All animals were treated in accordance with the guidelines and regulations from the Animal Care and Use of Laboratory of National Institute of Health. All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of City University of Hong Kong and Incheon National University.
1. In vivo field recording
2. In vitro field recording
We explored long-term synaptic plasticity of longitudinal CA1 pyramidal neurons of the hippocampus using extracellular field recordings both in vivo and in vitro. LTP and LTD are facets of long-term synaptic plasticity that have been demonstrated in the transverse axis of the hippocampus to be unidirectional.
We showed here that using longitudinal hippocampal brain slices, there is LTP in the CA1 longitudinal axis of the hippocampus. We prepared longitudinal slices of the hippocampus along the septotemporal axis, which is perpendicular to the transverse slices (Figure 1). Using recordings from the CA1 region of the hippocampus, we showed the presence of LTP that was not direction specific. There were no statistically significant differences in the recordings from the septal or temporal (Figure 2) side of the longitudinal hippocampal brain slice. We also showed the presence of LTP that was not layer specific; thus, recordings from both stratum radiatum and stratum oriens (Figure 2) showed successfully induced LTP in the longitudinal brain slice. We used D-AP5, an NMDAR antagonist to demonstrate that the LTP induced was dependent on NMDA receptors (Figure 3). What happens in vitro does not necessarily reflect in vivo conditions, so we investigated LTP in vivo. Figure 4a shows a schematic diagram of the stimulation and recording electrode positioned in the dorsal hippocampus along the longitudinal axis of CA1 region in vivo. The position of the electrodes used for the recording and stimulation was verified by lesion marks and crystal violet staining (Figure 4a). We demonstrated the presence of LTP in vivo in the longitudinal CA1 region (Figure 4b).
Using already established protocols for inducing LTD, we failed to successfully induce LTD both in vivo and in vitro (Figure 5).
Figure 1. A schematic drawing of transverse and longitudinal hippocampal brain slices. This figure is adapted and modified from Sun et al. 201821. Please click here to view a larger version of this figure.
Figure 2. LTP in longitudinal slices. Synaptic responses at S.R. (a) or S.O. (b) in longitudinal slices are potentiated right after tetanus stimulation with both temporal and septal inputs (S.R./temporal (n = 12, c), S.R./septal (n = 12, c), S.O./temporal (n = 10, d), S.O./septal (n = 9, d).
The n stands for the number of slices. Error bars represent SE. This figure is adapted and modified from Sun et al. 201821. Please click here to view a larger version of this figure.
Figure 3. NMDAR-dependent LTP in longitudinal slices. (a,b) LTP induction in temporal and septal direction is blocked by 50 μM D-AP5 (temporal, n = 6, a) (septal, n = 5, b). (c,d) LTP induction in temporal and septal direction is also blocked by D-AP5. The n stands for slices. Error bars represent SE. This figure is adapted and modified from Sun et al. 201821. Please click here to view a larger version of this figure.
Figure 4. In vivo LTP in the interlamellar network. (a) A schematic drawing of recording and stimulation electrodes in anesthetized animals. The loci of recording (on the septal side of CA1) and stimulating electrodes (on the temporal side of CA1) were identified by lesion marks. (b) LTP is induced in the interlamellar connection by 100 Hz high frequency stimulation (HFS) (n = 10 mice). Color traces: before (black) and after (red) HFS. Error bars represent SE. This figure is adapted and modified from Sun et al. 201821. Please click here to view a larger version of this figure.
Figure 5. Absence of in vivo and in vitro LTD in Interlamellar CA1 network. (a) 1 Hz-pp LFS does not induce in vivo LTD. (b) 1 Hz pp-LTP, (c) 5 Hz LFS, and (d) 1 Hz LFS do not produce LTD on either the temporal or septal sides of longitudinal brain slice. while LTD is induced by 1 Hz pp-LFS in transverse slices: temporal (n = 8), septal (n = 11) and transverse (n = 6) with 1 Hz pp-LFS; temporal (n = 3) and septal (n = 3) with 5 Hz LFS; temporal (n = 3) and septal (n = 3) with 1 Hz LFS. The n stands for slices. Error bars represent SE. This figure is adapted and modified from Sun et al. 201821. Please click here to view a larger version of this figure.
