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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.

Normal nervous system function is critically dependent on the balance of water and ions in the extracellular space. Pathological reduction in brain interstitial osmolarity results in osmotically-driven flux of water into cells, causing cellular edema which reduces the extracellular space and increases neuronal excitability and risk of seizures. Astrocytes are widely considered to be particularly susceptible to cellular edema due to selective expression of the water channel aquaporin-4 (AQP4). The apparent resistance of pyramidal neurons to osmotic swelling has been attributed to lack of functional water channels. In this study we report rapid volume changes in CA1 pyramidal cells in hypoosmolar ACSF (hACSF) that are equivalent to volume changes in astrocytes across a variety of conditions. Astrocyte and neuronal swelling was significant within 1 minute of exposure to 17 or 40% hACSF, was rapidly reversible upon return to normosmolar ACSF, and repeatable upon re-exposure to hACSF. Neuronal swelling was not an artifact of patch clamp, occurred deep in tissue, was similar at physiological vs. room temperature, and occurred in both juvenile and adult hippocampal slices. Neuronal swelling was neither inhibited by TTX, nor by antagonists of NMDA or AMPA receptors, suggesting that it was not occurring as a result of excitotoxicity. Surprisingly, genetic deletion of AQP4 did not inhibit, but rather augmented, astrocyte swelling in severe hypoosmolar conditions. Taken together, our results indicate that neurons are not osmoresistant as previously reported, and that osmotic swelling is driven by an AQP4-independent mechanism.

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.