Search tips
Search criteria 


Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Nat Neurosci. Author manuscript; available in PMC 2010 March 20.
Published in final edited form as:
PMCID: PMC2842592

Laminar and compartmental regulation of dendritic growth in mature cortex


Can dendrites grow in mature cortex? Here we address this question using chronic in vivo imaging to follow pyramidal neurons before and after cortical deletion of the Pten tumor suppressor gene in mature mice. We find that Pten/mTOR signaling uniquely regulates growth of layer 2/3 apical dendrites; no effects of gene deletion were observed on basal dendrites of these pyramidal neurons or along layer 5 apical dendrites.

Cortical dendrites are dynamic integrators of synaptic input to the neuronal soma. Their integrative properties are critically affected by the branching pattern of the dendritic tree1. In mice, the final form of the dendritic tree is laid down in the first two weeks of postnatal development during a period of maximum afferent innervation and synapse formation2, 3. Thereafter, large-scale dendritic structure is remarkably stable 46.

The PI-3 kinase/Pten signaling pathway regulates dendritic hypertrophy in developing cortex7, 8, but its action in mature cortex is unclear. To directly examine the potential, extent, dynamics, and molecular mechanisms of dendritic growth in the mature cortex in vivo we generated mice with a conditional cortical deletion of the Pten tumor suppressor gene (αCamKII-Cre+/−; Ptenloxp/loxp; Thy1-GFP). Experiments were approved by the University of California Los Angeles Office for Protection of Research Subjects and the Chancellor’s Animal Research Committee. Gene deletion significantly increases around the 8th postnatal week in these mice (Fig. S1).

We imaged pyramidal neurons in cortical layer 2/3 in their entirety in whole mount brains of 3.5–4 month old mice and reconstructed in three dimensions (Fig. 1). The apical dendrites of neurons in Pten−/− mice were much longer (Fig. 1a,c,e) and more tortuous (Fig. 1a,f) than those in controls. Total apical arbor length was on average 1.6mm longer in Pten−/− mice than in controls, representing an approximately 80% expansion of the apical dendritic tree relative to normal (Total arbor lengths: Pten−/− 3.1mm, 3.2mm, 4.6mm; WT 1.5mm, 2.1mm, 2.4mm). Notably, basal dendritic length (Fig. 1g) and tortuosity (Fig. 1h) from these same neurons were not measurably affected by Pten deletion. Nor did we find any significant difference in spine density between the two groups (density: 7.3±0.8 spines per 10µm in controls vs. 7.5±0.9 spines per 10µm in Pten−/−; P=0.29. n=1798 spines in control and 1989 in Pten−/− mice). All imaged neurons in Pten−/− mice showed signs of robust apical dendritic growth.

Figure 1
Compartment-specific dendritic growth

To examine the kinetics of apical dendritic growth we repeatedly imaged these dendrites in vivo. Imaging began at 7 or 8 weeks of age when cortical Cre expression was rapidly increasing in Cre+/− mice (Fig. S1b). In agreement with previous reports 6, the apical dendrites of layer 2/3 neurons in control mice (n=59 dendrites from 4 neurons in 3 mice) were quite stable when imaged over a 1 month interval (Fig. 2a,c). Although small elongations and retractions could be observed these amounted to a net loss of only 1–2% of apical dendritic length.

Figure 2
Dynamics of apical dendritic growth in vivo

In Pten−/− mice, the apical dendrites of pyramidal neurons imaged at 7 or 8 weeks of age were not measurably different from those in age-matched controls (Terminal branch point lengths: 91±46µm in controls, 104±52µm in Pten−/−; P=0.239; Tortuosity: 1.15±0.11 in controls, 1.19±0.14 in Pten−/−; P=0.20). Nor was there any significant difference in spine density between the two groups (control: 5.7±0.7 spines per 10µm; Pten−/−: 5.5±1.4 spines per 10µm; P=0.66). Over the next month these apical dendrites grew hundreds of microns, expanding the apical arborizations by 24% to 62% per imaged neuron (Fig. 2b,c). Again, this apical dendritic growth occurred in every layer 2/3 pyramidal neuron that we were able to locate in Pten−/− mice. This growth always occurred from the tips of existing dendrites and was characterized by the presence of filopodia-like protrusions at the growing tips (Fig. 2d). Spine density over the first 150µm of any newly grown dendrite was not significantly different from that along the original dendritic regions of these same neurons or in controls. However, spine density was significantly reduced along more distal dendritic regions (Fig. 2e). This new spine growth resulted in the addition of between 220 and 404 spines per imaged neuron. These newly formed spines were extremely labile; spine formation and elimination along newly grown dendrites was approximately two-fold greater than along pre-existing dendritic regions or on dendrites in control mice (Fig. 2f). This rate of growth and elimination is similar to that seen during early postnatal development when cortical circuits are refined by activity and experience 9.

Dendritic regions with spine densities less than 2 spines per 10µm ultimately retracted. This retraction was often associated with the presence of large swellings near the dendritic tips that were similar in appearance to “retraction bulbs” that have been described at the tips of retracting motor axons (10; Fig. S2). Thus, it appears that synaptic innervation, or at least the presence of spines is required for the maintenance of newly grown dendrites in mature cortex, much as in developing cortex 2.