Figure 6. Input-output curve presenting fEPSP slope in response to increasing stimulus input in hippocampal brain slice. Please click here to view a larger version of this figure.
Figure 7. Surgical tools used for hippocampal isolation during in vitro brain slicing. Please click here to view a larger version of this figure.
Figure 8. A longitudinal brain slice ready for recording. Stimulation electrode and recording pipette are inserted in the stratum radiatum. Please click here to view a larger version of this figure.
Figure 9. A transverse hippocampal brain slice ready for recording. Stimulation electrode is inserted at Schaffer collateral CA3 region and recording pippette is inserted at CA1 region. Please click here to view a larger version of this figure.
Compound | Slicing Solution(mM) | ACSF (mM) |
CaCl2.2H2O | 0.5 | 2 |
Glucose | 25 | 25 |
KCl | 2.5 | 2.5 |
MgCl2.6H2O | 7 | 1 |
NaCl | 87 | 125 |
NaH2PO4 | 1.3 | 1.3 |
NaHCO3 | 25 | 25 |
Sucrose | 75 |
Table 1: Concentrations of compounds in brain slice and artificial cerebrospinal fluid solutions.
Atropine Sulphate salt monohydrate, ≥97% (TLC), crystalline | Sigma-Aldrich | 5908-99-6 | Stored in Dessicator |
Axon Digidata 1550B | |||
Calcium chloride | Sigma-Aldrich | 10035-04-8 | |
Clampex 10.7 | |||
D-(+)-Glucose ≥ 99.5% (GC) | Sigma-Aldrich | 50-99-7 | |
Eyegel | Dechra | ||
Isoflurane | RWD Life Sciences | R510-22 | |
Magnesium chloride hexahydrate, BioXtra, ≥99.0% | Sigma-Aldrich | 7791-18-6 | |
Matrix electrodes, Tungsten | FHC | 18305 | |
Multiclamp 700B Amplifier | |||
Potassium chloride, BioXtra, ≥99.0% | Sigma-Aldrich | 7447-40-7 | |
Potassium phosphate monobasic anhydrous ≥99% | Sigma-Aldrich | 7778-77-0 | Stored in Dessicator |
Pump | Longer precision pump Co., Ltd | T-S113&JY10-14 | |
Silicone oil | Sigma-Aldrich | 63148-62-9 | |
Sodium Bicarbonate, BioXtra, 99.5-100.5% | Sigma-Aldrich | 144-55-8 | |
Sodium Chloride, BioXtra, ≥99.5% (AT) | Sigma-Aldrich | 7647-14-5 | |
Sodium phosphate monobasic, powder | Sigma-Aldrich | 7558-80-7 | |
Sucrose, ≥ 99.5% (GC) | Sigma-Aldrich | 57-50-1 | |
Temperature controller | Warner Instruments | TC-324C | |
Tungsten microelectrodes | FHC | 20843 | |
Urethane, ≥99% | Sigma-Aldrich | 51-79-6 | |
Vibratome | Leica | VT-1200S | |
Water bath | Grant Instruments | SAP12 |
The study of synaptic plasticity in the hippocampus has focused on the use of the CA3-CA1 lamellar network. Less attention has been given to the longitudinal interlamellar CA1-CA1 network. Recently however, an associational connection between CA1-CA1 pyramidal neurons has been shown. Therefore, there is the need to investigate whether the longitudinal interlamellar CA1-CA1 network of the hippocampus supports synaptic plasticity.
