The Use of Lentiviral Vectors and Cre/loxP to Investigate the Function of Genes in Complex Behaviors

Abstract

The use of conventional knockout technologies has proved valuable for understanding the role of key genes and proteins in development, disease states, and complex behaviors. However, these strategies are limited in that they produce broad changes in gene function throughout the neuroaxis and do little to identify the effects of such changes on neural circuits thought to be involved in distinct functions. Because the molecular functions of genes often depend on the specific neuronal circuit in which they are expressed, restricting gene manipulation to specific brain regions and times may be more useful for understanding gene functions. Conditional gene manipulation strategies offer a powerful alternative. In this report we briefly describe two conditional gene strategies that are increasingly being used to investigate the role of genes in behavior - the Cre/loxP recombination system and lentiviral vectors. Next, we summarize a number of recent experiments which have used these techniques to investigate behavior after spatial and/or temporal and gene manipulation. These conditional gene targeting strategies provide useful tools to study the endogenous mechanisms underlying complex behaviors and to model disease states resulting from aberrant gene expression.

Keywords: PTSD; amygdala; fear; gene therapy; hippocampus; inducible knockout; lentivirus.

Figure 1

Cre recombinase expressing transgenic mice. A DNA template in which the coding sequence for Cre recombinase was placed under the control of the CCK promoter (CCID) was used to create transgenic mouse lines. Three founder animals, with distinct transgene insertion sites, were generated and bred to RosaLacZ animals to assess Cre expression as revealed by LacZ staining. (A) Normal patterns of CCK mRNA expression, revealed with in situ hybridization for CCK mRNA. (B) Animals from transgene line A show low levels of Cre expression, but in appropriate CCK-specific regions. (C) Animals from line B relatively widespread expression, which was, with the exception of the dentate, similar to endogenous CCK mRNA expression. (D) Animals from line C showed high levels of Cre expression in the cortex, in a manner that was qualitatively similar to observed patterns of CCK mRNA expression but with virtually no Cre expression in hippocampus or subcortical regions. (E) Animals from transgenic line C were crossed with Td tomato/floxed stop GFP, to assess Cre mediated GFP expression. These mice express strong red fluorescence in all tissues and cell types examined. When bred to Cre recombinase expressing mice, the resulting offspring have the stop cassette deleted in the Cre expressing tissue, deleting Td tomato expression and allowing expression of the membrane-targeted GFP (red = Td tomato, green = eGFP, blue = DAPI; Left – GFP expression, Right – merged GFP + TdTomato expression). Panels (A–D) from Chhatwal et al. (2007).

Figure 2

In vivo validation of LV-Cre lentivirus. Robust Cre recombinase expression as labeled by LacZ when LV-Cre is injected into RosaLacZ reporter mice. Sections were processed for lacZ histochemistry with β-gal 14 days after LV-Cre injections to visualize Cre-dependent recombination in vivo in the (A,B) striatum and (C,E) dorsal hippocampus seen at ×10 and ×20 magnification. LV-Cre-infected cells within (D) CA1 and (E) DG, illustrating the dense β-gal and intact morphology of hippocampal neurons.

Figure 3

Inducible BDNF deletion in hippocampus with Cre lentivirus. (A) BDNF (top) and Cre-recombinase (middle) mRNA expression visualized with in situ hybridization 2 weeks after LV-Cre infection into BDNF-floxed mice. The bottom figure represents a pseudocolor overlay of the two in situ sections, demonstrating that Cre, but not BDNF, is now expressed where BDNF was previously expressed. (B) Qualitative figure showing BDNF in situ hybridization of dorsal hippocampus following a sham injection (top, −Cre) or following LV-Cre injection (bottom, +Cre). (C) Relative mRNA expression in dentate gyrus (DG), and CA1 and CA3 regions and the average of all regions (Avg) of dorsal hippocampus in LV-Cre (Cre)- or LV-GFP (GFP)-infected mice. (D) Morris water maze acquisition, measured as the average latency to find the platform over daily sessions of training. LV-Cre-infected mice demonstrated significantly slower acquisition (impairment) compared with LV-GFP-infected controls. (E) Percent of time spent exploring the new vs. old object during the test day for novel object recognition. LV-GFP-infected mice spent significantly more time exploring the novel compared to the previously habituated object. The LV-Cre-infected animals did not differentiate between the two, demonstrating their impairment on this task. Panels adapted from Heldt et al. (2007).

