06/27/2017

Myricetin modulates the ion currents of the hypothalamic paraventricular nucleus neurons: both presynaptic and postsynaptic actions medical student’s practical experi

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Myricetin modulates the ion currents of the hypothalamic paraventricular nucleus neurons: both presynaptic and postsynaptic actions.

——-A brief summary of the patch clamp experiments

Introduction:

The epidemiology studies showed that over 50% of the population suffers from sleep disorders and herbal medicine represents one of the alternative treatments. However, the pharmacological action of most of the herbal medicines remains largely unknown. Previous studies in our laboratory demonstrated that an herbal mixture, ECBRC-AG, could significantly prolong the sleep time in healthy rats; in which, an active compound, myricetin, is thought to be one of the active compounds. Myricetin is one of the flavonols. These flavoniod families show a wide range of biological activities including but not limited to modulation of neuronal oxidative metabolism, anticancer, anticonvulsant, sedative and anxiolytic effects. Some studies showed that several flavonols have a high affinity to the benzodiazepine binding site in γ-aminobutyric acid (GABA-A) receptors in vitro, so a benzodiazepine-like mechanism was proposed for flovoniod modulation of GABA-A receptors function. However, there is still no direct experimental evidence supporting this hypothesis. GABA is one of the predominant transmitters in the central nervous system, extensively involved in many physiological activities. Traditional anti-insomnia drugs such as benzodiazepine and phenobarbital both produce a hypnotic action by acting on the GABA-A receptors to modulate the GABA-A receptor mediated chloride currents. On the other hand, it has also been reported that myricetin could enhance L-type calcium channel currents. Calcium plays a very important role in the central nervous system; the elevation of intracellular calcium concentration is known to induce neuronal transmitter release such as GABA in the pre synapse. In the post synapse, elevation of calcium concentration may modulate receptor phosphoralation process by activating calcium dependent protein kinase and activate some calcium dependent channels such as calcium dependent potassium channel thereby regulate the membrane excitability of the neurons.

Based on these early observations, we hypothesized that the sedative effect of myricetin may be mediated by both its presynaptic and postsynaptic actions. On the one hand, myricetin may act on postsynaptic benzodiazepine biting site thereby modulating GABA-A receptor function; on the other hand, myricetin may activate calcium channels then enhance calcium mobilization thereby leads to presynaptic GABA release and postsynaptic signaling events leading to modulation of GABA-A receptor activities.

The brain slice techniques in vitro provide the feasibility to test these hypotheses, whereby a thin slice of specific areas of brain tissue can be maintained in physiological solution. Brain slice allows measurement of current on individual intact neurones both pre-synaptically and post-synaptically. Paraventricular nucleus (PVN) of the hypothalamus is the regulation center of the autonomic nervous and neuroendocrine systems; it is also involved in the modulation of the sleep-awake system. GABA is the major inhibitory transmitter in PVN. An immunohistochemical and electrophysilogical study showed that GABA and its receptor are functionally expressed in the PVN. Taken together, in the present study, we investigated the effects of myricetin on ion currents in PVN neurons using patch clamp techniques in hypothalamic brain slice to explore the possible mechanisms underlying the sedative effect of myticetin.

Method:

In vitro slice preparation:

Hypothalamic slices were obtained using techniques similar to those described previously. Briefly, male Sprague-Dawley rats (18-24 days old) were anaesthetized and decapitated. The brain was then immediately removed and placed into ice-cold artificial cerebrospinal fluid (ACSF), consisting of (mM): NaCl 125, KCl 2.0, MgSO4 1.2, CaCl2 2.5, KH2PO4 1.2, glucose 11 and NaHCO3 26, and bubbled with 95% O2 and 5% CO2. The hypothalamus block was isolated from the brain by making razor cuts rostral to optic chiasm, caudal to the median eminence, dorsal to the third ventricle and lateral to the fornix. 250μm slice containing PVN were sectioned using a vibrating microtome (integraslice 7550 MM, Camden Instrument). After equilibration for 1 hour at 35 in a tissue storage chamber containing ACSF saturated with 95% O2 and 5% CO2. A slice was transferred to a small volume chamber mounted on an upright microscope (Zeiss Axioskop), and superfused with oxygenated ACSF at a rate of 1-1.5 ml/min maintained at a temperature of 34-35. Neuronal soma and proximal dendrites were directly visualized by a combination of differential interference contrast optics and contrast-enhanced infrared video microscopy.

