Notch1 is present at the synapse and is induced by neuronal activity
Using an antibody that recognizes the active form of Notch1 (NICD1, S3 fragment), we found it present in the cell soma and dendrites of neurons in many regions of the brain, including the cerebral cortex and hippocampus (, and not shown). We also found that NICD1 and the activity-induced Arc were present in many of the same cells, suggesting that Notch1 signaling occurs in active neurons. Indeed, most EGFP+ neurons in a transgenic Notch reporter (TNR) mouse line (Mizutani et al., 2007
) expressed Arc (e.g, 73% of EGFP+ cells in the cortex, see ). In cultured neurons, Notch1 was enriched in the dendrites and cell soma, while the ligand Jagged1 (Jag1) was enriched in axons (, S1A
). Notch1, NICD1 and Jag1 co-localized with synaptic proteins ( Figure S1
), and NICD1 was enriched in synaptosomal fractions derived from cortical extracts ().
Notch1 is present at the synapse in mature neurons
Neuronal Notch signaling is disrupted in Arc mutants in vivo
Increased neuronal activity after treatment with NMDA () or bicuculline () led to higher NICD1 levels, while treatment with the NMDA-receptor blocker AP5 led to reduced NICD1 levels (). Neuronal activity also increased Notch1 protein levels (, see also ), including the pre-processed form of the receptor (), and Jag1 expression (). Activity-induced Notch1 expression occurred in the presence of the transcriptional inhibitor actinomycin-D, suggesting that pre-existing Notch1 transcript is translated in response to synaptic activity ().
Notch signaling occurs in neurons in response to activity
To test the effect of synaptic activity on Notch expression and processing further, we used acute hippocampal slices. The Schaffer collateral pathway was activated to induce LTP (), and increased Arc expression was observed in both CA3 and CA1 neurons (). In addition, somal Notch1 expression was increased in CA3 and CA1 (6.1-fold by pixel count, n=6, p<0.02)(), as was NICD1 staining (, and not shown). The increase in Notch expression in CA1 could be reduced by AP5 (Figure S2
Neuronal Notch signaling occurs in vivo in response to exploration
We next evaluated Notch expression and signaling in response to neuronal network activity in vivo
after exploration of a novel environment. Spatial exploration activates specific ensembles of hippocampal pyramidal neurons that can be identified by expression of Arc (Guzowski et al., 1999
). TNR mice were allowed to explore a novel environment for 5 min, and were sacrificed 1.5 or 8 hours later. Consistent with prior work (Ramirez-Amaya et al., 2005
), the number of Arc+ hippocampal CA1 neurons was increased ~3-fold at 1.5 hours, and ~2-fold at 8 hours (Figure S3A
). In addition, the number of Notch1+ CA1 neurons was elevated at both time points (~3-fold, Figure S3A,C
), as was EGFP expression (indicative of Notch activity) at 8 hours (Figure S3B,C
). Notably, nearly all (94–97%) of the Arc+ neurons also had Notch1 signal in the nucleus (e.g. see ), indicating that Arc induction and Notch signaling occur in the same neuronal networks in response to exploration. Some Notch1+ neurons did not express Arc (18% in controls and at 1.5 hours, and 29% at 8 hours). Thus, the temporal dynamics of Notch1 and Arc may be different, with Notch1 persisting longer than Arc, or not all neuronal Notch signaling occurs in Arc+ networks.
Neuronal Notch signaling is disrupted in Arc mutants in vivo
Both Notch signaling (Fortini and Bilder, 2009
; Vaccari et al., 2008
) and Arc function (Chowdhury et al., 2006
; Shepherd et al., 2006
) engage Dynamin-mediated endocytosis, raising the possibility that they might interact. Thus, we examined Notch activity in the adult brain of Arc
mutants using the TNR mouse line. Of fifteen Arc
mutants, twelve (80%) had reduced EGFP expression (Notch activity) throughout the cerebral cortex as compared to twenty-two non-mutants (). Arc
mutants also had reduced NICD1 levels, consistent with less Notch signaling in the absence of Arc ().
To test if Arc is required for Notch pathway recruitment in response to network activity in vivo, we compared Notch1 expression in the hippocampus of wild type and Arc mutants after exploration of a novel environment. In controls, we observed elevated expression of both Arc and Notch1, the latter of which was localized both to the cell soma and nucleus, in CA1 (not shown) and CA3 (). In contrast, no change in Notch1 expression or subcellular localization was observed in Arc mutants ().
