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.2014 Aug 12;111(32):E3343-52.
doi: 10.1073/pnas.1410893111. Epub 2014 Jul 28.

Astrocytes contribute to gamma oscillations and recognition memory

Affiliations

Astrocytes contribute to gamma oscillations and recognition memory

Hosuk Sean Lee et al. Proc Natl Acad Sci U S A..

Abstract

Glial cells are an integral part of functional communication in the brain. Here we show that astrocytes contribute to the fast dynamics of neural circuits that underlie normal cognitive behaviors. In particular, we found that the selective expression of tetanus neurotoxin (TeNT) in astrocytes significantly reduced the duration of carbachol-induced gamma oscillations in hippocampal slices. These data prompted us to develop a novel transgenic mouse model, specifically with inducible tetanus toxin expression in astrocytes. In this in vivo model, we found evidence of a marked decrease in electroencephalographic (EEG) power in the gamma frequency range in awake-behaving mice, whereas neuronal synaptic activity remained intact. The reduction in cortical gamma oscillations was accompanied by impaired behavioral performance in the novel object recognition test, whereas other forms of memory, including working memory and fear conditioning, remained unchanged. These results support a key role for gamma oscillations in recognition memory. Both EEG alterations and behavioral deficits in novel object recognition were reversed by suppression of tetanus toxin expression. These data reveal an unexpected role for astrocytes as essential contributors to information processing and cognitive behavior.

