Coupling of total hemoglobin concentration,oxygenation, and neural activity in rat somatosensory cortex

Recent advances in brain imaging techniques, including functional magnetic resonance imaging (fMRI), offer great promise for noninvasive mapping of brain function. However, the indirect nature of the imaging signals to the underlying neural activity limits the interpretation of the resulting maps. The present report represents the first systematic study with sufficient statistical power to quantitatively characterize the relationship between changes in blood oxygen content and the neural spiking and synaptic activity. Using two-dimensional optical measurements of hemodynamic signals, simultaneous recordings of neural activity, and an event-related stimulus paradigm, we demonstrate that (1) there is a strongly nonlinear relationship between electrophysiological measures of neuronal activity and the hemodynamic response, (2) the hemodynamic response continues to grow beyond the saturation of electrical activity, and (3) the initial increase in deoxyhemoglobin that precedes an increase in blood volume is counterbalanced by an equal initial decrease in oxyhemoglobin.     

A New Computational Model for Neuro-Glio-Vascular Coupling: Astrocyte Activation Can Explain Cerebral Blood Flow Nonlinear Response to Interictal Events

Developing a clear understanding of the relationship between cerebral blood flow (CBF) response and neuronal activity is of significant importance because CBF increase is essential to the health of neurons, for instance through oxygen supply. This relationship can be investigated by analyzing multimodal (fMRI, PET, laser Doppler…) recordings. However, the important number of intermediate (non-observable) variables involved in the underlying neurovascular coupling makes the discovery of mechanisms all the more difficult from the sole multimodal data. We present a new computational model developed at the population scale (voxel) with physiologically relevant but simple equations to facilitate the interpretation of regional multimodal recordings. This model links neuronal activity to regional CBF dynamics through neuro-glio-vascular coupling. This coupling involves a population of glial cells called astrocytes via their role in neurotransmitter (glutamate and GABA) recycling and their impact on neighboring vessels. In epilepsy, neuronal networks generate epileptiform discharges, leading to variations in astrocytic and CBF dynamics. In this study, we took advantage of these large variations in neuronal activity magnitude to test the capacity of our model to reproduce experimental data. We compared simulations from our model with isolated epileptiform events, which were obtained in vivo by simultaneous local field potential and laser Doppler recordings in rats after local bicuculline injection. We showed a predominant neuronal contribution for low level discharges and a significant astrocytic contribution for higher level discharges. Besides, neuronal contribution to CBF was linear while astrocytic contribution was nonlinear. Results thus indicate that the relationship between neuronal activity and CBF magnitudes can be nonlinear for isolated events and that this nonlinearity is due to astrocytic activity, highlighting the importance of astrocytes in the interpretation of regional recordings.     

Transporter mediated regulation of neural communication

A tight link exists between neuronal activity and energy metabolism. This relationship was first proposed by Roy and Sherrington who suggested that brain possesses intrinsic mechanisms to regulate the availability of energy substrates in register with local variations of functional activity. This concept was later confirmed by Sokoloff and colleagues who demonstrated that increased neuronal activity led to increased glucose utilization in almost any areas of the brain tested. Despite wide acceptance of this concept, the cellular and molecular mechanisms that underlie this close relationship between neuronal activity and energy metabolism have remained largely unknown. The extensive analysis carried out by our group will be discussed. Astrocytes appear to be the key cells that operate the coupling between synaptic activity and glucose utilization. Indeed both in vitro and in vivo evidences indicate that astrocytes can detect synaptically released glutamate through sodium-coupled uptake operated by glutamate transporters. Disruption of sodium homeostasis activates the energy-demanding Na-K-ATPase which promotes glucose uptake and lactate production. Relevance of these findings to functional brain imaging will be discussed.     

What does fMRI tell us about neuronal activity?

In recent years, cognitive neuroscientists have taken great advantage of functional magnetic resonance imaging (fMRI) as a non-invasive method of measuring neuronal activity in the human brain. But what exactly does fMRI tell us? We know that its signals arise from changes in local haemodynamics that, in turn, result from alterations in neuronal activity, but exactly how neuronal activity, haemodynamics and fMRI signals are related is unclear. It has been assumed that the fMRI signal is proportional to the local average neuronal activity, but many factors can influence the relationship between the two. A clearer understanding of how neuronal activity influences the fMRI signal is needed if we are correctly to interpret functional imaging data.     