Arc regulates the proteolytic processing of Notch1 in neurons
We next examined the status of Notch1 processing in Arc
mutant neuronal cultures. In the absence of Arc there was a reduction in the S3 cleaved form of Notch1 (NICD1)(), indicating that Arc positively regulates the γ-secretase-mediated cleavage of Notch1 in neurons. Treatment with bicuculline led to elevated Notch1 and NICD1 levels in control neurons but not in Arc
mutant neurons (), indicating that Arc is required for the activity-mediated recruitment of neuronal Notch signaling. No change in Jag1 expression was observed in Arc
mutant cultures (Figure S4
) in line with the idea that receptor processing, and not ligand availability, is defective in mutant cells.
In an effort to rescue Notch1 processing in Arc
mutant cells, we used Sindbis virus to introduce functional or non-functional Arc into mutant neurons in vitro
. Restoration of Arc expression rescued Notch1 processing (2.9-fold increase, n=3, p<0.001)(), suggesting that the Notch1 cleavage defect in Arc
mutant neurons is not caused by aberrant neuronal differentiation. A form of Arc lacking the ability to bind Endophilin and participate in endocytic trafficking (Δ91–100)(Chowdhury et al., 2006
) was unable to restore Notch1 processing in Arc
mutants neurons ().
Next, we found that Arc and Dynamin co-immunoprecipitated (IP) with Notch1 in protein preparations from adult cortical extracts (). In addition, Notch1 co-IP’d with Arc in protein extracts from wild type, but not Arc
mutant, cortical tissue (). Thus, Arc-mediated Dynamin-driven endocytosis of Notch1 may be important for activity-dependent Notch signaling in neurons. Interestingly, Arc is not required for Notch activation in embryonic forebrain progenitors (Figure S5
), indicating that Arc regulates Notch in a context-dependent manner.
Conditional deletion of Notch1 in the postnatal hippocampus
Having shown that Arc-dependent Notch signaling is activated in neuronal ensembles after spatial exploration, we next tested the function of Notch in such ensembles. To conditionally knock out (cKO) Notch1 in the postnatal hippocampus, we crossed Notch1flox/flox
(Radtke et al., 1999
) mice with the CamKII-cre (T29-1) driver line (Tsien et al., 1996
), and Notch1 deletion was confirmed at both the mRNA and protein levels (Figure S6
and , respectively; n=6 each). Golgi-Cox staining of CA1 pyramidal neurons revealed that loss of Notch1 postnatally did not affect dendritic length (). However, spine density on secondary and tertiary dendrites was reduced (), and spine morphologies were altered ().
Loss of Notch function in CA1 affects neuronal morphology and plasticity
To test the role of Notch in synaptic plasticity, the electrophysiological properties of Notch1 cKO animals were tested using hippocampal slices and field recordings. Basal transmission was the same for mutants and controls (10–11 slices)(), and paired-pulse facilitation (PPF), revealed that Notch1 cKO slices had presynaptic strength comparable to controls (). However, when we induced LTP in the Schaffer collateral pathway, the magnitude of LTP in the CA1 region was uniformly higher in controls (188.5 ± 23.1, n=6) than in Notch1 cKO slices (140.9 ± 20.6, n=5, p<0.05)(). Similarly, after low frequency stimulation, LTD in CA1 was uniformly reduced in Notch1 cKO mice (83.4 ± 11.2, n=6 slices) compared to controls (70.0 ± 11.5, n=5, p<0.05)(). Thus, Notch1 influences the magnitude of both the potentiation and depression of synaptic efficacy.
Notch1 cKO mice display deficits in learning and acquisition of new memory
Next we performed behavioral tests to evaluate the cognitive abilities of Notch1 cKO mice. During novel object recognition testing, mutants initially had a lower novel object preference than controls, and the next day, in contrast to controls, mutants had no preference (). Similarly, in a social interaction test unlike controls Notch1 cKO mice did not interact more with a new subject (), although like controls mutants preferred a subject to an object (not shown). In Y-maze testing, Notch1 cKO mice chose alternating arms at the same frequency as controls (not shown), but showed no preference for a previously hidden arm ().
Notch1 conditional ablation causes deficits in memory acquisition
Next, spatial reference memory was investigated using the Morris water maze. Performance improved over 5 days of learning in both Notch1 cKO and control mice (p<0.0001), although latency was greater in the mutants (, p<0.01), despite the average swim speed being comparable (p=0.4). A learning deficit was also seen in the Notch1 cKO mice when subjected to reversal learning (). In both cases, 24 hours after the last learning session, mutant and control mice spent more time in the target quadrant (Figure S7A,B
). Thus, Notch1 cKO mice can learn using spatial cues, although they do so more slowly than wild types.
In line with the previous report on the Notch1+/−
mice (Costa et al., 2003
), we could not detect any difference between Notch1 cKO and controls in contextual fear-conditioning 24 hours after a shock was delivered (Figure S7C
). In addition, Notch1 cKO mutant mice displayed normal motor coordination (rotarod test), motor activity (open field test), and anxiety levels (elevated plus maze)(Figure S7C