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The activation of Na/40Ca exchange in reverse (R) mode leading to neuronal swelling and muscle contraction has been suggested as a mechanism for generation of pain signals. However, the activation of R Na/45Ca exchange, having higher rate than R Na/40Ca exchange, brings to depression of pain sensation, the mechanism of which is not clear. The previous data that the 4 Hz mechanical vibration (MV) has pain-relieving effects by activation of cGMP-dependent Na/Ca exchange in forward (F) mode, which leads to muscle hydration and neuronal dehydration, as in case of R Na/45Ca exchange activation, allow us to suggest that the comparative study of the effects of 4 Hz MV and R Na/45Ca exchange on thermal pain thresholds, tissue hydrations in different experimental conductions will make it possible to evaluate the mechanisms of R Na/45Ca exchange-induced inhibition of pain sensation. The obtained data show that the R Na/45Ca exchange-induced depression of pain sensation is due to high [Ca2+]i-inactivation of Na/K pump which brings to further increase of [Ca2+]i, while 4 Hz MV-activated F Na/Ca exchange-induced pain-relieving effect is due to Na/K pump activation by low [Ca2+]i. It is suggested that the R Na/45Ca exchange inhibits pain sensation, which is due to high [Ca2+]i-induced depression inhibition of muscle contractility, while the 4 Hz MV-induced pain-relieving effect is due to activation of Na/K pump by cGMP-dependent decrease of [Ca2+]i leading to muscle relaxation and neuronal dehydration. Therefore, the 4 Hz MV has been suggested as a novel quantum-mechanical sensitive target for pain therapy.

Our lab aims to apply OCT to the study of the nervous system. In order to detect neural activity using this technology, we measure optical changes that occur in the neural tissue as a result of transient volume changes. Though these changes are critical to our studies, they are poorly understood and so, through analysis of the current literature on the subject, I narrowed down the possible mechanisms for this phenomenon. As such,our system needs to be able to resolve on the order of microns. We can increase the resolution by increasing the numerical aperture, but this leads to a characteristic trade-off with the depth of field. In order to maximize our resolution and depth of field, I implemented phase sensitive synthetic aperture technology into our OCT system which allows for an increase in the intensity and phase across a DOF beyond the Rayleigh range through modification of the wavefront curvature.

Nerve activity in a biological neural network is mostly characterized by generation and propagation of impulses through the nerves. These impulses, known as action potentials, are generated when the nerves are excited by a stimulus either as an external input or as a means of internal communication between nerves. This project is aimed to develop an optical imaging based minimally invasive technique for neural recording. Existing technologies largely limit the analysis of neuronal processing to a single or small cluster of neurons using different varieties of electrodes or the introduction of exogenous contrast agents and most of these techniques are invasive in some ways. Nerves undergo rapid transient thickness changes during propagation of action potentials. These activity associated structural changes are usually in the order of nanometers which is well beyond the usual limit of resolution of most common imaging technologies, including optical coherence tomography (OCT). However, recent advances in phase-resolved OCT (pr-OCT), a specific modality of OCT, have enabled the measurement of subnanometer changes in optical path length. This study demonstrates pr-OCT's capability to detect and measure rapid transient thickness changes in nerves during activity. Optically detected changes in nerve fiber thickness have timing and duration similar to the propagating electrical signal recorded in electrophysiology. Averaging of 8-10 impulses improves the SNR of detected optical signal. However, most of the results demonstrated here are single shot detections. Since OCT collects data from every depth points within a single A-line simultaneously, the changes in phase at every depth location are examined. Results demonstrate that these transient changes are present at different depths and this allows representing activity as a map of thickness changes. A custom-built cold block system has been used for switchable control of activation and deactivation of action potential propagation through the Limulus nerve. Optical recording has been compared with simultaneous electrical recording at every stages of cold block operation: activated nerve before deactivation, nerve after deactivation and nerve after reactivation. Hopefully, this study will serve as the basic ground work for future experiments and will have an overall significant impact on functional neuroimaging.