Bayesian comparison of neurovascular coupling models using EEG-fMRI

Functional magnetic resonance imaging (fMRI), with blood oxygenation level-dependent (BOLD) contrast, is a widely used technique for studying the human brain. However, it is an indirect measure of underlying neuronal activity and the processes that link this activity to BOLD signals are still a topic of much debate. In order to relate findings from fMRI research to other measures of neuronal activity it is vital to understand the underlying neurovascular coupling mechanism. Currently, there is no consensus on the relative roles of synaptic and spiking activity in the generation of the BOLD response. Here we designed a modelling framework to investigate different neurovascular coupling mechanisms. We use Electroencephalographic (EEG) and fMRI data from a visual stimulation task together with biophysically informed mathematical models describing how neuronal activity generates the BOLD signals. These models allow us to non-invasively infer the degree of local synaptic and spiking activity in the healthy human brain. In addition, we use Bayesian model comparison to decide between neurovascular coupling mechanisms. We show that the BOLD signal is dependent upon both the synaptic and spiking activity but that the relative contributions of these two inputs are dependent upon the underlying neuronal firing rate. When the underlying neuronal firing is low then the BOLD response is best explained by synaptic activity. However, when the neuronal firing rate is high then both synaptic and spiking activity are required to explain the BOLD signal.     

Energy on demand: a mechanism for astrocyte–neuron metabolic coupling

A tight link exists between neuronal activity and energy metabolism. This relationship was first proposed by Roy and Sherrington who suggested that brain possesses intrinsic mechanisms to regulate the availability of energy substrates in register with local variations of functional activity. This concept was later confirmed by Sokoloff and colleagues who demonstrated that increased neuronal activity led to increased glucose utilization in almost any areas of the brain tested. Despite wide acceptance of this concept, the cellular and molecular mechanisms that underlie this close relationship between neuronal activity and energy metabolism have remained largely unknown. The extensive analysis carried out by our group will be discussed. Astrocytes appear to be the key cells that operate the coupling between synaptic activity and glucose utilization. Indeed both in vitro and in vivo evidences indicate that astrocytes can detect synaptically released glutamate through sodium‐coupled uptake operated by glutamate transporters. Disruption of sodium homeostasis activates the energy‐demanding Na‐K‐ATPase which promotes glucose uptake and lactate production. Relevance of these findings to functional brain imaging will be discussed.     

Neuronal population activity and functional imaging.

Human functional brain imaging detects blood flow changes that are thought to reflect the activity of neuronal populations and, thus, the responses of neurons that carry behaviourally relevant information. Since this relationship is poorly understood, we explored the link between the activity of single neurons and their neuronal population. The functional imaging results were in good agreement with levels of population activation predicted from the known effects of sensory stimulation, learning and attention on single cortical neurons. However, the nature of the relationship between population activation and single neuron firing was very surprising. Population activation was strongly influenced by those neurons firing at low rates and so was very sensitive to the baseline or 'spontaneous' firing rate. When neural representations were sparse and neurons were tuned to several stimulus dimensions, population activation was hardly influenced by the few neurons whose firing was most strongly modulated by the task or stimulus. Measures of population activation could miss changes in information processing given simultaneous changes in neurons' baseline firing, response modulation or tuning width. Factors that can modulate baseline firing, such as attention, may have a particularly large influence on population activation. The results have implications for the interpretation of functional imaging signals and for cross-calibration between different methods for measuring neuronal activity.     

Exploring how extracellular electric field modulates neuron activity through dynamical analysis of a two-compartment neuron model

