Molecular Genetic Strategies in the Study of Corticohippocampal Circuits

Abstract

The first reproductively viable genetically modified mice were created in 1982 by Richard Palmiter and Ralph Brinster (Palmiter RD, Brinster RL, Hammer RE, Trumbauer ME, Rosenfeld MG, Birnberg NC, Evans RM. 1982. Dramatic growth of mice that develop from eggs microinjected with metallothionein-growth hormone fusion genes. Nature 300: 611-615). In the subsequent 30 plus years, numerous ground-breaking technical advancements in genetic manipulation have paved the way for improved spatially and temporally targeted research. Molecular genetic studies have been especially useful for probing the molecules and circuits underlying how organisms learn and remember—one of the most interesting and intensively investigated questions in neuroscience research. Here, we discuss selected genetic tools, focusing on corticohippocampal circuits and their implications for understanding learning and memory.

Figure 1.

The deletion of N-methyl-d-aspartate (NMDA) receptor NR1 subunit in specific subregions of the hippocampus. (A) Loss of NMDA receptor function in specific hippocampal areas can be achieved by using specific promoters to drive Cre recombinase. (B) In situ hybridization of NMDAR1 mRNA from wild types (WTs) (left panels) and subregion-specific NR1 knockout (KO) mice (right panels). (C) CA1-restricted deletion of the NR1 subunit impairs long-term potentiation (LTP) in the Schaffer collateral pathway (open symbols, WTs; closed circles, mutant). (D) CA1-specific knockout mice are impaired during training in the spatial version of the Morris water maze (open symbols, WTs; closed circles, mutant), and (E) during the probe test (black bars indicate mutants). (F) CA3-specific deletion of the NR1 subunit blocks LTP at the recurrent commissural/associational synapses. (G) CA3-specific knockout does not affect training in the spatial version of the Morris water maze, (H) but results in a reduced preference for the target quadrant during a probe trial with partial cues, indicating an impairment in pattern completion. (I) Perforant path LTP is lost as a consequence of ablation of the NR1 subunit in the dentate gyrus. (J) A design to test context discrimination in WT and dentate gyrus-specific NMDA receptor knockout mice. For the first 3 days of conditioning, mice visited only chamber A and each day received a single foot shock. Freezing was measured once in chamber A and once in chamber B over the subsequent 2 days. During days 6–17, 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. (K) Dentate gyrus (DG)-restricted ablation of NMDA receptors results in impaired pattern separation indicated by a reduced discrimination ratio between a shocked context (context A) and a nonshocked context (context B). adj., Adjacent; opp., opposite. (From Havekes and Abel 2009; reprinted, with permission, from Elsevier © 2009; original sources for data are Tsien et al. 1996b; Nakazawa et al. 2002; McHugh et al. 2007.)

Figure 2.

Regulation of the α-calcium-calmodulin-dependent protein kinase II (CaMKII) transgene with the tetracycline transactivator (tTA) system reversibly alters long-term potentiation (LTP) and spatial memory. (A) Strategy used to obtain forebrain-specific doxycycline (Dox)-regulated transgene expression. Two independent lines of transgenic mice are obtained, and the two transgenes are introduced into a single mouse through mating. (B) Quantification by RT-PCR Southern blot of CaMKII-Asp²⁸⁶ expression from the tetO promoter (Tg1, mouse carrying only the CaMKII promoter-tTA transgene; Tg2, mouse carrying only the tetO-CaMKII-Asp²⁸⁶ transgene; Tgl/Tg2, double transgenic mouse carrying both the CaMKII promoter-tTA transgene and tetO-CaMKII-Asp²⁸⁶ transgenes; Tg1/Tg2 + Dox, double transgenic mouse treated with doxycycline [2 mg/ml] plus 5% sucrose in the drinking water for 4 wk). (C) Double transgenic mice (B13) fail to potentiate following stimulation at 10 Hz for 1.5 min. Doxycycline treatment reversed the defect in B13 mice. (D) The Barnes circular maze. In this spatial memory task, mice are required to use distal cues to find the location of the escape hole. (E) Mean number of errors across session blocks composed of five sessions. Double transgenic mice (B22) show impaired performance on the Barnes maze. This impairment is reversed after doxycycline treatment. WT, Wild type. (From Mayford et al. 1996b; reprinted, with permission, from The American Association for the Advancement of Science © 1996.)

Figure 3.

Activation of the octopamine receptor in mouse forebrain neurons enhances long-term potentiation (LTP) and memory. (A) Aplysia octopamine receptor (Ap oa₁) is a G-protein-coupled receptor whose activation by octopamine stimulates adenylyl cyclase activity, which in turn synthesizes cyclic adenosine monophosphate (cAMP). (B) Hippocampal one-train LTP (100 Hz for 1 sec) is potentiated and lengthened in Ap oa₁ mice after octopamine application. Arrow indicates when the potentiation stimulus was delivered. (C) Training and 24-h long-term memory test during contextual fear conditioning in Ap oa₁ transgenic mice and wild-type (WT) littermates injected intraperitoneally with octopamine 30 min before training. No differences in freezing behavior are observed between Ap oa₁ transgenic mice and WT littermates before and after the shock during training. In contrast, Ap oa₁ transgenic mice show a significant increase in freezing behavior when reexposed to the fear-conditioned context 24 h after training. (D) Training and 24-h long-term memory testing during fear conditioning in Ap oa₁ transgenic mice and WT littermates receiving an intraperitoneal injection of octopamine 30 min before the retrieval session. Ap oa₁ transgenic mice and WT mice show similar levels of freezing during the training session (baseline). However, Ap oa₁ transgenic mice show significantly increased freezing behavior compared with WT mice during the contextual fear memory test 24 h after training. (Image reprinted from Isiegas et al. 2008 under the U.S. Fair Use Guidelines available from the Copyright Office at the Library of Congress.)

Figure 4.

Optogenetic inactivation of CA1 impairs recent (24 h) long-term memories, whereas optogenetic inactivation of anterior cingulate cortex (ACC) impairs remote (28 d) memory. (A) Double lentiviral injection resulted in eNpHR3.1 expression in CA1 only. (B) Bilateral in vivo light administration to CA1 (top). Illumination of CA1 neurons in eNpHR3.1-expressing mice resulted in a reversible reduction in spiking frequency without affecting average spike amplitude. A representative optrode recording trace is shown (bottom). (C) CA1 optogenetic inhibition prevented remote memory. This disruption was reversible, as when the same mice were reintroduced to the conditioning context with no illumination, they showed intact fear responses. Optogenetic inactivation of CA1 had no impact on cued fear memory, which is amygdala dependent (not shown). (D) CA1 optogenetic inhibition prevented remote fear recall only when light was administered precisely during testing (precise group, left), but not when the light was on continuously for 30 min before (as well as during) the test (prolonged group, middle). When the prolonged group mice were retested the next day with precise light, their recall was disrupted. (E) eNpHR3.0 expression in the ACC. (F) Precise light administration resulted in inhibition of remote but not recent memory recall. (From Goshen et al. 2011; reprinted, with permission, from Elsevier © 2011.)