Edema is an important target for clinical intervention after traumatic brain injury (TBI). We used in vivo cellular resolution imaging and electrophysiological recording to examine the ionic mechanisms underlying neuronal edema and their effects on neuronal and network excitability after controlled cortical impact (CCI) in mice. Unexpectedly, we found that neuronal edema 48 hours after CCI was associated with reduced cellular and network excitability, concurrent with an increase in the expression ratio of the cation-chloride cotransporters (CCCs) NKCC1 and KCC2. Treatment with the CCC blocker bumetanide prevented neuronal swelling via a reversal in the NKCC1/KCC2 expression ratio, identifying altered chloride flux as the mechanism of neuronal edema. Importantly, bumetanide treatment was associated with increased neuronal and network excitability after injury, including increased susceptibility to spreading depolarizations (SDs) and seizures, known agents of clinical worsening after TBI. Treatment with mannitol, a first-line edema treatment in clinical practice, was also associated with increased susceptibility to SDs and seizures after CCI, showing that neuronal volume reduction, regardless of mechanism, was associated with an excitability increase. Finally, we observed an increase in excitability when neuronal edema normalized by 1 week after CCI. We conclude that neuronal swelling may exert protective effects against damaging excitability in the aftermath of TBI and that treatment of edema has the potential to reverse these effects.

Spreading depolarization-induced focal dendritic swelling (beading) is an early hallmark of neuronal cytotoxic edema. Pyramidal neurons lack membrane-bound aquaporins posing a question of how water enters neurons during spreading depolarization. Recently, we have identified chloride-coupled transport mechanisms that can, at least in part, participate in dendritic beading. Yet transporter-mediated ion and water fluxes could be paralleled by water entry through additional pathways such as large-pore pannexin-1 channels opened by spreading depolarization. Using real-time in vivo two-photon imaging in mice with pharmacological inhibition or conditional genetic deletion of pannexin-1, we showed that pannexin-1 channels are not required for spreading depolarization-induced focal dendritic swelling.

Cortical spreading depression (CSD) is a self-propagating wave of cellular depolarization that has been implicated in migraine and in progressive neuronal injury after stroke and head trauma. Using two-photon microscopic NADH imaging and oxygen sensor microelectrodes in live mouse cortex, we find that CSD is linked to severe hypoxia and marked neuronal swelling that can last up to several minutes. Changes in dendritic structures and loss of spines during CSD are comparable to those during anoxic depolarization. Increasing O2 availability shortens the duration of CSD and improves local redox state. Our results indicate that tissue hypoxia associated with CSD is caused by a transient increase in O2 demand exceeding vascular O2 supply.

Aim Autophagy is an important process that balances cellular protein synthesis and degradation and is involved in many physiological and pathological conditions. However, the precise role of autophagy has not yet been defined in the model of spinal cord injury (SCI). Material and methods Here, we utilized a hemisection model of acute SCI to elucidate the role of autophagy in the pathological processes underlying SCI. Results LC3B-II, a well-known marker of autophagy, was immunohistochemically detected 4H after SCI, peaked at 3D, and decreased at 21D. Hematoxylin-eosin (HE) staining confirmed accurate spinal cord hemisection, which was accompanied by both neuronal swelling and shrunken neurons with darkly stained, condensed nuclei. These findings suggest that the process of autophagy is related with pathological changes following SCI. Conclusion Our results indicate autophagy is involved in the pathological changes after SCI, and potential therapies to promote neuronal regeneration following SCI should target the mechanism of autophagy.

As an immediate consequence of stroke onset, failure of the Na+-K+-ATPase pump evokes a propagating anoxic depolarization (AD) across gray matter. Acute neuronal swelling and dendritic beading arise within seconds in the future ischemic core, imaged as changes in light transmittance (ΔLT). AD is itself not a target for drug-based reduction of stroke injury because it is generated in the 1st min of stroke onset. Peri-infarct depolarizations (PIDs) are milder AD-like events that recur during the hours following AD and contribute to infarct expansion. Inhibiting PIDs with drugs could limit expansion. Two types of drugs, “caines” and σ1-receptor ligands, have been found to inhibit AD onset (and may also oppose PID initiation), yet their underlying actions have not been examined. Imaging ΔLT in the CA1 region simultaneously with whole cell current-clamp recording from CA1 pyramidal neurons reveal that the elevated LT front and onset of the AD are coincident. Either dibucaine or carbetapentane pretreatment significantly delays AD onset without affecting resting membrane potential or neuronal input resistance. Dibucaine decreases excitability by raising spike threshold and decreasing action potential (AP) frequency, whereas carbetapentane eliminates the fast afterhyperpolarization while accentuating the slow afterhyperpolarization to reduce AP frequency. Orthodromic and antidromic APs are eliminated by dibucaine within 15 min but not by carbetapentane. Thus both drugs reduce cortical excitability at the level of the single pyramidal neuron but through strikingly different mechanisms. In vivo, both drugs would likely inhibit recurring PIDs in the expanding penumbra and so potentially could reduce developing neuronal damage over many hours poststroke when PIDs occur.

