Hypertonic stimulation inhibits synaptic transmission in hippocampal slices through decreasing pre-synaptic voltage-gated calcium current

Neuroscience Letters 507 (2012) 106–111

Contents lists available at SciVerse ScienceDirect

Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet

Hypertonic stimulation inhibits synaptic transmission in hippocampal slices through decreasing pre-synaptic voltage-gated calcium current Lin Li a , Jun Yin a , Changjin Liu b , Lei Chen a,∗ , Ling Chen a,∗ a b

Department of Physiology, Nanjing Medical University, Nanjing 210019, PR China Department of Physiology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, PR China

a r t i c l e

i n f o

Article history: Received 24 October 2011 Received in revised form 18 November 2011 Accepted 23 November 2011 Keywords: Hypertonicity Synaptic transmission High voltage-gated calcium channel TRPV4 receptor AMPA-subtype glutamate receptor

a b s t r a c t Acute changes in the cerebrospinal fluid osmotic pressure modulate the brain excitability. The present study investigated the effect of hypertonic stimulation on the synaptic transmission in hippocampal slices. It was found that the slope of excitatory postsynaptic potential (EPSP) in hippocampal CA1 area was inhibited after the hypertonic treatment. Accompanied with the inhibition in EPSP slope, the paired-pulse facilitation (PPF) was increased by hypertonicity. Transient receptor potential vanilloid 4 (TRPV4 receptor) antagonists did not block hypertonicity-action. High voltage-gated calcium current (IHVA ) in hippocampal CA3 neurons was decreased by hypertonicity, whereas the ␣-amino-3hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-induced current in hippocampal CA1 neurons was unaffected. Additionally, inhibition of phosphatidylinositol 3-kinase (PI3K) or protein kinase A (PKA) markedly attenuated hypertonicity-induced decrease of IHVA , whereas antagonism of phosphorylated ERK1/2 mitogen-activated protein kinase (pERK1/2) had no effect. We conclude that hypertonic stimulation inhibits synaptic transmission in hippocampal slices through decreasing pre-synaptic voltage-gated calcium current. © 2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction The function of the brain is sensitive to changes in the osmolarity of cerebrospinal fluid. In the clinic, acute hypotonic condition (such as water intoxication) results in epileptiform seizures [13], while hypertonic condition (such as dehydration) leads to depression and coma [3]. Previous experiments have proved that both acute

Abbreviations: ACSF, artificial cerebrospinal fluid; AMPA, ␣-amino-3hydroxy-5-methyl-4-isoxazole propionic acid; AP, action potential; EPSP, excitatory postsynaptic potential; G–V curve, voltage-dependent activation curve; HS, hypertonic saline; H-89, N-[2-(p-bromocinnamylamino)ethyl]-5isoquinolinesulfonamide dihydrochloride; IAMPA , AMPA-induced current; IHVA , high voltage-gated calcium current; inactivation–voltage curve, voltage dependent inactivation curve; IPI, inter-pulse intervals; I/O curve, input/output curve; I–V curve, voltage–current relationship; LY290022, 2-(4-morpholinyl)-8-phenyl1(4H)-benzopyran-4-one hydrochloride; pERK1/2, phosphorylated ERK1/2 mitogen-activated protein kinase; PKA, protein kinase A; PI3K, phosphatidylinositol 3-kinase; PPF, paired-pulse facilitation; PPR, paired-pulse ratio; RR, ruthenium red; S1, the first stimulation; S2, the second stimulation; TRPV4 receptor, transient receptor potential vanilloid 4; TTX, tetrodotoxin; U0126, 1,4-diamino-2,3-dicyano1,4-bis(o-aminophenylmercapto)butadiene monoethanolate; VGCC, voltage-gated calcium channel; 4␣-PDD, 4␣-phorbol-12,13-didecanoate. ∗ Corresponding authors at: Department of Physiology, Nanjing Medical University, Nanjing, No. 140, Hanzhong Road, Nanjing 210019, PR China. Tel.: +86 25 8686 2878; fax: +86 25 8626 2878. E-mail addresses: [email protected] (L. Chen), [email protected] (L. Chen). 0304-3940/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2011.11.053

