Long Term Potentiation In Hippocampal Dentate Gyrus Astrocytes

In central nervous system, communication between neurons is mainly executed by chemical synaptic transmission occurring at the synapses, where presynaptic terminals and postsynaptic neurons are two functionally important elements. Astrocytes, the most numerous cell populations in the brain, have traditionally been considered as simple supportive elements that give structural and metabolic support to neurons. However, the growing evidence shows that astrocytes actively participate in the intracellular communication and modulate synaptic transmission and plasticity in the brain. It has been reported that astrocytes extend the processes to envelop the synapses, which form a tripartite synapse. It provided structural possibility for efficient and accurate modulation between neurons and astrocytes, which have been reported in brain cortex, hippocampus and cerebellum etc.In the recent decade, most attentions were focused on the elevated cytoplasmic free Ca~2+ (Ca~2+ wave and Ca~2+ oscillation) in astrocytes, which results from astrocytic response to presynaptically released neurotransmitters. As a new form of communication between neurons and astrocytes, the elevated cytoplasmic Ca~2+ leadsto release of several neurotransmitters and modulators from astrocytes such as glutamate, ATP, prostaglandin E2, TNFa and D-serine etc. These neuroactive factors act, in turn, on neuronal terminals to regulate the efficacy and strength of synaptic activities.It has been found that astrocytes also can express the various receptors and channels similar as those in neurons. It implies that astrocytes in vivo have the ability to respond to various neurotransmitters and display neuronal characteristics. The presence of ligand-gated ionotropic receptors such as AMPA receptors and glutamate transporters GLAST and GLT-1 enable astrocytes display directly electrical responses to neurotransmitters resulting from neuronal activity. Little is known, however, what biophysical properties of astrocytes are in hippocampal dentate gyrus area, how the astrocytes respond to synaptic inputs and whether astrocytic electrical responses can modulate synaptic transmission and plasticity.Long term potentiation (LTP) is a long lasting increase in synaptic strength evoked by high frequency stimulation (HFS) at input pathways. It is a form of synaptic plasticity that is believed to be neuron-specific. While neuronal LTP has been extensively investigated in distinct synapses and various regions, our understanding of the role of astrocytes in neuronal LTP is still primitive. It has been shown previously that LTP like response was detected in granule neuron-glial cell pairs in cerebellar culture, indicating that astrocytes are able to undertake behavioral changes in response to synaptic inputs. However, synaptic plasticity in astrocytes in response to HFS associated with LTP induction in neuron has not yet been established in various regions in vivo.LTP is first fully described in granule neurons of the dentate gyrus by Bliss and Lomo in 1973. However, little attention was put on the astrocytes activity and its modulation to granule neurons in this area. This is largely due to the fact that most of astrocytes mainly locate in granule cell body layer, and are difficult to be distinguishedbetween astrocytes and neurons in this area. The use of GFAP-GFP transgenic mice allows us to identify and visualize astrocytes with GFP fluorescence, and thus to characterize their biophysical properties and responses to synaptic inputs.In this study, the astrocytic potential or currents were recorded in dentate gyrus astrocytes in response to stimuli at perforant path in hippocampal slices of the GFAP-GFP transgenic mice using whole-cell patch clamp technique. Here, we will determine the electrophysiological characteristic in astrocytes, and will make clear whether astrocytes' responses to synaptic inputs are undergoing plasticity after high frequency stimulation (HFS) associated with neuronal LTP induction. The understanding of astrocyte plasticity and its modulation on neuron activity will help us to understand the mechanism of learn and memory, its role in releasing kinds of neuroactive factors associated with neurodegenerative diseases.Electrophysiological properties of astrocytes in hippocampal dentate gyrusTo determine the electrophysiological properties of astrocytes, which are different from those of neurons, whole-cell patch clamp were made on astrocytes located at granule cell layer of dentate gyrus in hippocampal slices. Electrophysiological recordings showed that these cells had a little more negative resting potential