Keywords: electroencephalogram; glia; glial fibrillary acidic protein; gliotransmitter; network oscillation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Intracellular calcium responses precede carbachol-induced oscillations. (A) Immunofluorescence-based profiling of the three cells imaged inB. (Top) Fura-2–loaded cells (white arrows) visualized using a 380-nm excitation wavelength. (Middle) The slice was fixed and immunostained using a rabbit anti-GFAP antibody. (Bottom) The merge of theLeft andCenter panels shows that the Fura-2–loaded cells were GFAP-positive astrocytes. To minimize neuronal loading, short Fura-2–loading time was used, and consequently only a few cells were loaded with the calcium dye. (B) Example traces of intracellular calcium dynamics in three astrocytes from stratum radiatum of the CA3 area following carbachol treatment (CCH). (C) Extracellular field potential from the pyramidal cell layer of the CA3 area recorded simultaneously with the calcium imaging shown inB. Note that the onset of the calcium response preceded the start of the oscillations. (D) Gamma oscillations from recording inC shown on expanded time scale.
Fig. 2.
Fig. 2.
Tetanus toxin expression in astrocytes eliminated the release of glutamate but did not significantly modify synaptic activity. The glutamate detectors (HEK-NMDA), the HEK cells with NR1 and NR2B, were seeded on top of primary astrocyte culture (A), and the HEK-NMDA cell responses to mechanical stimulation of astrocytes were measured using a fluorescent calcium indicator. (A) Merged bright-field and dsRed channel images. Transfected HEK cells are visualized by virtue of dsRed protein expression (red arrows). White arrow is the stimulation site. Numbered circles are astrocytes where the intracellular calcium dynamics were monitored. (B) Intracellular calcium concentration in astrocytes (blue and purple traces) and HEK-NMDA cells (red traces) following mechanical stimulation. (C) The experiment described inA andB was repeated in the presence of 50 μM AP-5. (D andE) Intracellular calcium concentration following transfection of astrocytes with TeNTΔ1-GFP cDNA. TeNTΔ1-GFP–transfected cells were visible with green fluorescent color (D); blue and purple traces are the astrocytes response to mechanical stimulation, whereas the HEK-NMDA cells did not detect any extracellular glutamate (E, red traces). (F) Schematic representation of the lentiviral vector constructs used in this study. (G andH) Infection of astrocytes with lenti-GFP. Red arrow indicates HEK-NMDA cell. Black arrow indicates stimulation sites. Intracellular calcium concentration (H) in astrocytes (black trace) and HEK-NMDA cells (red trace) following mechanical stimulation of the astrocyte. (I andJ) Infection of astrocytes with lenti-TeNTΔ1-GFP with HEK-NMDA. Note that HEK-NMDA did not detect glutamate from the astrocytes infected with TeNTΔ1-GFP. (K–M) Targeted expression of TeNT to astrocytes in slice cultures had no significant effect on synaptic activity. Traces show mEPSCs recorded from pyramidal neurons in the CA3 region infected with astrocyte-targeted lentivirus containing GFP (K,Upper) and TeNTΔ1-GFP (K,Lower). (L) Mean amplitude of mEPSCs was 17.03 ± 1.64 pA (n = 3) and 18.51 ± 1.67 pA (n = 3) for GFP and TeNTΔ1-GFP, respectively. (M) Mean frequency of mEPSCs was 0.23 ± 0.046 Hz (n = 3) and 0.22 ± 0.002 Hz (n = 3) for GFP and TeNTΔ1-GFP, respectively.
Fig. 3.
Fig. 3.
Astrocyte-specific expression of tetanus toxin-disrupted hippocampal oscillatory activity in vitro. (A) Example of a cultured hippocampal slice. (Left) Bright-field image of the organotypic culture (the recording electrode is visible in the pyramidal layer of the CA3 area). (Center) GFP fluorescence emitted by the slice under UV-light illumination. (Right) Merged images. (B) Extracellular field recording showing a carbachol-induced gamma oscillations in a lenti-GFP–infected slice. (C) Oscillatory activity elicited by carbachol in a slice infected with lenti-TeNTΔ1-GFP. (D) Oscillatory activity induced by coapplication of carbachol (30 μM) and AMPA (30 μM) in the lenti-TeNTΔ1-GFP–infected slice. (E) Example trace of beta oscillations induced by charbachol in a lenti-GFP–infected slice. (F) Quantitative comparison parameters for the gamma (F1–F4) and beta (F5–F8) oscillations under different conditions. The different experimental conditions are indicated at the bottom (in all cases oscillations were induced by 30 μM carbachol): Ctrl, uninfected slices; GFP, slices infected with lenti-GFP; TeNT, slices infected with lenti-TeNTΔ1-GFP; TeNT/AMPA, oscillations induced by coapplication of carbachol (30 μM) and AMPA (30 μM) in slices infected with lenti-TeNTΔ1-GFP. Asterisk indicatesP < 0.05 (ANOVA). (G) Shown in green is the region infected with lenti-TeNTΔ1-GFP. Field potentials recorded in the presence of 30 μM CCH from inside the infected area (Upper trace) or from outside the infected area (Lower trace).
Fig. 4.
Fig. 4.
