Figure 1.
Timm stain illustrating mossy fiber sprouting into the dentate gyrus inner molecular layer in pilocarpine-treated mice.
A. Dentate gyrus of a normal mouse. B. Dentate gyrus of a pilocarpine-treated mouse that survived status epilepticus (SE) showing mossy fiber sprouting. A1 and B1 are enlarged boxed regions of A and B. The arrows in B and B1 point to extensive mossy fiber sprouting into the inner molecular layer of the dentate gyrus. C. Graph showing the mean Timm score between controls and pilocarpine-treated mice that survived SE. Asterisk, significant difference between mean scores (P<0.05).
Figure 2.
Increase in synaptic activity in granule cells from pilocarpine-treated mice that survived SE and developed TLE.
A. sEPSCs recorded from granule cell of normal mouse. B. sEPSCs recorded from granule cell of a mouse that was injected with pilocarpine but did not develop SE. C. sEPSCs and bursting activity recorded from a granule cell of a pilocarpine-treated mouse that developed TLE. D. Trace showing that sEPSCs were blocked by application of ionotropic glutamate receptor antagonists, CNQX (10 µM) and AP-5 (50 µM). A1 and B1 are expanded boxed regions of A and B. C1, expansion of the sEPSC showing increased synaptic activity during the burst seen in C. Recordings were made in the absence of added Mg2+ and the presence of bicuculline (30 µM). E. Bar graph comparing the frequency of sEPSCs among the three groups of animals. Asterisk indicates significantly higher frequency in cells from mice with TLE (P<0.05).
Figure 3.
Action potential dependence of epileptiform EPSC bursts.
A. Spontaneous burst activity recorded from a granule cell in a pilocarpine-treated mouse that survived SE. B. The bursts were completely blocked by TTX (1 µM), indicating they were action potential-dependent. Action potential-independent mEPSCs were still seen in the presence of TTX. A1 and B1 are expanded segments of A and B respectively. The recordings were made in the absence of Mg2+ and presence of bicuculline (30 µM).
Figure 4.
Inhibitory effect of anandamide (AEA) on sEPSCs.
A. Spontaneous bursts of EPSCs observed in a granule cell from a pilocarpine-treated mouse that survived SE. A1 and A2 are expanded segments of the events indicated by the arrows. B. AEA (10 µM) suppressed the epileptiform bursts of activity. B1 and B2 are examples of individual events from B. C. Bursts were reinstated after 20 minute wash to normal ACSF. C1 and C2, expanded segments of C. Asterisks indicate currents preceding the largest amplitude EPSCs in some cases. The recordings were made in the absence of Mg2+ and the presence of bicuculline (30 µM). D. Cumulative graph of normalized sEPSC frequency from control mice and pilocarpine-treated mice that survived SE and developed TLE before and after application of AEA. Asterisk in D indicates significant reduction in frequency by AEA (P<0.05). In mice with TLE, the effect of AEA was prevented by preapplication of the CB1 receptor antagonist AM-251 (10 µM). Number of cells for each experiment is in parentheses.
Figure 5.
Effect of cannabinoid agonists on TTX-independent mEPSCs in pilocarpine-treated mice that survived SE.
A. mEPSCs recorded from a granule cell in the presence of TTX (1 µM). B. Trace showing the reduction of mEPSC frequency after application of AEA (10 µM). C. Wash to TTX alone (20 min) showing that the effect was reversible. A1, A2, B1, B2 and C1, C2 are expanded segments of A, B and C, respectively. Recordings were made in the presence of bicuculline (30 µM). D. Cumulative probability plot showing a change in the frequency of mEPSCs in this cell. E and F. Cumulative charts showing effect of AEA, WIN 55,212-2 and 2-AG on mEPSC frequency (E) and amplitude (F) on pilocarpine-treated (TLE) and control mice. Asterisk indicates significant change in mEPSC frequency (p<0.05); number of recordings shown in parentheses.
Figure 6.
Anandamide attenuated secondary population activity that followed antidromic stimulation of granule cell axons.
A. Trace showing granule cell response to mossy fiber stimulation in a mouse with TLE. B. Antidromically-evoked secondary afterdischarges were reduced in the presence of 1 µM AEA. C. The effect of AEA was reversible after 30 min wash to control ACSF. Recordings were made in the absence of Mg2+ and the presence of 30 µM bicuculline; stimulus intensity 0.1 mA.
Figure 7.
Cannabinoid-mediated attenuation of EPSC bursts evoked by glutamate ‘uncaging’ in the granule cell layer.
Sets of 3 overlapping traces showing evoked EPSCs after photolytic release of caged glutamate (250 µM) at the same location in the granule cell layer of control mice and mice with TLE. Arrows indicate the time of uncaging. A. Uncaging glutamate in the granule cell layer did not evoke a synaptic response in control mice. B. EPSC bursts evoked in pilocarpine-treated mice that survived SE. C. In the same cell as B, EPSC bursts were attenuated by WIN 55,212-2 (10 µM). A1, B1 and C1 show a single expanded trace from the group of traces shown in A, B and C. D. Grouped data from 6 cells showing the effect of WIN 55,212-2 on EPSC bursts evoked by photolysis of caged glutamate. Glutamate uncaging increased EPSC frequency over baseline; ** indicate significant difference in EPSC frequency before and after photolysis (p<0.05). * indicate a significant reduction in EPSC frequency after application of WIN 55,212-2 during comparable post-photolysis 100 ms time periods (p<0.05).
Figure 8.
Western blot detection of cannabinoid type 1 receptor (CB1R) expression.
A. Diagram of dentate gyrus illustrating the area that was micro-dissected (box) for analysis. B. Western blot showing CB1R expression in pilocarpine-treated mice that survived SE and developed TLE compared to untreated mice. Actin was used as the loading control, which did not change significantly. C. Graph showing cumulative (60%) increase in CB1R expression in mice with TLE versus controls (n = 6; p<0.05). * indicates significance. IML, inner molecular layer; GCL, granule cell layer; PML, polymorphic layer.