Patch clamp recording:

Patch electrodes were pulled in multiple stages on a Flaming/Brown micropipette puller (P-97, Sutter Instruments Co. Navoto, CA) from borosilicate glass (o.d. 1.5 mm, i.d. 0.86, Sutter Instrument, Navoto, CA). It typically had a resistance of 3-5 MΩ when filled with an internal solution. Whole-cell patch clamp recordings from PVN neurons were obtained using a MultiClamp 700A amplifier (Axon Instruments). Electrodes were advanced through the tissue using a MP-285 micromanipulator (Sutter Instrument Co.) and positioned on the soma of the PVN neurons under visual control. Normally, the liquid junction potential was adjusted automatically, no series resistance compensation was applied but the cell was rejected if the series resistance increased significantly (>20%) during recording. Data were low-pass filtered at 3kHz with the amplifier and sampled using compex 8.2 software. Selected traces were saved to the hard drive using a Digidata 1322A interface (Axon Instrument) for off-line analysis.

Results:

Part one: Effects of myricetin on the GABA-A receptor mediated inhibitory post-synaptic currents in PVN neurons

In this section, an internal solution consisting of (mM): KCl 140, EGTA 1.0, MgCl2 2.0, HEPES 10, Na2ATP 2.0, Tris GTP 0.4 and pH was adjusted to 7.25-7.35 with 1 M KOH was used. The inclusion of 140 mM of KCl in the recording pipette reversed the polarity of GABA-A receptor mediated currents from outward to inward and enhanced their detection presumably by increasing the driving force on the chloride ions. The post-synaptic currents were recorded at a holding potential of –70mV. (±)-2-Amino-5-phosphonopentanoic acid (AP-5, 50μM) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 20μM) were included in the bath solution to eliminate glutamate-mediated synaptic currents. When isolating miniature inhibitory post-synaptic currents (mIPSCs), tetrodotoxin (TTX) of 0.5μM was added into the bath solution.

The data were represented as mean ± SEM. Kolmogorov-Smirnov test (K-S test) was used to compare two distributions of synaptic currents inter-event intervals, amplitudes and decay time; otherwise paired Student’s t test was applied. Probability (P) value of 0.05 was considered to be significant.

1.1 myricetin prolongs the half decay time of both mIPSCs and spontaneous inhibitory post-synaptic currents (sIPSCs) in PVN neurons

As shown in figure 1, in 14 cells studied, the decay time of mIPSCs were significantly increased from 8.264 ± 0.456 to 9.544 ± 0.603 ms (P < 0.001, paired t test), in which, 5 neurons showed an increase of frequency of mIPSCs (K-S test, P < 0.05), these data indicated that myricetin might enhance GABA release by presynaptic mechanisms in addition to a post-synaptic modulating effect. In addition, 3 showed a decrease and 1 showed an increase of mIPSCs amplitude (K-S test, P < 0.05) in the tested 14 neurons, these might be due to the heterogeneous of GABA-A receptor subunits. Further investigation of the sIPSCs also detected a prolongation of decay time (Fig. 2, from 7.529 ± 0.625 to 8.861 ± 0.716, n=8, P < 0.001, paired t test).

A

**

B

Fig.1 Effects of myricetin on the mIPSCs in PVN neurons

A. An example of mIPSCs in PVN neuron before and during myricentin (5μg/ml) application.

B. Bar graph showing the effects of myricetin on the mean decay time of mIPSCs. (n=14; *** P<0.001)

C. Cumulative probability plot of the amplitude, decay time and inter-event intervals of mIPSCs for the cell shown in A. The probability distribution of decay time was shifted to right direction, indicated an increase of decay time by myricetin. The probability distribution of inter-event intervals was sifted to the left direction indicated an increase of frequency by myricetin. (** P<0.01; ns not significant)

Fig.2 Effects of myricetin on the decay time of sIPSCs in PVN neurons.

