(a) Schematic of the excitatory hippocampal circuit with a paired-patch recording configuration in the CA3 region. CA3 pyramidal cells receive mossy fiber (M.F.) inputs from the dentate gyrus (DG) granule cells as well as recurrent collaterals (R.C.) from neighboring CA3 pyramidal cells. The Schaffer collaterals (S.C.) are the output of CA3 pyramidal neurons onto CA1 pyramidal neurons.
(b) Example IR-DIC image of a paired-recording from two CA3 neurons that were connected. Scale bar represents 10 μm.
(c) Example paired-recordings from a mono-synaptically coupled (left) and synaptically unconnected (right) pair of CA3 neurons. APs (gray, bottom trace) were generated with brief depolarizing current injections in the presynaptic neuron under I-clamp. The resulting evoked excitatory currents (EPSCs) were recorded in the postsynaptic neuron in V-clamp as it allowed the isolation of monosynaptic EPSCs without contaminating di-synaptic inhibitory postsynaptic current when the postsynaptic neuron was held at the reversal potential for chloride (~ −60 mV under our recording conditions).
(d) Percentage of connected CA3 pyramids in control (black, N=90) and TTX-treated (gray, N=131) slices. Percentages of unconnected pairs in each group are represented in white.
(e) The connectivity in control (black) and TTX-treated (gray) slices was ~45% and ~20% respectively, whether cell bodies of the neuron pairs were adjacent to one another or ≥3 cell bodies away (P >0.5; χ²-test for both groups in control and TTX-treated slices).

(a) Example simultaneous recordings from a synaptically coupled pair of CA3 neurons in control (left) and TTX-treated (right) slices.
(b) Cumulative plot of average synaptic strength of individual connected pairs in control (black) and TTX-treated (gray) slices. For each connected pair, the average amplitude of the EPSC was calculated from at least 30 trials, including measurements of zero amplitude when synaptic transmission failed. The average EPSC amplitude in TTX-treated recurrent connections (N=26) was significantly larger than in control connections (N=41; **P <0.005, K-S test).
(c) Synaptic strength (EPSC amplitude) as function of inter somatic distance between the preand postsynaptic CA3 pyramids in control (black) and TTX-treated (gray) slices. Error bars represent s.e.m.
(d–f) Cumulative plot showing a significant increase in the average EPSC rise times (d) and rate of rise (e) of individual connected pairs in chronically silenced slices but no change in response latency (f). Control (black) and TTX-treated (gray) slices (*P <0.05, ***P <0.001, K-S test).

(a) Example traces from a connected CA3 pair with synaptic transmission failures (arrow). The incidence of failures (zero amplitude events) was determined for synaptically connected neurons from at least 100 consecutive trials.
(b) Average percentage of failures was significantly decreased in connected CA3 pairs from activity-deprived slices (control, black, N=13; TTX-treated, gray, N=12; P <0.05, K-S test).
(c) Example traces of evoked EPSCs in response to pairs of AP at 30, 50 and 150 ms intervals.
(d) Average paired pulse ratio in control (black, N=12) and TTX-treated (gray, N=8) connections at different inter-pulse intervals ranging from 20 ms to 250 ms. Connections in TTX-treated slices show significant paired pulse depression in the 50–100ms interval (*P <0.05, ***P <0.001, 2-way ANOVA). Error bars represent s.e.m.
(e) Example traces showing excitatory responses in the post-synaptic CA3 neuron evoked by repeated stimulation. Control (top) and TTX-treated (middle) slices. For a given condition in a pair of neurons, 10 traces were averaged. Bottom trace (gray) shows a 20 Hz train of five APs in the presynaptic neuron.
(f) Ratio of the EPSC response evoked by the fifth action potential to that evoked by the first action potential demonstrates a greatly enhanced short-term depression in activity deprived slices indicating an increase in P_r in TTX-treated slices (gray, N=6) as compared to control slices (black, N=10; *P <0.005, unpaired two-tailed t-test). Error bars represent s.e.m.

(a) Five consecutive traces showing responses to a pair of AP from a connected CA3 pair in three different ratios of external Ca⁺²/Mg⁺² concentrations (0.3, magenta; 1, black; 12.3, blue). Presynaptic AP traces shown in gray.
(b) Variance-mean analysis for the paired recording depicted in (a) plotting the variance and mean EPSC size in response to the first and second AP in three different external Ca⁺²/Mg⁺² concentrations and fitted with a second order polynomial (parabola).
(c–e) Variance-mean analysis showed a significant increase in P_r in TTX-treated slices (c; P <0.03, unpaired two-tailed t-test), but no significant change in quantal amplitude (d) or the total number of synapses between synaptically coupled neuron pairs (e). Control, black, N=11; TTX-treated, gray, N=11. Error bars represent s.e.m.

