Deletion of TrkB in adult progenitors alters newborn neuron integration into hippocampal circuits and increases anxiety-like behavior

Abstract

New neurons in the adult dentate gyrus are widely held to incorporate into hippocampal circuitry via a stereotypical sequence of morphological and physiological transitions, yet the molecular control over this process remains unclear. We studied the role of brain-derived neurotrophic factor (BDNF)/TrkB signaling in adult neurogenesis by deleting the full-length TrkB via Cre expression within adult progenitors in TrkB(lox/lox) mice. By 4 weeks after deletion, the growth of dendrites and spines is reduced in adult-born neurons demonstrating that TrkB is required to create the basic organization of synaptic connections. Later, when new neurons normally display facilitated synaptic plasticity and become preferentially recruited into functional networks, lack of TrkB results in impaired neurogenesis-dependent long-term potentiation and cell survival becomes compromised. Because of the specific lack of TrkB signaling in recently generated neurons a remarkably increased anxiety-like behavior was observed in mice carrying the mutation, emphasizing the contribution of adult neurogenesis in regulating mood-related behavior.

Fig. 1.

Deletion of TrkB affects the survival of newborn neurons. (A) Schematic diagram showing the experimental paradigm used for tamoxifen-induced Cre recombination in TrkB^lox/lox-Cre R26R mice and control littermates. Histograms depict the density distribution of reporter-positive cells (βgal+) expressing GFAP, Dcx, or NeuN in TrkB^wt/wt-Cre (wt/wt) and TrkB^lox/lox-Cre (lox/lox) mice. (B) Density of reporter-positive cells coexpressing Dcx and NeuN as in A. Ten slices per hemisphere and two mice for each time point were analyzed (*, P < 0.05). (C) Representative confocal images depict the reporter marker βgal colocalized with either the neuronal marker NeuN (βgal/NeuN) or Dcx (βgal/Dcx). Colocalization signals (red) were superimposed on NeuN or Dcx immunoreactivity (gray). Labeling of each single marker from which colocalization was obtained is shown as SI Materials and Methods (see Fig. S2). (Scale bar, 20 μm.)

Fig. 2.

TrkB is required for dendritic arborization in adult-born neurons. (A) Schematic diagram showing the experimental paradigm used for retrovirus injection into tamoxifen-induced TrkB^lox/lox-Cre R26R and control mice. Histograms show the total number of branches and the dendritic length of reporter-positive (βgal+) or -negative (βgal−) cells virally transduced with GFP (n = 20 neurons; 3 animals per genotype) (*, P < 0.05). (B) Graph depicts Sholl analysis of the dendritic arbors in the same neurons as in A (n = 20 neurons; 3 animals per genotype) (*, P < 0.05). (C) Representative confocal images depicting the dendritic morphology of reporter-positive (2–4; βgal+) or -negative (1; βgal−) cells transduced with GFP expressing retrovirus in TrkB^lox/lox-Cre R26R mice. Two-dimensional projection of tridimensional reconstruction of the cells is shown. (Scale bar, 10 μm.) (D) Schematic diagram showing the experimental paradigm. Graph depicts Sholl analysis of the dendritic arbor of cells single transduced (Cre or TrkB) or cotransduced (Cre/TrkB or Cre/TrkB FFF) with the retrovirus (n = 10; 3 animals for each experimental group) (*, P < 0.05). Representative confocal images of transduced cells are presented. Insets show the expression of the reporter marker GFP (green) and/or DsRed (red). (Scale bar, 20 μm.)

Fig. 3.

Spine density is regulated by TrkB in adult-born neurons. (A) Graphs depict the quantification of protrusions in dendritic segments of newborn neurons transduced with retrovirus expressing GFP in tamoxifen-induced TrkB^lox/lox-Cre R26R (n = 24 dendritic segments; 18 βgal+; 11 βgal−; 3 animals) and control mice (n = 60 dendritic segments; 26 βgal+; 3 animals) mice. The density of protrusions is expressed as the number of protrusions per micrometer of dendritic length (*, P < 0.01). Representative images show spine morphology in reporter-positive (βgal+) or -negative (βgal−) cells 28 dpi. (Scale bar, 2 μm.) (B) Representative electron micrographs showing synaptic contacts of newborn neurons in tamoxifen-induced TrkB^lox/lox-Cre Z/EG and control mice. Reporter-positive cells (GFP+) show GFP fluorescence as photoconverted electron-dense signals at postsynaptic sites. A synapse from a reporter-negative cell (GFP−) is shown as a reference. (Scale bar, 50 nm.)

Fig. 4.

TrkB is required for neurogenesis-dependent LTP. (A) LTP evoked in tamoxifen-induced TrkB^lox/lox-Cre and control mice. Experiments were conducted in hippocampal slices of TrkB^wt/wt-Cre mice in the absence (n = 8 slices, 4 animals) or presence (n = 6 slices, 2 animals) of ifenprodil (3 μM) and in slices of TrkB^lox/lox-Cre mice (n = 7 slices, 3 animals). Each point in the graph shows the average fEPSP slope elicited in response to a test stimulus before (−20–0 min) and after (0–60 min) tetanic stimulation of the MPP. Representative fEPSP traces recorded before (−5 min) and after (60 min) tetanus are shown on the top. (B) LTP evoked as in A in the presence of bicuculline (20 μM) (n = 4 slices, 4 animals).

Fig. 5.

Deletion of TrkB in newborn neurons affects exploratory activity in mice exposed to a novel environment. (A) Exploratory behavior of tamoxifen-induced TrkB^lox/lox-Cre and control mice in the open-field test. Histograms show the number of entries and the percentage of time spent in the center, corner, or the entire periphery of the arena (n = 20) (*, P < 0.05). The total distance moved is shown in the right panel. (B) Exploratory behavior in the elevated plus maze test. Histograms show the proportion of entries and time spent in the open arms (n = 20) (*, P < 0.05). The total number of entries in the four arms is shown in the lower panel. (C) Digital tracking of mice exposed to the open-field (upper panels) and the elevated plus maze (lower panels) behavioral tests. Representative traces of mice pattern activity (red) resulting from video tracking are depicted. Each panel is representative of individual mice activity.