Genetic constructs used for the generation of the mouse model for temporally and spatially controlled expression of TeNTΔ1-GFP. (A) Schematic representation of the 3×TG (triple transgenic) mouse genotype:GFAP-tTA;GFAP-Cre-ERt2;TRE-STOP-TeNT-GFP. (B) TeNT is expressed only when the STOP sequence is removed following tamoxifen administration, which activates the Cre-ER protein required to excise the STOP sequence by loxP sequences. (C) The tTA is inactivated by doxycycline, resulting in the complete suppression of TeNT expression. Therefore, in the triple transgenic mouse, TeNT toxin expression is induced by tamoxifen injection and suppressed by feeding doxycycline.
Fig. 5.
Fig. 5.
Synaptic responses were preserved in astrocytic TeNT-expressing transgenic mice. (A) Input/output relationship between the fiber volley amplitude and fEPSP slope was determined over a range of stimulus intensities. Each point represents the mean of all slices for each stimulus intensity. Error bar illustrates SEM for both axes (n2xTG = 15 slices from seven mice,n3xTG = 17 slices from eight mice). (B) Paired-pulse facilitation induced by two consecutive stimuli (S1 and S2) delivered at different time intervals. Facilitation is represented as the S2/S1 ratio (one slice per mouse,n = 6 mice each). (C) Synaptic fatigue induced by 12 consecutive stimuli at 25-ms interpulse intervals (one slice per mouse,n = 6 mice each). (D) Long-term potentiation induced by high frequency (100 Hz for 1 s repeated three times at 5-min intervals, as indicated by the arrows; one slice per mouse,n = 5 mice each). (E) Long-term depression induced by low-frequency stimulation (1 Hz for 15 min,n2xTG = 4 slices from three mice,n3xTG = 3 slices from three mice). (F) mEPSCs obtained in the presence of TTX (0.5–1 μM) and picrotoxin (100 μM) with demonstrative recordings (Upper). (Scale bar, 5 s and 10 pA.) (Lower) Mean amplitude/frequency ± SEM. (n2xTG = 14 cells from five mice,n3xTG = 9 cells from three mice). Demonstrative fEPSP traces are illustrated asInsets inB–E. (Scale bar, 10 ms and 0.5 mV.)
Fig. 6.
Fig. 6.
EEG power spectrum was altered by astrocyte-specific TeNT expression in a behavioral state-dependent manner. (A) Percentage of duration of awake (green), non-REM sleep (purple), and REM sleep (orange) during light (Upper) and dark (Lower) phases for 2xTG (blue,n = 10) and 3xTG (red,n = 8) under different treatments. Light-colored red and blue horizontal bars represent SD of population. (B) Behavioral state-dependent EEG power spectra for control animals (2xTG) without drug treatment. Solid green (awake), purple (non-REM sleep), and orange (REM sleep) curves represent different behavioral states of the animal. Light-colored shades: mean ± SEM (may not be visible because the width of the shade is comparable or less than the thickness of the curves). (C–E) Effects of tamoxifen and subsequent doxycycline treatments on EEG power spectra on 3×TG (red) and control (2×TG, blue) animals in awake (C), non-REM sleep (D), and REM sleep (E) state. (Lower Left) C3-4, D3-4, and E3-4 show mean (solid curve) and SEM (shade) of power spectra in response to tamoxifen (Left) and then doxycycline (Right) treatment. (Upper) C1-2, D1-2, and E1-2 show ANOVAP values between 3×TG and control groups as a function of frequency (solid line); shaded regions at the bottom mark statistical significance (P < 0.05). (Right) C5-6, D5-6, and E5-6 show ANOVAP values of comparison before and after doxycycline treatment for 3×TG (Upper: C5, D5, and E5) and control (Lower: C6, D6, and E6) animals. The reduction in spectral power density was statistically significant only in low-gamma range (20–40 Hz, green arrows) and only while the animals were awake.
Fig. 7.
Fig. 7.
Astrocyte-specific TeNT expression in vivo provoked a task-specific cognitive decline, manifested as a significant deficit in recognition memory. Y-maze–evaluated (A) and FC-evaluated memories (B) showed no gamma oscillation-dependent impairments. (A,Left) No group differences in general exploratory levels were observed as assessed by the number of arm entries (ANOVA:F(4,17) = 2.5,P = 0.07). (A,Right) Bars represent the percentage of spontaneous alternations (unique triplets) ± SEM. The TeNT-expressing mice (3×TG-T) showed no changes compared with the control groups (ANOVA:F(4,17) = 2.3,P = 0.10). (B) Bars represent the time (in seconds) freezing for animals trained to associate a context or a tone cue with an aversive stimulus (foot shock) ± SEM. The 3×TG-T mice showed similar freezing levels to the control groups in both the contextual (ANOVA:F(4,17) = 1.0,P = 0.41) and conditioned stimulus tests (ANOVA:F(4,17) = 1.5,P = 0.24). (C) Gamma-oscillation–defective mice suffered a significant memory deficit in a NOR test. Bars represent the discrimination index value [DI = (TNOVEL-TFAMILIAR)/(TNOVEL+TFAMILIAR)] ± SEM (ANOVA:F(4,17) = 4.03,P = 0.01). Although the control mice (2×TG,ncontrol = 5,n2xTG-T = 3,n2xTG-TD = 3) showed significant preference for the novel object, the 3xTG-T mice (n = 5) did not discriminate between the familiar and the novel object, and therefore these groups were different [Fisher’s protected least significant difference (PLSD),P = 0.02, labeled with an asterisk]. Such defect was restored in mice with suppression of the TeNT by doxycycline (3xTG-T+D.n = 6; Fisher’s PLSD,P = 0.003, labeled with “#”), suggesting the need for a normal expression of gamma oscillations for the regular processing of recognition memory.
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