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Originally published in Science Express on 7 June 2007
Science 6 July 2007:
Vol. 317. no. 5834, pp. 94 - 99
DOI: 10.1126/science.1140263
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Research Articles
Dentate Gyrus NMDA Receptors Mediate Rapid Pattern Separation in the Hippocampal Network
Thomas J. McHugh1,2*,
Matthew W. Jones1* ,
Jennifer J. Quinn3 ,
Nina Balthasar4,
Roberto Coppari4 ,
Joel K. Elmquist4,
Bradford B. Lowell4,
Michael S. Fanselow3,
Matthew A. Wilson1 and
Susumu Tonegawa1,2||
1 The Picower Institute for Learning and Memory, RIKEN–MIT Neuroscience Research Center, Department of Biology and Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
2 Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
3 Department of Psychology, University of California–Los Angeles, Los Angeles, CA 90095, USA.
4 Division of Endocrinology, Harvard Medical School, Beth Israel Deaconess Medical Center, Boston, MA 02215, USA.
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Fig. 1. Basic features of POMC-Cre transgenic mice. (A) Image (1x magnification) of ß-galactosidase (ß-Gal) expression in a 12-week-old POMC-Cre/ROSA26 double-transgenic mouse stained with X-Gal and nuclear fast red. (B to D) Anti–ß-Gal immunohistochemical (1x, images visualized with Cy3, red) staining showing expression in a 16-week-old POMC-Cre/ROSA26 double-transgenic mouse in three coronal sections taken along the rostro-caudal axis of the forebrain. (E to K) Immunofluorescence staining of coronal sections of a 20-week-old POMC-Cre/ROSA26 double-transgenic mouse. Single staining with (E) anti-NeuN (neuronal marker; AlexaFlour 555, red, 4x), (F) anti–ß-Gal (marker of Cre recombination; aminomethylcoumarin, blue, 4x), and (G) anti-s100ß (glial cell marker; fluorescein isothiocyanate, green, 4x). (H) A 4x merge of (E), (F), and (G) indicating that the Cre-loxP recombination is restricted to the neurons in the DG. Inset is a 20x image of the DG. (I to K) Single staining with (I) an anti–ß-Gal (AlexaFlour488, green, 20x) and (J) anti-GAD67 (marker of inhibitory neurons; Cy3, red, 20x). (K) A merge of (I) and (J), indicating no recombination in GAD-67–positive inhibitory neurons. In the bottom right corner of each figure is a magnified image of several cells in the upper blade of the DG showing the separation of the green and red signals. (L to N) After injection of bromodeoxyuridine (BrdU) into a 12-week-old male POMC-Cre/ROSA26 double-transgenic mouse, brain sections were imaged by confocal microscopy. To the right of each 10x image are three single cells imaged at 63x at the positions in the 10x image labeled with the corresponding number. Single staining with (L) anti–ß-Gal (Cy3, red) and (M) anti-BrdU (marker of newly born cells; AlexaFlour488, green). (N) The overlap of the green and red signal in the cells imaged indicates that recombination occurs in newly born neurons after they reach the GC layer.
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Fig. 2. Basic features of DG-NR1 KO mice. (A to F) Images of coronal sections after in situ hybridization with a 33P-labeled NR1 cDNA probe. (A) Dark-field image (1x magnification) of a midbrain coronal section from a 20-week-old fNR1 control male; (B) 1x image of a mid-brain coronal section from a 20-week-old DG-NR1 KO male. (C) Image (4x magnification) of the hippocampus from the control mouse; (D) 4x image of the mutant mouse hippocampus. The NR1 transcript is deleted specifically in the DG granule cell layer in the mutant mouse. Light-field image of (E) the fNR1 hippocampus and (F) the DG-NR1 KO hippocampus reveal no changes in the gross structure of the hippocampus. (G and H) Immunohistochemical labeling of the NR1 protein (visualized with AlexaFlour 488, 4x) in the hippocampus of (G) a fNR1 animal at 16-weeks of age and (H) a DG-NR1 KO littermate. There is complete and specific loss of the receptor in the dentate gyrus of the KO mouse. (I and J) Timms staining of the mossy fiber pathway in (I) a 20-week-old fNR1 mouse and (J) a mutant littermate revealed no changes in the structure of the DG outputs as a result of the mutation. (K to N) In situ hybridization with the NR1 probe did not indicate a reduced NR1 mRNA level elsewhere in the brains of the knockout mice. Examination of 4x dark-field images of (K) the habenular nucleus of a 20-week-old fNR1 control animal and (L) a DG-NR1 KO littermate, and (M) the arcuate nucleus of control and (N) DG-NR1 KO mouse, found no difference in the abundance of the transcript in either region in the mutant mice.