To investigate how extracellular electric field modulates neuron activity, a reduced two-compartment neuron model in the presence of electric field is introduced in this study. Depending on neuronal geometric and internal coupling parameters, the behaviors of the model have been studied extensively. The neuron model can exist in quiescent state or repetitive spiking state in response to electric field stimulus. Negative electric field mainly acts as inhibitory stimulus to the neuron, positive weak electric field could modulate spiking frequency and spike timing when the neuron is already active, and positive electric fields with sufficient intensity could directly trigger neuronal spiking in the absence of other stimulations. By bifurcation analysis, it is observed that there is saddle-node on invariant circle bifurcation, supercritical Hopf bifurcation and subcritical Hopf bifurcation appearing in the obtained two parameter bifurcation diagrams. The bifurcation structures and electric field thresholds for triggering neuron firing are determined by neuronal geometric and coupling parameters. The model predicts that the neurons with a nonsymmetric morphology between soma and dendrite, are more sensitive to electric field stimulus than those with the spherical structure. These findings suggest that neuronal geometric features play a crucial role in electric field effects on the polarization of neuronal compartments. Moreover, by determining the electric field threshold of our biophysical model, we could accurately distinguish between suprathreshold and subthreshold electric fields. Our study highlights the effects of extracellular electric field on neuronal activity from the biophysical modeling point of view. These insights into the dynamical mechanism of electric field may contribute to the investigation and development of electromagnetic therapies, and the model in our study could be further extended to a neuronal network in which the effects of electric fields on network activity may be investigated.     

Simultaneous BOLD fMRI and fiber-optic calcium recording in rat neocortex

Functional magnetic resonance imaging (fMRI) based on blood oxygen level-dependent (BOLD) contrast is widely used for probing brain activity, but its relationship to underlying neural activity remains elusive. Here, we combined fMRI with fiber-optic recordings of fluorescent calcium indicator signals to investigate this relationship in rat somatosensory cortex. Electrical forepaw stimulation (1-10 Hz) evoked fast calcium signals of neuronal origin that showed frequency-dependent adaptation. Additionally, slower calcium signals occurred in astrocyte networks, as verified by astrocyte-specific staining and two-photon microscopy. Without apparent glia activation, we could predict BOLD responses well from simultaneously recorded fiber-optic signals, assuming an impulse response function and taking into account neuronal adaptation. In cases with glia activation, we uncovered additional prolonged BOLD signal components. Our findings highlight the complexity of fMRI BOLD signals, involving both neuronal and glial activity. Combined fMRI and fiber-optic recordings should help to clarify cellular mechanisms underlying BOLD signals.     

Regulation of oxygen transport during brain activation: stimulus-induced hemodynamic responses in human and animal cortices

BACKGROUND: The correlation between regional changes in neuronal activity and changes in hemodynamics is a major issue for noninvasive neuroimaging techniques such as functional magnetic resonance imaging (fMRI) and near-infrared optical imaging (NIOI). A tight coupling of these changes has been assumed to elucidate brain function from data obtained with those techniques. In the present study, we investigated the relationship between neuronal activity and hemodynamic responses in the occipital cortex of humans during visual stimulation and in the somatosensory cortex of rats during peripheral nerve stimulation. METHODS: The temporal frequency dependence of macroscopic hemodynamic responses on visual stimuli was investigated in the occipital cortex of humans by simultaneous measurements made using fMRI and NIOI. The stimulus-intensity dependence of both microscopic hemodynamic changes and changes in neuronal activity in response to peripheral nerve stimulation was investigated in animal models by analyzing membrane potential (fluorescence), hemodynamic parameters (visible spectra and laser-Doppler flowmetry), and vessel diameter (image analyzer). RESULTS: Above a certain level of stimulus-intensity, increases in regional cerebral blood flow (rCBF) were accompanied by a decrease in regional cerebral blood volume (rCBV), i.e., dissociation of rCBF and rCBV responses occurred in both the human and animal experiments. Furthermore, the animal experiments revealed that the distribution of increased rCBF and O2 spread well beyond the area of neuronal activation, and that the increases showed saturation in the activated area. CONCLUSIONS: These results suggest that above a certain level of neuronal activity, a regulatory mechanism between regional cerebral blood flow (rCBF) and rCBV acts to prevent excess O2 inflow into the focally activated area.     

Cellular mechanisms of brain energy metabolism and their relevance to functional brain imaging.

