a manipulation that attenuates physiological activity in the bulb. However, olfactory restriction from P1 dramatically reduces immunoreactivity for antibody AP18, which recognizes MAP2 only when phosphorylated on Ser136. Quantification of staining in the granule cell layer indicates that the greatest difference (64%) between control and experimental bulbs occurs after occlusion from P1 to P30 compared with animals deprived from P1 to P10 or P1 to P20. The shift in MAP2 phosphorylation occurs even when deprivation is delayed until P30, after the sensitive period for experience-dependent changes in bulb volume. Thus, the degree of the phosphorylation shift depends on the duration but not the time of onset of naris closure. Because staining for phosphorylation-independent forms of MAP2 is unchanged by naris closure, the total amount of the protein per unit area is probably not significantly altered. However, the large reductions of AP18-immunoreactivity in the bulb after olfactory restriction suggest that there is an activity-dependent stimulation of MAP2 phosphorylation.