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Fig. 3. In vivo synaptic transmission and plasticity of the DG-NR1 KO mice. PP-GC input/output curves of fNR1 control (open circles) and mutant (filled circles) mice, showing similar fEPSP slopes (A) and population spike (PS) amplitudes (B) at all stimulation intensities. (C) Paired-pulse facilitation at PP-GC synapses also appeared normal in the mutant mice. (D) Theta-burst stimulation of the PP input to the DG induced potentiation of fEPSP in fNR1 (open circles) and POMC-cre control animals (squares), but not in DG-NR1 KO mice (filled circles). In contrast, high-frequency stimulation of Schaffer commissural input induced (E) CA1 LTP in both DG-NR1 KO mice and fNR1 controls. Error bars are the SEMs.
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Fig. 4. DG NRs are important for the discrimination of similar contexts. (A and B) Contextual fear was measured 48 hours after conditioning with a single 2-s 0.75-mA footshock. Both fNR1 mice (n = 12) and DG-NR1 KO littermates (n = 12) showed (A) elevated total freezing in the conditioned context, as well as (B) identical kinetics of freezing across the 5-min test. (C) Generalized freezing behavior to a second, very different context was low in both genotypes. Separate groups of mice were subjected to a protocol (D) designed to test contextual discrimination. For the first 3 days of conditioning, mice visited only chamber A and each day received a single footshock (2 s, 0.65 mA). Freezing was measured once in chamber A and once in chamber B over the subsequent 2 days, and (E) control and mutant mice displayed equal amounts of freezing in both chambers. During days 6 to 17, mice visited each chamber daily (receiving a shock in one of the two), and freezing was assessed during the first 3 min in each chamber. (F) fNR1 mice (open circles; n = 12) showed significantly greater discrimination than the DG-NR1 KO mice (filled circles; n = 12) across most of the acquisition. (G and H) Freezing in chamber A and chamber B for the control (open bars) and mutant (filled bars) on (G) day 10 (middle of discrimination) and (H) day 17 (end of discrimination) demonstrated that the initial DG-NR1 KO discrimination deficit was rescued with additional training. Error bars in (B) to (H) are the SEMs.
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Fig. 5. DG NRs are important for context-specific modulation of firing rate in CA3. (A) Examples of firing-rate maps showing the activity of 10 fNR1 control (left) and 10 DG-NR1 KO (right) CA3 place cells as mice successively explored a white circular box and a black square box in the same location. Colors are scaled to maximum firing rates given by the numbers (red, maximum; blue, silent). (B) The rate difference in average firing rate in the two boxes was calculated for each cell [(high rate – low rate)/(high rate + low rate)]. fNR1 control rate differences were larger for CA3 than for CA1, whereas DG-NR1 KO rate differences were similar in CA3 and CA1. Mutant CA3 rate differences were significantly smaller than those of control mice (*P < 0.05, **P < 0.01). Red lines indicate rate differences expected given independent firing in the two boxes. (C) Cumulative probability histograms of the overlap [(low rate)/(high rate)] values for both genotypes and subregions showing significantly greater rate remapping (leftward shift) in control mice. (D) When the two box/one room experiment was repeated 24 hours later, DG-NR1 KO also showed significant rate remapping in CA3 (**day 1 P < 0.01; day 2 P = 0.57). Error bars in (B) and (D) are the SEMs.
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