hypo- and hypertonic treatment can stimulate C-fiber afferent nerve [1,2] and increase the AP generation in trigeminal ganglion neurons [5]. Additionally, osmotic disturbances affect the excitability in neocortical slices [18] and modulate the firing patterns in hippocampal neurons [4,13]. In the nervous system, information, processed at the synapses, is believed to be the basis for the higher functions of the brain. Hypertonic treatment depresses orthodromically transmitted population spikes and extracellular synaptic potentials [9]. Besides this, hypertonic treatment during hypoxia prevents depolarization and improves the subsequent functional synaptic recovery [8]. On the other hand, there is evidence that the hypertonic shock increases spontaneous quantal neurotransmitter release at many synapses in a calciumindependent manner [11,23]. It is reported that TRPV4 receptor is necessary for the response to changes in osmotic pressure and to function as a cellular osmotic sensor in the peripheral and central nervous system [15,16]. TRPV4 receptors are present in hippocampal neurons [20]. Previous studies have reported the important role of TRPV4 receptor in the hypotonic-action such as in various types of nociception [2,5,14]. However, there is implication of TRPV4 in hyper- as well as hypoosmotic regulation in the central nervous system [15,16]. Herein, we firstly studied the effect of hypertonic stimulation on the synaptic transmission in hippocampal slices and then examined the role of TRPV4 receptor in hypertonicity-action. We further explored the action site of hypertonic stimulation by examining the changes

L. Li et al. / Neuroscience Letters 507 (2012) 106–111

in IHVA in hippocampal CA3 pyramidal neurons and IAMPA in hippocampal CA1 area upon hypertonic treatment.

107

I/Imax = 1/(1 + exp (V0.5 − Vm )/k), with V0.5 being membrane potential at which 50% of activation or inactivation was observed and k being the slope of the function.

2. Materials and methods 3. Results Care of animals conformed to standards established by the National Institutes of Health. All animal protocols were approved by the Nanjing Medical University Animal Care and Use Committee. All efforts were made to minimize animal suffering and to reduce the number of animals used. Male Sprague–Dawley rats (3–4 weeks old) were used throughout the study. The coronal brain slices (400 ␮m) were cut using a vibrating microtome (Microslicer DTK 1500, Dousaka EM Co, Kyoto, Japan) in ice-cold modified ACSF composed of (in mM) NaCl 126, CaCl2 1, KCl 2.5, MgCl2 1, NaHCO3 26, KH2 PO4 1.25, and d-glucose 20 oxygenated with a gas mixture of 95% O2 and 5% CO2 . After a 1 h recovery, the hippocampal slices were transferred to a recording chamber. All experiments were performed at room temperature (22–24 ◦ C). For recording EPSP, the slices were perfused continually with the oxygenated bath solution composed of (in mM) NaCl 84, CaCl2 2, KCl 2.5, MgCl2 1, NaHCO3 26, KH2 PO4 1.25, d-glucose 20 and d-mannitol 80 at osmolarity 300 mOsm. The Schaffer collateral/commissural pathway was stimulated by a stimulator (SEN-3301, Nihon Kohden, Japan). EPSPs were recorded from the stratum radiatum using glass microelectrodes with the resistance of 4–5 M when filled with 0.9% NaCl. Signals were obtained using an Axoclamp 2B amplifier (Axon Instruments, Foster City, CA, USA), sampled at 20 kHz and filtered at 10 kHz, and the output was digitized with a Digidata 1200 converter (Axon Instruments). Pulses (0.1 ms duration/each) with the same stimulus intensity were delivered every 15 s and the amplitudes of four EPSPs were averaged as a recording data. For whole-cell patch clamp recording, hippocampal neurons were viewed with an upright microscope equipped with infraredsensitive camera (DAGE-MTI, IR-1000) and perfused continually with the oxygenated bath solution added with 0.001 ␮M TTX. IHVA and IAMPA were recorded using an EPC-10 amplifier (HEKA Elektronik, Lambrecht/Pfalz, Germany). Data were acquired at a sampling rate of 10 kHz and filter (Bessel) of 2.9 kHz. For recording IHVA , the resistance of glass pipettes was 4–5 M filled with the pipette solution composed of (in mM) CsCl 140, MgCl2 2, Na2 –ATP 5, TEA–Cl 2, HEPES 10, EGTA 10 at pH 7.2. IHVA was elicited by depolarizing to −40 mV (200 ms) from a holding potential of −80 mV (50 ms) and then depolarized to 0 mV (200 ms). IHVA was activated by the second depolarization. I–V curve was measured by a series of depolarizing pulses (200 ms) from −60 to +40 mV stepping by 10 mV with interval time of 5 s. Inactivation–voltage curve was measured by double pulses: precondition pulses (3 s) ranging from −80 to +20 mV by stepping 10 mV and following +10 mV test pulse (200 ms) with internal time of 5 s. For recording IAMPA , the glass pipette was filled with (in mM) KCl 140, Tris–ATP 2, HEPES 10, EGTA 10 at pH 7.2. The hippocampal pyramidal neuron was hold at −70 mV and AMPA (10 ␮M) was added in the bath solution and applied for 1.5 s using a rapid drug delivery system. Data obtained from neurons in which uncompensated series resistance resulted in voltage-clamp errors >5 mV were not taken in further analysis. Hypertonic solution was obtained by adjusting the concentration of d-mannitol. The osmolality was measured using the Advanced Micro Osmometer, model 3300 (Advanced instruments Inc., Norwood, MA). Data were expressed as means ± S.E.M., and analyzed with pClamp (Axon Instruments), PulseFit (HEKA Elektronik) and SigmaPlot (SPSS Inc., Chicago, IL, USA) software. Paired or unpaired t test was used for statistical analysis with the significance level set at p < 0.05. G–V curve and inactivation–voltage curve were fitted by Boltzmann functions, which G/Gmax = 1/(1 + exp (V0.5 − Vm )/k) or