A. Raw trace showed the changes of sIPSCs in the absence and presence of myricetin.

B. Summarized data demonstrated that myricetin prolonged the decay time of sIPSCs (n=8; P < 0.001).

1.2 Effects of flumazenil on myricetin induced prolongation of mIPSCs decay time

To investigate that if a benzodiazipine-like modulation of GABA-A receptor function by myricetin exists, the actions of flumazenil, the specific benzodiazipine biting site antagonist, were also tested. As shown in figure 3, consistently to the previous report, flumazenil of 10μM itself did not show any influence to mIPSCs , however, it also could not antagonize myricetin induced increase of mIPSCs decay time. These data suggested that the mechanisms underlying myricetin modulated GABA-A receptors function might differ from described for a classic benzodiazipine-binding site.

C

Fig 3 Effects of flumazenil on myricetin induced prolongation of mIPSCs. Flumazenil alone has no significant effects on mIPSCs decay time; it also could not antagonize the myricetin actions. (n=11, * P < 0.05)

1.3 Effects of calcium/calmodulin dependent protein Kinase II (CaM-KII) pathway on myricetin induced decay time prolongation of mIPSCs

It has been shown that the protein phosphorylation process is extensively involved in the regulation of receptor and channel activity. Many studies have reported that CaM-KII plays a key role in the modulation of GABA-A receptors functions. The role of CaM-KII on myricetin-induced changes of mIPSCs was further investigated in this experiment. KN-62, a CaM-KII inhibitor, (15μM) was added into the internal solution to inhibit the activity of CaM-KII. After whole-cell mode was established for 5 min, the mIPSCs were then recorded. In the 7 tested neurons, the myricetin-induced changes in mIPSCs were abolished when KN-62 was included in the internal solution (the decay time of control: 8.352 ± 0.933 pA; myricetin: 8.641 ± 0.859 pA) These data suggested that myricetin actions on the GABA-A receptors functions might mediated by enhancing the activity of CaM-KII.

Fig. 4 Effects of KN-62 on myricetin induced

prologation of mIPSCs.

The CaM-KII inhibitor KN-62 could

completely prevent the increase of decay

time induced by myticetin. (n=7; ns, not

significant)

Part two: Effects of myricetin on voltage-dependent calcium channel (VDCC) currents in PVN neurons

Calcium is a very important signal molecule in central nervous system, widely involved in transmitter release, synaptic plasticity, signal transduction and modulation of enzymic activity. The changes in intracellular calcium concentration will induce a sequence of actions thereby infect the synaptic currents.

In this section, the internal solution was composed of: (mM) CsCl 135, MgCl2 1.0, HEPES 10, EGTA 10, Na2-ATP 4, Tris GTP 0.4 and pH adjusted to 7.3-7.4 with 1 M CsOH. The perfusion solution consists of: (mM) NaCl 100, TEA-Cl 40, KCl 2.5, BaCl2 5, HEPES 10, glucose 10, 4-AP 5, and pH adjusted to 7.3-7.4 with 1 M HCl. TTX of 1μM was also included in the superfused solution.

When traditional whole-cell recording was established, the fast and slow membrane capacity were compensated automatically by the amplifier, the membrane potential was hold at –80mV, after stabled for several minute, currents were elicited according to a series of pulse protocols.

2.1 Myricetin enhances the VDCC currents in PVN neurons.

Figure 5A shows a typical recording of calcium current under control conditions and after addition of 5μg/ml myricetin. The currents were elicited in response to a 100 ms depolarization from a holding potential of –80mV to –20mV. These currents could be blocked by 200μM cadmium chloride, indicating they are calcium currents. After perfusion with myricetin, the peak current was increased. Figure 5B shows the averaged peak current before and during myricetin perfusion. The peak current was significantly increased compare with that of control condition. (n=8, P <0.05) To further test effects of myricetin on calcium channels in PVN neurons, the current-voltage relationship was also examined. Figure 6A shows the current-voltage relationship measured with or without myricetin with a 500ms voltage ramp from –80mV to +60mV in a typical neuron. As it indicated myricetin increased the inward current and shifted the peak current to the hyperpolarizing direction. The averaged current-voltage curves in response to a 100 ms step pulse from –80mV to +20mV with an increment of 10mV were also investigated. As shown in figure 6B, myricetin significantly enhanced the peak currents in the range between –30mV and –10mV and shifted the averaged peak current about 10mV to hyperpolarizing direction.