Despite striking advances in functional brain imaging, the cellular and molecular mechanisms that underlie the signals detected by these techniques are still largely unknown. The basic physiological principle of functional imaging is represented by the tight coupling existing between neuronal activity and the associated local increase in both blood flow and energy metabolism. Positron emission tomography (PET) signals detect blood flow, oxygen consumption and glucose use associated with neuronal activity; the degree of blood oxygenation is currently thought to contribute to the signal detected with functional magnetic resonance imaging, while magnetic resonance spectroscopy (MRS) identifies the spatio-temporal pattern of the activity-dependent appearance of certain metabolic intermediates such as glucose or lactate. Recent studies, including those of neurotransmitter-regulated metabolic fluxes in purified preparations and analyses of the cellular localization of enzymes and transporters involved in energy metabolism, as well as in vivo microdialysis and MRS approaches have identified the neurotransmitter glutamate and astrocytes, a specific type of glial cell, as pivotal elements in the coupling of synaptic activity with energy metabolism. Astrocytes are ideally positioned to sense increases in synaptic activity and to couple them with energy metabolism. Indeed they possess specialized processes that cover the surface of intraparenchymal capillaries, suggesting that astrocytes may be a likely site of prevalent glucose uptake. Other astrocyte processes are wrapped around synaptic contacts which possess receptors and reuptake sites for neurotransmitters. Glutamate stimulates glucose uptake into astrocytes. This effect is mediated by specific glutamate transporters present on these cells. The activity of these transporters, which is tightly coupled to the synaptic release of glutamate and operates the clearance of glutamate from the extracellular space, is driven by the electrochemical gradient of Na+. This Na(+)-dependent uptake of glutamate into astrocytes triggers a cascade of molecular events involving the Na+/K(+)-ATPase leading to the glycolytic processing of glucose and the release of lactate by astrocytes. The stoichiometry of this process is such that for one glutamate molecule taken up with three Na+ ions, one glucose molecule enters an astrocyte, two ATP molecules are produced through aerobic glycolysis and two lactate molecules are released. Within the astrocyte, one ATP molecule fuels one 'turn of the pump' while the other provides the energy needed to convert glutamate to glutamine by glutamine synthase. Evidence has been accumulated from structural as well as functional studies indicating that, under aerobic conditions, lactate may be the preferred energy substrate of activated neurons. Indeed, in the presence of oxygen, lactate is converted to pyruvate, which can be processed through the tricarboxylic acid cycle and the associated oxidative phosphorylation, to yield 17 ATP molecules per lactate molecule. These data suggest that during activation the brain may transiently resort to aerobic glycolysis occurring in astrocytes, followed by the oxidation of lactate by neurons. The proposed model provides a direct mechanism to couple synaptic activity with glucose use and is consistent with the notion that the signals detected during physiological activation with 18F-deoxyglucose (DG)-PET may reflect predominantly uptake of the tracer into astrocytes. This conclusion does not question the validity of the 2-DG-based techniques, rather it provides a cellular and molecular basis for these functional brain imaging techniques.     

The ventilation, lactate and electromyographic thresholds during incremental exercise tests in normoxia, hypoxia and hyperoxia

These experiments examined the effect of hypoxia and hyperoxia on ventilation, lactate concentration and electromyographic activity during an incremental exercise test in order to determine if coincident chances in ventilation and electromyographic activity occur during an incremental exercise test, despite an enhancement or reduction of peripheral chemoreceptor activity. In addition, these experiments were completed to determine if electromyographic activity and ventilation are enhanced or reduced in response to the inspiration of oxygen-depleted and oxygen-enriched air, respectively. Seven subjects performed three incremental exercise tests, until volitional exhaustion was achieved, while inspiring air with a fractional concentration of oxygen of either 66%, 21% or 17%. In addition, another single subject completed two tests while inspiring air with a fractional concentration of either 17% or 21%. During the tests, ventilation, mixed expired oxygen and carbon dioxide, arterialized venous blood and the electromyographic activity from the vastus lateralis were sampled. From these values ventilation, electromyographic and lactate thresholds were detected during normoxia, hypoxia and hyperoxia. The results showed that although ventilation and lactate concentration were significantly less during hyperoxia as compared to normoxia or hypoxia, the carbon dioxide production values were not significantly different between the normoxic, hypoxic and hyperoxic conditions. For a particular condition, the time, carbon dioxide production and oxygen consumption values that corresponded to the ventilation and electromyographic thresholds were not significantly different, but the values corresponding to the lactate threshold were significantly less than those for the electromyographic and ventilation thresholds. Comparisons between the three conditions showed that the time, carbon dioxide production and oxyen consumption values corresponding to each of these thresholds were not significantly different. These findings have led us to conclude that the changes in lactate concentration observed during exercise may not be directly related to the fractional concentration of inspired oxygen, and that the peripheral chemoreceptors may not be the sole mediators of the first ventilatory threshold. It is suggested that this threshold may be mediated by an increase in neural activity originating from higher motor centers or the exercising limbs, induced in response to the need to progressively recruit fast twitch muscle fibers as exercise power output is increased and as individual muscle fibers begin to fatigue.     