The synaptic transmission in hippocampal CA1 area was assessed by examining the response to the stimulation ranging from 0.1 to 1.0 mA (I/O curve). The basal synaptic transmission recording was obtained by delivering single pulse at an intensity of yielding half-maximal EPSP slope for a given slice. As EPSP slope provides a better measure of the excitatory component of the synaptic input, this study mainly examined the effect of hypertonic stimulation on EPSP slope. It was found that the slope of EPSP was inhibited by 42.87 ± 4.46% (n = 14, paired t test, p < 0.01) when the isotonic bath solution (300 mOsm) was changed into the hypertonic solution (360 mOsm) (Fig. 1A and B). The inhibition of EPSP was largely reversible after the hypertonic stimulation was washed out. We also found that the inhibition of EPSP was dependent on the osmotic pressure gradient (Fig. 1C) and 360 mOsm was used in the following experiments. Here, it was noted that EPSP was almost unaffected in isotonic bath solution when fructose replaced mannitol to adjust the osmolarity (Fig. 1C-first histogram, n = 7, paired t test, p > 0.05). In order to determine the site of hypertonicity-action on synaptic transmission, the probability of pre-synaptic glutamate release was examined by measuring PPF, an index of pre-synaptic facilitation. PPF data were expressed as a ratio (i.e. PPR) of the second response slope relative to the first. In the present study, PPF was measured with various IPI of 25–100 ms. As shown in Fig. 1D, PPR with IPI of 25–50 ms in hypertonic solution was larger than that in isotonic solution (p < 0.01), indicating that pre-synaptic factor was responsible for the hypertonicity-inhibition of synaptic transmission. As an important cellular osmotic sensor, we tested the role of TRPV4 receptor in hypertonicity-induced inhibition of EPSP. The results showed that pre-application of TRPV4 antagonist RR (10 ␮M) or GdCl3 (100 ␮M) did not block the inhibition of EPSP in hypertonic solution (p > 0.05) (Fig. 1E), though both of them slightly decreased EPSP in isotonic solution (p < 0.05, data not shown). These results indicated that TRPV4 receptor was not involved in hypertonicity-induced inhibition of synaptic transmission. Pre-synaptic glutamate release is triggered by the calcium influx, through which VGCC is one of the most important candidates. We then tested the effect of hypertonic stimulation on IHVA in pyramidal neurons of hippocampal CA3 area. Fig. 2 shows that the amplitude of IHVA was markedly inhibited from −16.47 ± 2.19 pA/pF to −10.16 ± 1.01 pA/pF (n = 15, paired t test, p < 0.01) when the bath solution was changed from isotonicity (300 mOsm) into hypertonicity (360 mOsm) (Fig. 2A and B). After the hypertonic solution was washed out for 5 min, IHVA recovered to −14.10 ± 1.17 pA/pF. We also found that G–V curve did not shift before and during hypertonicity treatment (p > 0.05) (Fig. 2D). When the extracellular osmolarity was changed from 300 mOsm to 360 mOsm, the inactivation–voltage curve of IHVA markedly shifted (11 mV) to the hyperpolarizing direction (Fig. 2E). We continued to explore some intracellular pathways to determine whether they were responsible for the inhibition of IHVA by hypertonicity. Here, three protein kinase antagonists (LY294002 for PI3K, H-89 for PKA and U0126 for pERK1/2) were used for these kinases have been reported to modulate voltage-gated calcium channels. As shown in Fig. 2F and G, pre-application of LY294002 (10 ␮M) or H-89 (10 ␮M) markedly attenuated hypertonicityinduced inhibition of IHVA (unpaired t test, p < 0.01 in each case). However, pre-application of U0126 had no effect on hypertonicitydecreased IHVA (unpaired t test, p > 0.05) (Fig. 2H).