than granule neurons. Striking characteristics of these cells are no firing upon depolarizing current injection and lower input resistance when compared to that of granule neurons. Upon the perforant path stimulus, a monophasic excitatory postsynaptic potentials (EPSPs) was elicited in granule neurons, but a biphasic excitatory postsynaptic astrocytic potentials (EPAPs) was detected in astrocytes. The EPAPs exhibited an initial hyperpolarizing phase following by a long-lasting depolarizing phase. The initially hyperpolarized potential appeared to result from the extracellular field potential due to neuron depolarization because of the nature of low input resistance of astrocytes. Moreover, EPAPs had relative small amplitude and slowkinetics such as slow rise time and delay time.The constitution of EPAPsTo understand the difference between the EPAPs and EPSPs, stimulation evoked astrocytic currents were pharmacologically isolated in Mg2+-free external solution. Application of AMPA/kainate receptor antagonist NBQX (20 uM) resulted in a reduction of astrocytic currents to 47.7 ± 6.9%. Subsequently, the glutamate transporter inhibitors THA (300 uM) and DHK (300 uM) were applied simultaneously, and the astrocytic currents were reduced to 40.4 ± 7.1%. The remaining current was completely eliminated by 50 uM APV or 30 uM MK-801. It suggested that the EPACs mainly consisted of AMPA/kainate receptor-, NMDA receptor- and glutamate transporters mediated currents.Identification of NMDA receptor subunits using single cell rt-PCR techniqueIt is still uncertain whether the NMDA receptor is expressed in astrocytes. In our study, however, the NMDA receptor mediated currents were recorded in astrocytes. To further confirm the expression of the NMDA receptor in astrocytes, single cellular mRNA harvested from astrocyte was isolated;reverse transcripted;amplified and probed using single cell rt-PCR technique. The data showed that the significant amplification signals for NMDA receptor subunits NR1, NR2A and NR2B were detected. It indicates that the NMDA receptor can be expressed in astrocytes.Long term potentiation in astrocytesThe HFS-induced LTP induction at perforant path-dentate granule cell synapses has been well established. To test whether the similar paradigm could elicit an LTP like event in astrocytes in response to HFS at perforant path, EPAPs were recorded in astrocytes under whole-cell current clamp configuration. EPAPs were significantlypotentiated following HFS without simultaneously paired depolarizing current injection in astrocytes, and this potentiation was lasted for more than 30 minutes. The magnitudes of the enhancement of EPAPs were more pronounced when compared to those of EPSPs during neuronal LTP induced by HFS at perforant path.Effect of extracellular field potential on LTP induction in astrocytesIt is possible that the synaptically evoked astrocytic potentials contain the field potential component resulting from the activity of the neighboring neurons. To eliminate this contamination, the internal K+ was replaced with Cs+ and NMG+;the gap junction inhibitor was included in the pipette solution. However, these treatments did not significantly alter LTP induction although the input resistance is slightly elevated. In addition, the stimulation evoked inward currents that were less affected by field potential also significantly increased after HFS. It indicates that the induction of astrocytic LTP was not caused by field potentials.Glutamate transporters did not contribute to LTP induction in astrocytesAs demonstrated previously the induction of LTP at perforant path-dentate gyrus granule neuron synapses is accompanied with the increased release of glutamate. Since the transporters are highly sensitive to the amount of the released glutamate, it is possible that the increased transporter currents during neuronal LTP contribute to the potentiation of EPAPs. To address this issue, a combination of 150 uM THA (GLAST and GLT-1 inhibitor) and 150 uM DHK (GLT-1 inhibitor) were applied to astrocytes. It appeared that EPAPs were still enhanced following HFS in the presence of transporter inhibitors. There were no significant differences in the magnitudes of the EPAPs after HFS in the presence of transporter inhibitors when compared to that in control. To determine the percentage of the glutamate transporter mediated currents contribute to EPAPs, we recorded the remained transporter current after blockade of ionotropicglutamate receptor before and after HFS, respectively. It showed that the transporter current is increased by -60% during LTP. But the transporter current only remained 11.3 ± 1.9% and 6.7 ± 0.6% before and after