Hemodynamic responses evoked by neuronal stimulation via channelrhodopsin-2 can be independent of intracortical glutamatergic synaptic transmission

Maintenance of neuronal function depends on the delivery of oxygen and glucose through changes in blood flow that are linked to the level of ongoing neuronal and glial activity, yet the underlying mechanisms remain unclear. Using transgenic mice expressing the light-activated cation channel channelrhodopsin-2 in deep layer pyramidal neurons, we report that changes in intrinsic optical signals and blood flow can be evoked by activation of a subset of channelrhodopsin-2-expressing neurons in the sensorimotor cortex. We have combined imaging and pharmacology to examine the importance of glutamatergic synaptic transmission in this form of neurovascular coupling. Blockade of ionotropic glutamate receptors with the antagonists CNQX and MK801 significantly reduced forepaw-evoked hemodynamic responses, yet resulted in no significant reduction of channelrhodopsin-evoked hemodynamic responses, suggesting that stimulus-dependent coupling of neuronal activity to blood flow can be independent of local excitatory synaptic transmission. Together, these results indicate that channelrhodopsin-2 activation of sensorimotor excitatory neurons produces changes in intrinsic optical signals and blood flow that can occur under conditions where synaptic activation of neurons or other cells through ionotropic glutamate receptors would be blocked.     

Coupling between neuronal firing rate, gamma LFP, and BOLD fMRI is related to interneuronal correlations

BACKGROUND: To what extent is activity of individual neurons coupled to the local field potential (LFP) and to blood-oxygenation-level dependent (BOLD) functional magnetic resonance imaging (fMRI)? This issue is of high significance for understanding brain function and for relating animal studies to fMRI, yet it is still under debate. RESULTS: Here we report data from simultaneous recordings of isolated unit activity and LFP by using multiple electrodes in the human auditory cortex. We found a wide range of coupling levels between the activity of individual neurons and gamma LFP. However, this large variability could be predominantly explained (r = 0.66) by the degree of firing-rate correlations between neighboring neurons. Importantly, this phenomenon occurred during both sensory stimulation and spontaneous activity. Concerning the coupling of neuronal activity to BOLD fMRI, we found that gamma LFP was well coupled to BOLD measured across different individuals (r = 0.62). By contrast, the coupling of single units to BOLD was highly variable and, again, tightly related to interneuronal-firing-rate correlations (r = 0.70). CONCLUSIONS: Our results offer a resolution to a central controversy regarding the coupling between neurons, LFP, and BOLD signals by demonstrating, for the first time, that the coupling of single units to the other measures is variable yet it is tightly related to the degree of interneuronal correlations in the human auditory cortex.     

Variable threshold as a model for selective attention, (de)sensitization,and anesthesia in associative neural networks

We study the influence of a variable neuronal threshold on fixed points and convergence rates of an associative neural network in the presence of noise. We allow a random distribution in the activity levels of the patterns stored, and a modification to the standard Hebbian learning rule is proposed for this purpose. There is a threshold at which the retrieval ability, including the average final overlap and the convergence rate, is optimized for patterns with a particular activity level at a given noise level. This type of selective attention to one class of patterns with a certain activity level may be obtained at the cost of reducing the retrieval ability of the network for patterns with different activity levels. The effects of a constant threshold independent of noise, time, and pattern are discussed. For high-(low-) activity patterns, the average final overlap is shown to be increased at high noise levels and decreased at low noise levels by a negative (positive) constant threshold, whereas a positive (negative) threshold always reduces the final average overlap. When the magnitude of the constant threshold exceeds a critical value, there is no retrieval. Rates of convergence towards the stored pattern with negative (positive) thresholds are greater than those with positive (negative) thresholds. These results are related to (de)sensitization and anesthesia. For certain threshold values and patterns with certain activity levels, hysteresis appears in the plot of the average final overlap versus the noise level, even for first order interactions. We make the analogy between the pattern-dependent neuronal threshold proposed in the present paper and the task-related modulation in neuronal excitability determined by cognitive factors, such as the attentional state of a higher animal. A constant threshold is associated with overall changes in neuronal excitability caused, e.g., by various drugs and physical injuries. Neurophysiological evidence of a dynamically variable neuronal threshold, such as accommodation and potentiation, is presented.     