Several physical phenomena accompany the firing of electrical impulses by axons. Some of these, such as the microscopic swelling of axons, alter the transmission of light through axons. This produces what are called “intrinsic optical signals” because optical methods can be used to see axons fire without adding voltage-sensitive dyes or using electronic amplifiers. These physical changes allow neurons to communicate through nonsynaptic signals to adjacent cells, such as other neurons or glia. Two of the three videos in this Teaching Resource show the optical manifestations of the microscopic swelling of axons that accompanies the firing of action potentials in cultured neurons, and one shows the nonsynaptic release of ATP that occurs through membrane channels that are stimulated by neuronal swelling.

Neurons are mechanically robust. During prolonged swelling, molluscan neurons can triple their apparent membrane area. They gain surface area and capacitance independent of extracellular Ca concentration ([Ca]e), but it is unknown if an increase in intracellular Ca concentration ([Ca]i) is necessary. If Ca for stimulating exocytosis is unnecessary, it is possible that swelling-induced membrane tension changes directly trigger surface area readjustments. If, however, Ca-mediated but not tension-mediated membrane recruitment is responsible for surface area increases, swelling neurons should sustain elevated levels of [Ca]i. The purpose of this investigation is to determine if the [Ca]iin swelling neurons attains levels high enough to promote exocytosis and if any such increase is required. Lymnaeaneurons were loaded with the Ca concentration indicator fura 2. Calibration was performed in situ using 4-bromo-A-23187 and Ca-ethylene glycol-bis(β-aminoethyl ether)- N, N, N′, N′-tetraacetic acid (EGTA), with free Ca concentration ranging from 0 to 5 μM. Swelling perturbations (medium osmolarity reduced to 25% for 5 min) were done at either a standard [Ca]eor very low [Ca]elevel (0.9 mM or 0.13 μM, respectively). In neither case did the [Ca]iincrease to levels that drive exocytosis. We also monitored osmomechanically driven membrane dynamics [swelling, then formation and reversal of vacuole-like dilations (VLDs)] with the [Ca]iclamped below 40 nM via 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid (BAPTA). [Ca]idid not change with swelling, and VLD behavior was unaffected, consistent with tension-driven, [Ca]i-independent surface area adjustments. In addition, neurons with [Ca]iclamped at 0.1 μM via an ionophore could produce VLDs. We conclude that, under mechanical stress, neuronal membranes are compliant by virtue of surface area regulatory adjustments that operate independent of [Ca]i. The findings support the hypothesis that plasma membrane area is regulated in part by membrane tension.

SUMMARY A 50–year-old woman showing psycho-organic syndrome, cerebellar symptoms, choreatic involuntary movements, Romberg's sign and epileptic fits over a period of 14 years, was neuropathologically studied. The calcarine and postcentral cortex, the medio-dorsal nucleus of the thalamus and the cerebellar cortex were affected with neuronal loss and astrocytic gliosis. Neurons in various regions showed chromatolytic neuronal swelling. Ultrastructurally, these chromatolytic neurons contained a large hollow core with numerous mitochondria, dispersed rough endoplasmic reticulum, degenerated cisternal structures and a few neurofilaments. Some neuronal mitochondria showed membranous trans-somal bridges composed of about 60 A parallel filaments with a spacing of about 80 A.

Action potentials of single cells, recorded by means of extracellular microelectrodes from the cat's cerebral cortex after topical application of penicillin, undergo characteristic changes in the course of seizure episodes. In individual neurons, these changes in spike configuration are fully reversible and are repeatedly observed with each of such episodes. Two alternative interpretations offered for these findings appear to be critically dependent upon the relationship between the position of the microelectrode and the cell itself: there might be a transitory failure of the spike to invade part of the neuronal membrane or transitory neuronal swelling.