108

L. Li et al. / Neuroscience Letters 507 (2012) 106–111

Fig. 1. Effect of hypertonic stimulation on the synaptic transmission in hippocampal slices. (A) Typical recordings show that EPSP (including the amplitude and the slope) was reversibly inhibited by hypertonic stimulation. (B) Slopes of EPSP are plotted against stimulus intensity ranging from 0.1 to 1.0 mA. The slope (b) of the regression line for I/O curve was 1.21 and 0.59 in isotonic and hypertonic solution, respectively (n = 8, paired t test, p < 0.01). The maximal response was smaller in hypertonic solution (n = 8, paired t test, p < 0.01). (C) Hypertonicity-induced inhibition of EPSP was prominent with larger osmotic pressure gradient. EPSP changed insignificantly in the isotonic solution when fructose was used to adjust the osmolarity. (D) PPF evoked by IPI at 25–50 ms was increased significantly in hypertonic solution (25 ms: 290.83 ± 9.34%; 50 ms: 242.29 ± 17.99%), compared with that in isotonic solution (25 ms: 121.13 ± 6.86%; 50 ms: 168.45 ± 15.73%) (n = 10, paired t test, **p < 0.01 in each case). (E) Pre-application of RR or GdCl3 , EPSP was inhibited 49.25 ± 5.19% (n = 11) and 47.90 ± 6.14% (n = 10) by hypertonicity, respectively, which was similar to the inhibition by hypertonicity alone (unpaired t test, p > 0.05 in each case). (F) In the presence of RR, hypertonicity increased PPF from 108.00 ± 10.98% to 323.43 ± 14.53% (n = 6) at 25 ms and from 147.87 ± 11.74% to 300.10 ± 16.78% (n = 6) at 50 ms, respectively (paired t test, p < 0.01 in each case). Pre-incubation of GdCl3 , hypertonicity increased PPF from 105.05 ± 12.98% to 313.84 ± 22.53% (n = 6) at 25 ms and from 142.69 ± 13.74% to 283.11 ± 17.78% (n = 7) at 50 ms, respectively (paired t test, **p < 0.01 in each case).

In the present study, we also explored the effect of hypertonic stimulation on IAMPA in pyramidal neurons of hippocampal CA1 area to determine whether post-synaptic factor was involved in the depression of synaptic transmission. It was found that IAMPA was almost unchanged by hypertonic stimulation and the amplitude was −14.11 ± 2.15 pA/pF and −13.16 ± 2.08 pA/pF before and during hypertonic treatment, respectively (n = 14, paired t test, p > 0.05). 4. Discussion Changes in the osmolarity disturb the function of nervous system. In the peripheral system, both hyper- and hypotonic stimuli

increase the number of APs generated in trigeminal ganglion neurons, which might be responsible for the anisotonicity-induced nociception [5]. In the central nervous system, hypotonicity increases the endogenous burst firing in hippocampal neurons, while hypertonicity produces the opposite effect [4]. The hippocampus contains neural circuitry that is crucial for higher brain function, such as learning and memory. Besides the effect of anisotonicity on the non-synaptic interactions within the neuronal network, the present study found that the basal synaptic transmission in hippocampal slices was inhibited by hypertonic treatment (Fig. 1A–C). This result was consistent with the previous study [9]. It is reported that rapid decrease of plasma osmolarity promotes seizure, while raising osmolarity affords a protection