Pten deficiency leads to increased activity of the mTOR kinase and a concomitant increase in the activity of S6 kinase (Fig. S1c), resulting in increased protein translation and cell growth 11. Daily intraperitoneal injections of rapamycin antagonized cortical mTOR signaling in vivo (Fig. 3a,b) and completely prevented the robust dendritic growth normally seen following Pten deletion (Fig. 3c). In control mice rapamycin had no effect on dendrite length (Fig. 3c) or spine kinetics (Fig. 3d), indicating that the effects of rapamycin are specific to growing dendrites.

Figure 3
Effect of rapamycin

Having shown that rapamycin prevents dendritic growth; we next investigated how rapamycin affects growing dendrites. To examine this we performed an in vivo wash-in experiment. Dendrites were first imaged weekly for 4 weeks (PW8–11). At the period of most rapid growth (PW11 in this mouse) we began rapamycin treatment. Imaging continued weekly for 4 additional weeks (PW 12–15). One week after the onset of treatment all dendritic growth had stopped (7 of 7 imaged dendrites), and all filopodial protrusions were gone (Fig. 3e; compare 2nd and 3rd panels). Rapamycin also reduced spine density on the newly grown dendrites (Fig. 3f) by inhibiting spine gain (Fig. 3g). Prolonging rapamycin treatment did not reverse dendritic growth significantly (cf. last panel Fig. 3e). Mild growth resumed when rapamycin was withdrawn.

Notably, the apical dendrites of layer 5 pyramidal neurons in Pten−/− mice displayed no measurable differences in dendritic growth, spine density, or spine kinetics relative to controls. Laser capture rtPCR and immunostaining confirmed the absence of Pten RNA and protein in layer 5 pyramidal neurons in Pten−/− mice. Rapamycin had no measurable effect on these neurons in control mice or in Pten−/− mice. These data (Fig. S3), suggest that Pten/mTOR signaling does not regulate dendritic growth of layer 5 pyramidal neurons at this age.

We draw three major conclusions from this study. First, dendrites in mature cortex retain the capacity for large-scale growth. Second, growing dendrites appear to behave much like dendrites in younger cortex. Third, the regulation of this growth by the Pten/mTOR signaling pathway is restricted to layer 2/3 apical dendrites in mature cortex. Lastly, our results suggest that growth-promoting pathways are constitutively active in adult cortical neurons; the growth we observed was induced not by activating a dormant growth pathway, but by removing a barrier to an active pathway.

Supplementary Material

Fig S1

Fig S2

Fig S3



We thank EM Callaway and K Bochmann for providing the ZnG reporter mice. This work was supported by the US National Eye Institute (EY016052), The Esther and Joseph Klingenstein Foundation, and by the US National Institute for Mental Health (MH068172).


Author Contributions:

DKC, MG, and JTT designed the experiments. DKC and JTT performed all imaging experiments, analyzed the data, and wrote the manuscript. MP performed the pS6 immunostaining. MM and STC performed the laser capture qrtPCR. MP, and XL provided the mice.


1. Hausser M, Spruston N, Stuart GJ. Diversity and dynamics of dendritic signaling. Science. 2000;290:739–744. [PubMed]
2. Wong RO, Ghosh A. Activity-dependent regulation of dendritic growth and patterning. Nat Rev Neurosci. 2002;3:803–812. [PubMed]
3. Cline HT. Dendritic arbor development and synaptogenesis. Curr Opin Neurobiol. 2001;11:118–126. [PubMed]
4. Trachtenberg JT, et al. Long-term in vivo imaging of experience-dependent synaptic plasticity in adult cortex. Nature. 2002;420:788–794. [PubMed]
5. Holtmaat A, Wilbrecht L, Knott GW, Welker E, Svoboda K. Experience-dependent and cell-type-specific spine growth in the neocortex. Nature. 2006;441:979–983. [PubMed]
6. Lee WC, et al. Dynamic remodeling of dendritic arbors in GABAergic interneurons of adult visual cortex. PLoS Biol. 2006;4:e29. [PMC free article] [PubMed]
7. Fraser MM, Bayazitov IT, Zakharenko SS, Baker SJ. Phosphatase and tensin homolog, deleted on chromsome 10 deficiency in brain causes defects in synaptic structure, transmission and plasticity, and myelination abnormalities. Neuroscience. 2008;151:476–488. [PMC free article] [PubMed]
8. Kwon CH, et al. Pten regulates neuronal arborization and social interaction in mice. Neuron. 2006;50:377–388. [PubMed]
9. Holtmaat A, et al. Transient and persistent dendritic spines in the neocortex in vivo. Neuron. 2005;45:279–291. [PubMed]
10. Bishop DL, Misgeld T, Walsh MK, Gan WB, Lichtman JW. Axon branch removal at developing synapses by axosome shedding. Neuron. 2004;44:651–661. [PubMed]
11. Guertin DA, Sabatini DM. An expanding role for mTOR in cancer. Trends Mol Med. 2005;11:353–361. [PubMed]