We designed a protocol to investigate the presence or absence of long-term synaptic plasticity in the interlamellar hippocampal CA1 network using electrophysiological field recordings both in vivo and in vitro. For in vivo extracellular field recordings, the recording and stimulation electrodes were placed in a septal-temporal axis of the dorsal hippocampus at a longitudinal angle, to evoke field excitatory postsynaptic potentials. For in vitro extracellular field recordings, hippocampal longitudinal slices were cut parallel to the septal-temporal plane. Recording and stimulation electrodes were placed in the stratum oriens (S.O) and the stratum radiatum (S.R) of the hippocampus along the longitudinal axis. This enabled us to investigate the directional and layer specificity of evoked excitatory postsynaptic potentials. Already established protocols were used to induce long-term potentiation (LTP) and long-term depression (LTD) both in vivo and in vitro. Our results demonstrated that the longitudinal interlamellar CA1 network supports N-methyl-D-aspartate (NMDA) receptor-dependent long-term potentiation (LTP) with no directional or layer specificity. The interlamellar network, however, in contrast to the transverse lamellar network, did not present with any significant long-term depression (LTD).
The study of synaptic plasticity in the hippocampus has focused on the use of the CA3-CA1 lamellar network. Less attention has been given to the longitudinal interlamellar CA1-CA1 network. Recently however, an associational connection between CA1-CA1 pyramidal neurons has been shown. Therefore, there is the need to investigate whether the longitudinal interlamellar CA1-CA1 network of the hippocampus supports synaptic plasticity.
We designed a protocol to investigate the presence or absence of long-term synaptic plasticity in the interlamellar hippocampal CA1 network using electrophysiological field recordings both in vivo and in vitro. For in vivo extracellular field recordings, the recording and stimulation electrodes were placed in a septal-temporal axis of the dorsal hippocampus at a longitudinal angle, to evoke field excitatory postsynaptic potentials. For in vitro extracellular field recordings, hippocampal longitudinal slices were cut parallel to the septal-temporal plane. Recording and stimulation electrodes were placed in the stratum oriens (S.O) and the stratum radiatum (S.R) of the hippocampus along the longitudinal axis. This enabled us to investigate the directional and layer specificity of evoked excitatory postsynaptic potentials. Already established protocols were used to induce long-term potentiation (LTP) and long-term depression (LTD) both in vivo and in vitro. Our results demonstrated that the longitudinal interlamellar CA1 network supports N-methyl-D-aspartate (NMDA) receptor-dependent long-term potentiation (LTP) with no directional or layer specificity. The interlamellar network, however, in contrast to the transverse lamellar network, did not present with any significant long-term depression (LTD).
The study of synaptic plasticity in the hippocampus has focused on the use of the CA3-CA1 lamellar network. Less attention has been given to the longitudinal interlamellar CA1-CA1 network. Recently however, an associational connection between CA1-CA1 pyramidal neurons has been shown. Therefore, there is the need to investigate whether the longitudinal interlamellar CA1-CA1 network of the hippocampus supports synaptic plasticity.
We designed a protocol to investigate the presence or absence of long-term synaptic plasticity in the interlamellar hippocampal CA1 network using electrophysiological field recordings both in vivo and in vitro. For in vivo extracellular field recordings, the recording and stimulation electrodes were placed in a septal-temporal axis of the dorsal hippocampus at a longitudinal angle, to evoke field excitatory postsynaptic potentials. For in vitro extracellular field recordings, hippocampal longitudinal slices were cut parallel to the septal-temporal plane. Recording and stimulation electrodes were placed in the stratum oriens (S.O) and the stratum radiatum (S.R) of the hippocampus along the longitudinal axis. This enabled us to investigate the directional and layer specificity of evoked excitatory postsynaptic potentials. Already established protocols were used to induce long-term potentiation (LTP) and long-term depression (LTD) both in vivo and in vitro. Our results demonstrated that the longitudinal interlamellar CA1 network supports N-methyl-D-aspartate (NMDA) receptor-dependent long-term potentiation (LTP) with no directional or layer specificity. The interlamellar network, however, in contrast to the transverse lamellar network, did not present with any significant long-term depression (LTD).