L. Li et al. / Neuroscience Letters 507 (2012) 106–111

109

Fig. 2. Effect of hypertonic stimulation on IHVA . (A) The typical recordings show that IHVA was reduced from −835.57 pA to −480.59 pA when the bath solution was changed from 300mOsm to 360mOsm for 10 min and recovered to −710.66 pA after washout. (B) On the average, IHVA was −16.47 ± 2.19 pA/pF and −10.16 ± 1.01 pA/pF (n = 15, paired t test, p < 0.01) in isotonic and hypertonic solution, respectively. (C) I–V curve did not change before and during hypertonic treatment. (D) G–V curve was assessed using the data transformed from the I–V data shown in (C). G–V curve did not shift after hypertonic treatment, in which V0.5 was −15.77 ± 1.81 mV and −16.55 ± 1.07 mV (n = 6, paired t test, p > 0.05), k was 8.44 ± 2.14 and 9.86 ± 1.19 (n = 6, paired t test, p > 0.05), for 300 mOsm and 360 mOsm respectively. (E) In the presence of hypertonicity, inactivation–voltage curve shifted to the hyperpolarizing direction. The Boltzmann parameters V0.5 was −46.23 ± 2.03 mV and −57.09 ± 2.43 mV (n = 6, paired t test, p < 0.05); k was −13.67.4 ± 1.93 and −16.13 ± 2.1 for 300 mOsm and 360 mOsm respectively (n = 6, paired t test, p > 0.05). (F) and (G) In the presence of LY294002 or H-89, the inhibition of IHVA by was reduced from 43.68 ± 7.48% to 10.64 ± 3.92% (n = 22, unpaired t test, **p < 0.01), and to 15.04 ± 9.11% (n = 28, unpaired t test, **p < 0.01), respectively. (H) In the presence of U0126, IHVA was decreased by 26.59 ± 8.13% upon hypertonic treatment, which was not significantly different from the inhibition by hypertonicity alone (n = 25, unpaired t test, p > 0.05).

against seizure [8]. Therefore, hypertonicity-induced decrease of EPSP might be involved in the above protection. PPF is generally considered to reflect the pre-synaptic factor in which EPSP to S2 applied just after the first one becomes larger compared with that to S1 due to an additive effect of residual calcium in the pre-synaptic nerve terminal originally caused by S1. Here, it was noted that the PPF was increased by hypertonic stimulation (Fig. 1D), implying that hypertonicity might decrease the pre-synaptic neurotransmitter release. Calcium influx into the presynaptic nerve terminal triggers the release of neurotransmitter. This study reported that IHVA in the hippocampal CA3 pyramidal neurons area was inhibited by hypertonic stimulation (Fig. 2A and B). Additionally, hypertonicity shifted the inactivation–voltage curve to the hyperpolarizing direction indicative of fast inactivation of IHVA (Fig. 2E), which might be responsible for hypertonicityinhibition of IHVA . The molecular mechanism underlying the effect of hypertonicity on IHVA was explored by using the antagonists of several intracellular signaling pathways. It was found that preincubation of antagonists of PKA and PI3K markedly attenuated hypertonicity-induced inhibition of IHVA , implying that PKA and PI3K signaling pathways were likely involved in hypertonicityaction (Fig. 2F and G). There is evidence that many voltage-gated ion channels (including calcium, potassium and sodium channels) are modulated by pERK1/2 [6,7,22]. In this study, inhibition of pERK1/2

did not affect the hypertonicity-action on IHVA (Fig. 2H), excluding the role of pERK1/2 pathway in the hypertonicity-induced inhibition. The increase of spontaneous quantal transmitter release by hypertonic shock has been reported at neuromuscular junctions in a calcium-independent manner [11]. The present result of evoked release was not consistent. Here, we should note the difference between the present study and the previous ones: in this study hippocampal slices were incubated for 10 min in the hypertonic solution to find the inhibition of basal synaptic transmission and IHVA , whereas hypertonic stimulation was applied for only a few second to find the increase of spontaneous transmitter release [11,23]. Rosenmund and Stevens [19] reported the transient potentiation and the depression of hypertonicity on transmitter release evoked by action potential in cultured hippocampal neurons. Therefore, it is possible that the hypertonic stimulation initially increases the neurotransmitter release within a few second to deplete the readily releasable pool, leading to the subsequent decrease of neurotransmitter release evoked by stimulation in hypertonic solution. The efficiency of synaptic transmission depends on both preand postsynaptic processes involving vesicular release and recruitment as well as postsynaptic response to the neurotransmitter. The present study also explored the effect of hypertonic stimulation on the function of AMPA-subtype glutamate receptor. Here, it was

110

L. Li et al. / Neuroscience Letters 507 (2012) 106–111

of choice. HS is reported to have favorable effect in treating cerebral edema and intracranial hypertension in animal models and suggested to be more effective than mannitol in reducing intracranial pressure and have a longer duration of action [24]. There is also evidence supports the use of HS in clinical practice [10]. The osmotherapy with HS is promising, but due to the limited number of clinic cases, further studies are required to provide the definitive evidence to support its routine use in the clinic. Our data also showed that the mechanism underlying the depression of synaptic transmission was probably attributed to the inhibition of voltagegated calcium current. Concerning the important role of calcium channels in cellular function, this should be considered in the application of hypertonic therapy.

