Abstract
Although much is known about the functional expression of the neuronal nicotinic acetylcholine receptors (nAChRs) in various neuronal populations in the brain and elsewhere, much less is known about their expression and functional relevance in glial cells. The expression of functional nAChRs has been reported for cultured astrocytes; however, previous work has failed to detect nAChR-mediated responses in astrocytes in acute slices. In the current study, functional α7 nAChRs on astrocytes in the CA1 region of the rat hippocampus were studied in situ using whole-cell patch-clamp recording and two-photon calcium imaging techniques in acute slices. We found that astrocytes and the chondroitin sulfate proteoglycan NG2-expressing (i.e., NG2) cells did express functional α7 nAChRs. Although the amplitudes of the responses were small, they could be enhanced by the α7-selective positive allosteric modulator PNU-120596. Under these conditions, we found that in comparing the properties of these responses between astrocytes, NG2, and interneurons, there were differences in the kinetics and increases in intracellular calcium levels. This is the first demonstration of functional α7 nAChR-mediated current responses in astrocytes in acute hippocampal slices, data which may shed light on the role of α7 nAChRs in neuroprotection.
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📋 Methods
Slice Preparation
All experiments were carried out in accordance with guidelines approved by the NIEHS Animal Care and Use Committee, which includes minimizing the number of animals used and their suffering. Standard techniques were used to prepare 310 μm thick acute hippocampal slices from 14–21-day-old rats ( Shen et al. 2009 ). Briefly, rats were anesthetized with halothane (Sigma) and decapitated. Brains were quickly removed and placed into an ice-cold oxygenated artificial cerebral spinal fluid (ACSF) containing (in mM): 126 NaCl, 3.5 KCl, 1.3 MgCl 2 , 2 CaCl 2 , 1.2 NaH 2 PO 4 , 25 NaHCO 3 , and 11 glucose. Slices were placed onto nylon mesh immersed in oxygenated ACSF at room temperature (23–25°C) and then used for recordings after at least 1 h of recovery period and within about 6 h.
Electrophysiology
Whole-cell patch-clamp recordings were performed on hippocampal CA1 stratum radiatum layer astrocytes, interneurons, or NG2-expressing (i.e., NG2) cells in acute slices. Patch pipettes (Garner 8250 glass, with resistances of 3–7 MΩ for interneurons, and 6–10 MΩ for astrocytes and NG2 cells) were filled with an intracellular solution (ICS) that contained (in mM): 120 potassium gluconate, 2 NaCl, 2 MgATP, 0.3 Na 2 GTP, 1 EGTA and 10 HEPES; pH was adjusted to 7.2–7.3 with KOH (1 M), and osmolarity was adjusted to ~285 mOsm with KCl (2 M). Slices were superfused at room temperature with oxygenated ACSF. Using an Eclipse E600FN upright microscope (Nikon, Japan) with a×40 fluorescent water-immersion objective and an iXon cooled 14-bit digital camera (Andor Technology, UK), real-time infrared differential interference contrast (IR-DIC) images were acquired with iQ imaging software (Andor). Cells were clamped using an Axopatch 200B amplifier (Axon Instruments, Union City, CA) at a holding potential of –70 mV (corrected for a 10 mV junction potential) for interneurons and –90 mV for astrocytes and NG2 cells using pClamp 10 software (Axon Instruments). Action potentials were recorded under current-clamp conditions, and current was injected to depolarize neurons to induce spiking; neurons were defined as regular-spiking if the rate of action potential firing evoked by minimal depolarization was
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Slice Preparation
All experiments were carried out in accordance with guidelines approved by the NIEHS Animal Care and Use Committee, which includes minimizing the number of animals used and their suffering. Standard techniques were used to prepare 310 μm thick acute hippocampal slices from 14–21-day-old rats ( Shen et al. 2009 ). Briefly, rats were anesthetized with halothane (Sigma) and decapitated. Brains were quickly removed and placed into an ice-cold oxygenated artificial cerebral spinal fluid (ACSF) containing (in mM): 126 NaCl, 3.5 KCl, 1.3 MgCl 2 , 2 CaCl 2 , 1.2 NaH 2 PO 4 , 25 NaHCO 3 , and 11 glucose. Slices were placed onto nylon mesh immersed in oxygenated ACSF at room temperature (23–25°C) and then used for recordings after at least 1 h of recovery period and within about 6 h.
Electrophysiology
Whole-cell patch-clamp recordings were performed on hippocampal CA1 stratum radiatum layer astrocytes, interneurons, or NG2-expressing (i.e., NG2) cells in acute slices. Patch pipettes (Garner 8250 glass, with resistances of 3–7 MΩ for interneurons, and 6–10 MΩ for astrocytes and NG2 cells) were filled with an intracellular solution (ICS) that contained (in mM): 120 potassium gluconate, 2 NaCl, 2 MgATP, 0.3 Na 2 GTP, 1 EGTA and 10 HEPES; pH was adjusted to 7.2–7.3 with KOH (1 M), and osmolarity was adjusted to ~285 mOsm with KCl (2 M). Slices were superfused at room temperature with oxygenated ACSF. Using an Eclipse E600FN upright microscope (Nikon, Japan) with a×40 fluorescent water-immersion objective and an iXon cooled 14-bit digital camera (Andor Technology, UK), real-time infrared differential interference contrast (IR-DIC) images were acquired with iQ imaging software (Andor). Cells were clamped using an Axopatch 200B amplifier (Axon Instruments, Union City, CA) at a holding potential of –70 mV (corrected for a 10 mV junction potential) for interneurons and –90 mV for astrocytes and NG2 cells using pClamp 10 software (Axon Instruments). Action potentials were recorded under current-clamp conditions, and current was injected to depolarize neurons to induce spiking; neurons were defined as regular-spiking if the rate of action potential firing evoked by minimal depolarization was
📊 Figures
Fig. 1
Electrophysiological properties of interneurons, astrocytes, and NG2 cells in acute CA1 rat hippocampal slices. a Firing pattern of a representative interneuron (current-clamp configuration). Action p...
Fig. 2
Morphological features of astrocytes and NG2 cells in acute hippocampal slices. IR-DIC ( left panels ) and two-photon ( right panels ) images of an interneuron ( a ), astrocytes ( b ), and NG2 cell ( ...
Fig. 3
Functional u03b17 nAChR-mediated responses on interneurons, astrocytes, and NG2 cells in slices. a Representative rapidly activating and decaying inward current u03b17 nAChR-mediated responses in an i...
Fig. 4
Activation of the u03b17 nAChRs increases intracellular calcium levels in astrocytes. a, b Representative time-courses showing global calcium increases in an astrocyte ( a ) and interneuron ( b ) afte...
Figure images are served from the NIH/NLM PubMed Central Open Access Subset or Europe PMC; copyright remains with the publishers and authors.
💬 Discussion
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