Figure 4

Impaired fear extinction in mice with hippocampus-specific BDNF deletions. (A) Acquisition of cue conditioned fear, as measured with freezing after the onset of the auditory CS, during the conditioned fear acquisition session. There was no difference in acquisition of fear in animals receiving bilateral hippocampal injections of LV-Cre or LV-GFP. (B) Animals were tested within the same context in which training occurred for the presence of contextual fear as measured with freezing. There was no difference between the groups on level of contextual fear. (C,D) Extinction of conditioned fear is impaired in mice with dorsal hippocampus BDNF deletions. (C) Percent FPS is graphed for the first post-fear training test (pre-extinction) vs. the last test (post-extinction). Mice infected with LV-GFP demonstrate significant decreases in their level of conditioned fear as measured with %FPS compared with LV-Cre-infected mice. (D) Impaired extinction of fear, measured with %FPS, is stable across multiple testing sessions. Panels adapted from Heldt et al. (2007).

Figure 5

Effect of TrkB. T1 in the amygdala on the acquisition of conditioned fear and extinction. (A–C) Histological examination of viral infection in the BLA following behavioral studies. (A) No amygdala damage was seen following infection as visualized with Cresyl violet staining. LA, lateral amygdala; CeA, central amygdala. (B) Expression of TrkB.t1 was assessed using immunocytochemistry (ICC) for a hemagglutinin (HA) epitope tag incorporated into the TrkB.t1 coding sequence. (C) GFP expression directly visualizing under an epifluorescence microscope (scale bar, 1 mm). Level of fear-potentiated startle (FPS) following (D,E) acquisition and (F,G) extinction of fear. (D) Mean startle amplitude on startle-alone trials, light-startle trials, and the difference between the two are shown for animals receiving lentivirus infusion into the amygdala. Mean difference scores of LV-TrkB.T1-infused animals were significantly lower than difference scores of LV-GFP-infused animals. (E) Effect of amygdala infection with LV-TrkB.T1 on the expression of fear-potentiated startle. When LV-TrkB.T1 is present during expression, but not acquisition, of fear learning there is no difference between FPS with LV-TrkB.T1 animals compared with LV-GFP animals. (F) Averaging across all trials, TrkB.t1-infected rats showed a deficit in extinction as compared to GFP-infected rats. (G) Examining extinction within the testing session suggested that the TrkB.t1-infected rats had normal within-session extinction, but lacked extinction retention across the 2-day interval between tests TrkB.t1 and GFP groups. Panels (A–C, F,G) adapted from Chhatwal et al. (2006) and panels (D,E) from Rattiner et al. (2004a).

Figure 6

LV-CRF injection into the central nucleus of the amygdala (CeA) significantly increased corticotrophin releasing hormone (CRF) protein production site-specifically. (A) Number of positively labeled CRF cells in CeA of LV-GFP (closed bars) and LV-CRF treated rats (open bars) determined by immunohistochemistry. (B) Cresyl violet stained section representing a section parallel to the sections used to quantify the number of CRF positive neurons in CeA. (C) Representative section with an additional ×20 magnification inset showing the effects of LV-CRF injection into CeA on the number of positively labeled CRF neurons. (D) Representative section with an additional ×20 magnification inset showing the amount of CRF staining observed in the control, LV-GFP-treated rats. *P < 0.05. Panels adapted from Keen-Rhinehart et al. (2009).

Figure 7

Effect of LV-CRF injection on HPA axis feedback, anxiety-and depression-related behaviors. (A) HPA axis feedback as assessed by the dexamethasone suppression test. Corticosterone levels before and following a dexamethasone injection (shown by arrow) for LV-GFP control (closed symbol) and LV-CRF rats (open symbol). (B–D) LV-GFP-injected control (closed bars) and LV-CRF-injected rats (open bars). (B) Baseline acoustic startle response, (C) amount of time animals spent actively trying to escape and (D) and time spent floating in the forced swim test. Mean ± SEM. *P < 0.05. Panels adapted from Keen-Rhinehart et al. (2009).

Figure 8

In vivo validation of cell-type specific LV-CCK lentivirus. CCK mRNA expression. CCK mRNA was examined using in situ hybridization, demonstrating high levels of expression within the hippocampal formation [low power (A), high power (B,C)]. (B) Intense CCK mRNA expression is normally present within the CA3 subfield and within the interneuron-rich region (PoDG) separating the granule cell layers of the dentate gyrus, and (C) in the fasciola cinereum medial to CA1. (D) Low power image showing LacZ expression parallels CCK mRNA expression in RosaLacZ Cre-reporter mice injected with LV-CCK-Cre. LacZ expression (blue precipitate) and CCK mRNA expression (silver grains) were assessed in the same sections. (E) High-power images showing a high degree of overlap in mRNA expression was observed in the polymorph layer of the dentate (high power). High and low panels depict images of low and high CCK mRNA expression in the polymorph layer of the dentate, with correspondingly low and high numbers of LacZ-positive cells. (F) Similar co-expression was seen in the fasciola cinereum. (G–I) Virus encoding CCK-GFP injected into the dentate gyrus of adult mice. (G) Fluorescence was assessed on sectioned, fixed tissue. (H) Hoechst-stained photomicrographs of the same section shown in (G). (I) Overlays of (G) and (H). Panels adapted from Chhatwal et al. (2007).