Acknowledgements This work was supported by National Natural Science Foundation of China (No. 30900577) and Science and Technology Projects of Jiangsu Province (No. BK2009416).

Fig. 3. Effect of hypertonic stimulation on IAMPA . Hypertonic stimulation almost had no effect on IAMPA . IAMPA was −876.14 pA and −856.11 pA in the typical recordings (A); −14.11 ± 2.15 pA/pF and −13.16 ± 2.08 pA/pF on the average (B) in the presence of isotonicity and hypertonicity, respectively.

found that IAMPA in hippocampal CA1 pyramidal neurons was unaffected by hypertonic stimulation (Fig. 3). Therefore, this subtype of glutamate receptor is probably not involved in the inhibition of EPSP by hypertonicity. TRPV4 is widely expressed in the nervous system, including sensory nerve terminals, osmosensory neurons and hippocampus, etc. In hippocampus, TRPV4 has been proved to regulate the neural excitability at the body temperature (∼37 ◦ C) [20]. To avoid the effect of temperature, all experiments in the present study were performed at room temperature (∼22 ◦ C). As a cellular osmotic sensory, TRPV4 plays a role in the hyper- or hypo-osmotic regulation in the central nervous system. However, the present study found TRPV4 antagonists (RR and GdCl3 ) failed to block hypertonicityinduced inhibition of basal synaptic transmission in hippocampal slices (Fig. 1E and F), indicating that TRPV4 is probably not responsible for hypertonicity-action. As generally accepted, cells shrink when the osmolarity of the extracellular solution increases. Therefore, it is possible that the cell shrinkage mediates the action of hypertonicity on intracellular pathways to modulate calcium current, like the mechanism of the stress-induced change in enzyme activity or channel state [17]. However, it has been shown that the volume of acute dissociated pyramidal cells changed insignificantly during rapid and severe hypertonic stimulation [21] and the resting membrane potential or input resistance did not change much in the hippocampal slices, either [4]. Therefore, the question how the present mild hypertonic stimulation is transmitted into the neurons and has effect on intracellular signaling pathways, if not through TRPV4 receptor, needs to be explored in the future study. On the other hand, although it is difficult to determine whether the volume regulation happens in glial cells, neurons or both [12], the increase of interstitial volume is detected in hypertonic media [9]. The increase of synaptic cleft and the synaptic transmission distance caused by the hypertonic stimulation might also contribute to the depression of synaptic transmission to some degree. The present study demonstrated that the hippocampal synaptic transmission was depressed in hypertonic media, which supports the clinical use of some osmotic agents to raise plasma osmolality in the treatment of cerebral edema and other condition. Mannitol is effective and safe and is recommended as the idea osmotic drug