Extracellular Brain Glucose Levels Reflect Local Neuronal Activity: A Microdialysis Study in Awake, Freely Moving Rats

The relationship between brain extracellular glucose levels and neuronal activity was evaluated using microdialysis in awake, freely moving rats. The sodium channel blocker tetrodotoxin and the depolarizing agent veratridine were administered through the dialysis probe to provoke local changes in neuronal activity. The extracellular glucose content was significantly increased in the presence of tetrodotoxin and decreased sharply following veratridine application. The systemic injection of a general anaesthetic, chloral hydrate, led to a large and prolonged increase in extracellular glucose levels. The brain extracellular glucose concentration was estimated by comparing dialysate glucose efflux over a range of inlet glucose concentrations. A mean value of 0.47 mM was obtained in five animals. The results are discussed in terms of the coupling between brain glucose supply and metabolism. The changes observed in extracellular glucose levels under various conditions suggest that supply and utilization may be less tightly linked in the awake rat than has previously been postulated.     

SIRT3 deregulation is linked to mitochondrial dysfunction in Alzheimer's disease

Alzheimer's disease (AD) is the leading cause of dementia in the elderly. Despite decades of study, effective treatments for AD are lacking. Mitochondrial dysfunction has been closely linked to the pathogenesis of AD, but the relationship between mitochondrial pathology and neuronal damage is poorly understood. Sirtuins (SIRT, silent mating type information regulation 2 homolog in yeast) are NAD‐dependent histone deacetylases involved in aging and longevity. The objective of this study was to investigate the relationship between SIRT3 and mitochondrial function and neuronal activity in AD. SIRT3 mRNA and protein levels were significantly decreased in AD cerebral cortex, and Ac‐p53 K320 was significantly increased in AD mitochondria. SIRT3 prevented p53‐induced mitochondrial dysfunction and neuronal damage in a deacetylase activity‐dependent manner. Notably, mitochondrially targeted p53 (mito‐p53) directly reduced mitochondria DNA‐encoded ND2 and ND4 gene expression resulting in increased reactive oxygen species (ROS) and reduced mitochondrial oxygen consumption. ND2 and ND4 gene expressions were significantly decreased in patients with AD. p53‐ChIP analysis verified the presence of p53‐binding elements in the human mitochondrial genome and increased p53 occupancy of mitochondrial DNA in AD. SIRT3 overexpression restored the expression of ND2 and ND4 and improved mitochondrial oxygen consumption by repressing mito‐p53 activity. Our results indicate that SIRT3 dysfunction leads to p53‐mediated mitochondrial and neuronal damage in AD. Therapeutic modulation of SIRT3 activity may ameliorate mitochondrial pathology and neurodegeneration in AD.     

Morphological and electrophysiological changes in mouse dorsal root ganglia after partial colonic obstruction

There is evidence that sensitization of neurons in dorsal root ganglia (DRG) may contribute to pain induced by intestinal injury. We hypothesized that obstruction-induced pain is related to changes in DRG neurons and satellite glial cells (SGCs). In this study, partial colonic obstruction was induced by ligation. The neurons projecting to the colon were traced by an injection of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate into the colon wall. The electrophysiological properties of DRG neurons were determined using intracellular electrodes. Dye coupling was examined with an intracellular injection of Lucifer yellow (LY). Morphological changes in the colon and DRG were examined. Pain was assessed with von Frey hairs. Partial colonic obstruction caused the following changes. First, coupling between SGCs enveloping different neurons increased 18-fold when LY was injected into SGCs near neurons projecting to the colon. Second, neurons were not coupled to other neurons or SGCs. Third, the firing threshold of neurons projecting to the colon decreased by more than 40% (P < 0.01), and the resting potential was more positive by 4-6 mV (P < 0.05). Finally, the number of neurons displaying spontaneous spikes increased eightfold, and the number of neurons with subthreshold voltage oscillations increased over threefold. These changes are consistent with augmented neuronal excitability. The pain threshold to abdominal stimulation decreased by 70.2%. Inflammatory responses were found in the colon wall. We conclude that obstruction increased neuronal excitability, which is likely to be a major factor in the pain behavior observed. The augmented dye coupling between glial cells may contribute to the neuronal hyperexcitability.