(a) Individual CA3 neurons in hippocampal slices were electroporated with EGFP at DIV 14 and imaged at DIV 24. Left, montage of two-dimensional z-projections of three-dimensional confocal image stacks in control (top) and TTX-treated (bottom) slices. Right, three-dimensional Neurolucida reconstruction of dendrites: basal (magenta), apical (cyan). Scale bar represents 100 μm.
(b) Activity-deprivation increased the average number of branches in the basal (control, black, N=5; TTX-treated, gray, N=4; P<0.05 unpaired two-tailed t-test) but not in the apical dendrites. Error bars represent s.e.m.
(c) Total length of basal and apical dendrites was similar in CA3 neurons from control and TTX-treated slices.
(d) Average number of branches for different branch orders in neurons from control and TTX-treated slices. Left, chronic inactivity caused an increase in branching for branch order 6–8 in the basal dendrites (control, black, N=5; TTX-treated, gray, N=4; P <0.05 2-way ANOVA). No significant difference was observed for apical dendrites (right). Error bars represent s.e.m.
(e) Representative segment of basal dendrite from a EGFP-expressing neuron in control (top, left) and TTX-treated slice (bottom, left). Activity deprivation did not significantly change the average spine density in either basal or apical dendrites (control, black, N=5; TTX-treated, gray, N=9; right). Scale bar=10 μm.
(f) Example axon segment in stratum oriens from a CA3 neuron in control (top, left) and TTX-treated slice (bottom, left). Activity-deprivation did not significantly alter the density of axonal varicosities in the strata oriens or radiatum (control, N=5; TTX-treated, N=9; right). Scale bar=10 μm.

(a) Pie-chart depicting percentage of connected CA3 pyramids in control (black) and TTX-treated (gray) slices alone (top), after 50μM forskolin treatment (middle), and in the presence of 50μM roscovitine (bottom), respectively. Unconnected pairs, white. Numbers reflect percentages for each group.
(b) Average postsynaptic response from a synaptically connected neuron pair in control (black) and TTX-treated (gray) slices alone (top), after 50μM forskolin treatment (middle), and in the presence of 50 μM roscovitine (bottom), respectively; presynaptic AP not shown. Each trace represents an average of 25 sweeps.
(c) Left, western blot of CA3 tissue lysates from control and TTX-treated slices immunoblotted with the Anti-Cdk5 and Anti-Tuj1 (normalizing protein). Right, total Cdk5 protein levels in control (black) and TTX-treated (gray) slices were similar (control, n=42, N=4; TTX-treated, n=42, N=4; where `n' is total number of slices and `N' is number of organotypic culture batches). Error bars represent s.e.m.
(d) Western blot of a co- immunoprecipitation (Co-IP) assay used to identify Cdk5 physically associated with p35/p25. Right, activity-deprivation showed a significant increase in Cdk5 physically associated with p35/p25 (control, black; n=42, N=4; TTX-treated, gray; n=42, N=4, P<0.03, t-test). Error bars represent s.e.m.
(e) Converse Co-IP assay used to probe the amount of p35/p25 physically associated with Cdk5. Activity-deprivation showed a significant increase in both p35 (middle) and p25 (right) physically associated with CDK5 (control, black; n=42, N=4; TTX-treated, gray; n=42, N=4, P<0.01, unpaired two-tailed t-test). Error bars represent s.e.m.

Schematic representation of homeostatic changes in the CA3 excitatory recurrent microcircuit caused by chronic activity-deprivation and its acute reversion by CDK5 inhibition.
(a) In control conditions, CA3 pyramidal neurons are functionally connected to ~45% of the neighboring CA3 neurons. CA3 pyramidal cell body and basal dendrites are in green and axon in red. Filled circles denote active connections. After chronic activity-deprivation, there is a reduction in connectivity to ~20% due to presynaptic silencing of connections (empty circles). The synaptic strength of the remaining connections is increased due to a significant increase in P_r, depicted by the larger size of the red, filled circles. There is also an increase in basal dendritic branching. Acute application of roscovitine reverses the affect of chronic TTX-treatment on functional connectivity and average strength of the connections to that of control conditions.
(b–c) Average functional connectivity (b) and synaptic strength (c) of CA3 neuron pairs in slices from control, activity-deprived, and activity-deprived in the presence of roscovitine conditions.