B

Fig. 5 Effects of myricetin on voltage-dependent calcium currents in PVN neurons

A. Row traces showed the calcium current changes in the absence and presence of myricetin in a typical neuron. B. The statistic data demonstrated a significant increase of peak current after treatment with myricetin (n=8, * P < 0.05).

Fig. 6 Effects of myricetin on the calcium channel properties in PVN neurons.

a. The current was recorded in response to a 500 ms ramp pulse depolarization from a holding potential of –80mV to +60mV, the peak current was increased and shifted to hyperpolarizing direction after myricetin treatment. b. Summarized current-voltage relationship curves showed the currents amplitude were increased in the region between –30mV and –10mV; the peak currents were shifted about 10mV to negative direction (n=10, * P < 0.05, ** P < 0.01).

2.2 Effects of myricetin on the different types of VDCC currents in PVN neurons

In general, The PVN consists predominantly of two types of neurons: the magnocellular neuron and parvocellular neurons. Several studies showed that in spite of high voltage dependent calcium channel, the T type calcium channel were also functionally expressed in the parvocellular neurons and even a few magnocellular neurons, so the effects of myricetin on different types of VDCC currents were further investigated. As shown in figure 7, the calcium currents were elicited with the following pulse: first depolarized to –40mV for 100 ms from –80mV then give a brief hyperpolarizing pulse to –45mV for 100 ms; and then further depolarized to 0mV. According to this pulse, two parts of currents were recorded. The first part inactivated quickly and sensitive to 300μM NiCl2, showed a T-type calcium channel properties. The second part inactivated slowly and sensitive to 30μM nifedipine, confirming a high voltage dependent L-type calcium currents. In the studied 13 cells, 4 neurons only contain high voltage dependent calcium currents, other neurons are all expressed a T-type calcium currents, however, the variations are very large (from 30pA to 900pA). Figure 7A shows the changes of calcium channel currents after myricetin treatment in a typical neuron. Both of the two parts currents were increased in the presence of myricetin. However, the summarized data could not show a significantly increase (fig. 7B), probably due to the large variance of the currents.

Fig.7 Effects of myricetin on the different types of calcium currents in PVN neurons.

A. Row traces showed that myricetin enhanced both T-type and L-type calcium currents. The currents were recorded in response to a pulse showed in this figure. The first part current can be blocked by 300μM NiCl2 (n=3) and the second part can be blocked by 30μM nifedipine (n=3). B. The pooled data summarized effects of myricetin on calcium currents when depolarized to both –40mV and 0mV from a holding potential of –80mV compared that of control condition (ns, not significant).

Part three: Effects of myricetin on the membrane potential in PVN neurons.

In the present study, we found myricetin can modulate GABA-A receptor mediated synaptic currents and enhance calcium channel currents. However, effects of myricetin on the neurons excitability are still uncertain. In this section, the membrane potential was examined after myricetin treatment. The internal solution we used in this part was consisted of (mM): Kglu 130, KCl 10, MgCl2 2, HEPES 10, EGTA 1, Na2ATP 2, Tris GTP 0.4 and pH adjusted to 7.3 with 1 M KOH.

3.1 Effects of myticetin on the membrane potential in PVN neurons.

After whole cell recording was established, the current-clamp mode was chosen to observe the membrane potential and the action potentials. We have tested total 24 neurons, in which 10 have no spontaneous firing. The other 14 neurons have a spontaneous firing however exhibit a different fire mode. As shown in figure 8, 9 neurons generate a tonic fire (fig.8A); and 5 generate a low-threshod depolarizing potential which gave rise to bursts of several action potentials (fig.8B). The silent neurons normally have a membrane potential of –47.28 ± 2.15 mV, after superfused with myricetin, the membrane has a slight hyperpolarization to –51.73 ± 1.55 mV (Fig.9 n=6). In the tonic fire neurons, we observed the frequency of action potential was inhibited in the presence of myricetin (Fig.10, n=7).