References [1] N. Alessandri-Haber, E. Joseph, O.A. Dina, W. Liedtke, J.D. Levine, TRPV4 mediates pain-related behavior induced by mild hypertonic stimuli in the presence of inflammatory mediator, Pain 118 (2005) 70–79. [2] N. Alessandri-Haber, J.J. Yeh, A.E. Boyd, C.A. Parada, X. Chen, D.B. Reichling, J.D. Levine, Hypotonicity induces TRPV4-mediated nociception in rat, Neuron 39 (2003) 497–511. [3] R.D. Andrew, Seizure and acute osmotic change: clinical and neurophysiological aspects, J. Neurol. Sci. 101 (1991) 7–18. [4] R. Azouz, G. Alroy, Y. Yaari, Modulation of endogenous firing patterns by osmolarity in rat hippocampal neurons, J. Physiol. 502 (1997) 175–187. [5] L. Chen, C. Liu, L. Liu, Osmolality-induced tuning of action potentials in trigeminal ganglion neurons, Neurosci. Lett. 452 (2009) 79–83. [6] E.M. Fitzgerald, Regulation of voltage-dependent calcium channels in rat sensory neurons involves a Ras-mitogen-activated protein kinase pathway, J. Physiol. 527 (2000) 433–444. [7] H.J. Hu, K.S. Glauner, R.W. Gereau 4th, ERK integrates PKA and PKC signaling in superficial dorsal horn neurons. I. Modulation of A-type K currents, J. Neurophysiol. 90 (2003) 1671–1679. [8] R. Huang, P.G. Aitken, G.G. Somjen, Hypertonic environment prevents depolarization and improves functional recovery from hypoxia in hippocampal slices, J. Cerebr. Blood F. Metab. 16 (1996) 462–467. [9] R. Huang, G.G. Somjen, The effect of graded hypertonia on interstitial volume, tissue resistance and synaptic transmission in rat hippocampal tissue slices, Brain Res. 702 (1995) 181–187. [10] H. Kamel, B.B. Navi, K. Nakagawa, J.C. Hemphill 3rd, N.U. Ko, Hypertonic saline versus mannitol for the treatment of elevated intracranial pressure: a meta-analysis of randomized clinical trials, Crit. Care Med. 39 (2011) 554–559. [11] A.H. Kashani, B.M. Chen, A.D. Grinnell, Hypertonic enhancement of transmitter release from frog motor nerve terminals: Ca2+ independence and role of integrins, J. Physiol. 530 (2001) 243–252. [12] O. Kempski, S. von Rosen, H. Weigt, F. Staub, J. Peters, A. Baethmann, Glial ion transport and volume control, Ann. N.Y. Acad. Sci. 633 (1991) 306–317. [13] W. Kilb, P.W. Dierkes, E. Syková, L. Vargová, H.J. Luhmann, Hypoosmolar conditions reduce extracellular volume fraction and enhance epileptiform activity in the CA3 region of the immature rat hippocampus, J. Neurosci. Res. 84 (2006) 119–129. [14] L. Li, C. Liu, L. Chen, L. Chen, Hypotonicity modulates tetrodotoxinsensitive sodium current in trigeminal ganglion neurons, Mol. Pain 7 (2011) 27–32. [15] W. Liedtke, J.M. Friedman, Abnormal osmotic regulation in trpv4−/− mice, Proc. Natl. Acad. Sci. U.S.A. 100 (2003) 13698–13703. [16] A. Mizuno, N. Matsumoto, M. Imai, M. Suzuki, Impaired osmotic sensation in mice lacking TRPV4, Am. J. Physiol. Cell Physiol. 285 (2003) C96–C101. [17] B. Poolman, P. Blount, J.H. Folgering, R.H. Friesen, P.C. Moe, T. van der Heide, How do membrane proteins sense water stress? Mol. Microbiol. 44 (2002) 889–902. [18] A.S. Rosen, R.D. Andrew, Osmotic effects upon excitability in rat neocortical slices, Neuroscience 38 (1990) 579–590. [19] C. Rosenmund, C.F. Stevens, Definition of the readily releasable pool of vesicles at hippocampal synapses, Neuron 16 (1996) 1197–1207. [20] K. Shibasaki, M. Suzuki, A. Mizuno, M. Tominaga, Effects of body temperature on neural activity in the hippocampus: regulation of resting membrane potentials by transient receptor potential vanilloid 4, J. Neurosci. 27 (2007) 1566–1575.

L. Li et al. / Neuroscience Letters 507 (2012) 106–111 [21] G.G. Somjen, G.C. Faas, M. Vreugdenhil, W.J. Wadman, Channel shutdown: a response of hippocampal neurons to adverse environments, Brain Res. 632 (1993) 180–189. [22] S. Stamboulian, J.S. Choi, H.S. Ahn, Y.W. Chang, L. Tyrrell, J.A. Black, S.G. Waxman, S.D. Dib-Hajj, ERK1/2 mitogen-activated protein kinase phosphorylates sodium channel Nav1.7 and alters its gating properties, J. Neurosci. 30 (2010) 1637–1647.

111

[23] K. Suzuki, A.D. Grinnell, Y. Kidokoro, Hypertonicity-induced transmitter release at Drosophila neuromuscular junctions is partly mediated by integrins and cAMP/protein kinase A, J. Physiol. 538 (2002) 103–119. [24] W.C. Ziai, T.J. Toung, A. Bhardwaj, Hypertonic saline: firstline therapy for cerebral edema? J. Neurol. Sci. 261 (2007) 157–166.