LTP, respectively. The results suggest that the transporter current contributes a small percentage to EPAPs. It indicates that EPAPs are mainly composed of ionotropic glutamate receptor mediated components, especially after induction of LTP. This is in harmony with the results reported by others.mGluR was not involved in LTP induction in astrocytesmGluR can induce or enhance neuronal LTP. Group I mGluR have been shown to be expressed in astrocytes. It is possible that the synaptic released glutamate may act on mGluR in astrocytes that contributes to the induction of LTP. However, application of group I mGluR antagonist AIDA (500 fjM) did not affect HFS induced LTP in astrocytes suggesting that mGluR may not participate in the astrocytic LTP. Application of group I mGluR agonist DHPG (30 uM) reduced LTP induction in 50% astrocytes, while enhanced LTP induction in the other 50% astrocytes. CHPG, a group I mGluR5 agonist, did not affect the induction of LTP in astrocytes.NMDA receptor antagonist blocked LTP induction in astrocytesIt will be determined whether the ionotropic glutamate receptors are responsible for the LTP induction in astrocytes. AMAP/kainate receptors antagonist DNQX (10 uM), or NMDA receptor antagonist APV (50 uM), or ionotropic glutamate receptor antagonist Kyn (1.5 mM) was applied for 5 min before HFS and were immediately washout after HFS. The EPAPs were gradually potentiated to 280.5 ± 31.6% of baseline after washout of DNQX for 25 min following HFS. However, the HFS-induced enhancement of EPAPs was completely inhibited upon washout of APV or Kyn. These results indicate that the activation of the NMDA receptor is required forLTP induction in astrocytes.Intracellular calcium was not involved in the LTP inductionIn hippocampal dentate gyrus, NMDA receptor dependent LTP in granule neurons requires a rise in intracellular Ca2+ via activation of NMDA receptor. Since the NMDA receptor is functionally expressed in astrocytes, we asked whether the induction of LTP in astrocytes also requires an increase in intracellular Ca2+ through activation of the NMDA receptor. To test this hypothesis, the patch pipettes were filled with the internal solution containing 40 mM BAPTA (a Ca2+ chelator). However, the presence of BAPTA failed to block the induction of LTP in astrocytes.Inhibition of some kinases did not affect LTP induction in astrocytesCaMKII, PKA and PKC are all key molecules involved in LTP induction. It has been shown that these kinases can phosphorylate various target proteins, include AMPA receptor, which results in an increase of single channel conductance in AMPA receptor and helps trafficking of AMPA receptors. However, Application of CaMKII inhibitor KN93 (2 mM), or PKA inhibitor H89 (10 uM), or PKC inhibitor chelerythrione (10 [iM) had little effect on LTP induction in astrocytes. It suggests that the mechanism of LTP induction in astrocytes is different from that in neurons.BAPTA still failed to block LTP after blockade of gap junctionSince gap junctions between astrocytes make the astrocytes tightly connected that create a large buffer pool. It is possible that the intracellular application of BAPTA and kinase inhibitors could not reach to an enough high concentration because of dissipation via gap junction coupling. To test this hypothesis, the effect of intracellular BAPTA in the presence of gap junction inhibitor octanol or carbenoxolone was examined. Bath application of octanol (300 uM) or carbenoxolone (100 uM) for atleast 20 minutes resulted in significant reduction of astrocytic LTP after HFS when compared to that of control. However, octanol or CBX per se inhibited EPAPs, and reduced LTP in absence of intracellular BAPTA. These results indicate that the attenuated LTP is attributed to the gap junction inhibitors, but not to BAPTA.Conclusions1. In hippocampal dentate gyms, the electrophysiological properties of astrocytes are different from those of neurons. The stimulation evoked EPACs are mainly consisted of AMPA/kainate receptor-, NMD A receptor- and glutamate transporter-mediated currents.2. LTP can be induced in hippocampal dentate gyrus astrocytes, and it is independent on potentiated glutamate transporter current and extracellular field potentials. The AMPA/kainate receptor mediated potential contributes to most of the potentiated potential.3. The induction of LTP in astrocytes requires activation of the NMD A receptor, but it does not need a rise in intracellular Ca2+ and activation of key kinases. It indicates that the increased presynaptic release may be responsible for astrocytic LTP. But it is also possible that the induction of astrocytic LTP has different mechanism to neuronal LTP.