Fetal hippocampal grafts containing CA3 cells restore host hippocampal glutamate decarboxylase-positive interneuron numbers in a rat model of temporal lobe epilepsy

Abstract

Degeneration of CA3-pyramidal neurons in hippocampus after intracerebroventricular kainic acid (KA) administration, a model of temporal lobe epilepsy, results in hyperexcitability within both dentate gyrus and the CA1 subfield. It also leads to persistent reductions in hippocampal glutamate decarboxylase (GAD) interneuron numbers without diminution in Nissl-stained interneuron numbers, indicating loss of GAD expression in a majority of interneurons. We hypothesize that enduring loss of GAD expression in hippocampal interneurons after intracerebroventricular KA is attributable to degeneration of their CA3 afferent input; therefore, fetal CA3 grafts can restore GAD interneuron numbers through graft axon reinnervation of the host. We analyzed GAD interneuron density in the adult rat hippocampus at 6 months after KA administration after grafting of fetal mixed hippocampal, CA3 or CA1 cells into the CA3 region at 45 d after lesion, in comparison with "lesion-only" and intact hippocampus. In dentate and CA1 regions of the lesioned hippocampus receiving grafts of either mixed hippocampal or CA3 cells, GAD interneuron density was both significantly greater than lesion-only hippocampus and comparable with the intact hippocampus. In the CA3 region, GAD interneuron density was significantly greater than lesion-only hippocampus but less than the intact hippocampus. Collectively, the overall GAD interneuron density in the lesioned hippocampus receiving either mixed hippocampal or CA3 grafts was restored to that in the intact hippocampus. In contrast, GADinterneuron density in the lesioned hippocampus receiving CA1 grafts remained comparable with lesion-only hippocampus. Thus, grafts containing CA3 cells restore CA3 lesion-induced depletions in hippocampal GAD interneurons, likely by reinnervation of GAD-deficient interneurons. This specific graft-mediated effect is beneficial because reactivation of interneurons could ameliorate both loss of functional inhibition and hyperexcitability in CA3-lesioned hippocampus.

Fig. 1.

Nissl-stained sections of the hippocampal formation from a control rat (A₁) and a KA-treated rat at 6 months after lesion (B₁). Intracerebroventricular kainic acid induced CA3 pyramidal cell loss (B₁, asterisks) appears total in CA3b and CA3c subregions. A2 and B2, respectively, show a magnified view of the boxed CA1 area in A₁and B₁. A₃,B₃, Enlarged view of boxed CA3 area in A₁ and B₁.Arrowheads in A₂,B₂, A₃, andB₃ point to interneurons. Note that the distribution of interneurons in the stratum radiatum (SR) of the CA1 subfield and strata oriens (SO) and radiatum of the CA3 subfield appear similar between the control hippocampus and the KA-lesioned hippocampus. In contrast, the stratum oriens of the CA1 subfield in KA-lesioned hippocampus exhibits a far fewer interneurons (B₂). Asterisks inB₃ denote the degenerated CA3 cell layer.DG, Dentate gyrus; SP, stratum pyramidale. Scale bars: A₁,B₁, 400 μm; A₂,B₂, A₃,B₃, 200 μm.

Fig. 2.

GAD-67-immunostained sections of the hippocampal formation from a control rat (A₁) and a KA-treated rat (B₁).A₂–A₄, Magnified views of dentate, CA1, and CA3 regions from A₁;B₂–B₄, enlarged views of dentate, CA1, and CA3 regions from B₁. Note that the density of GAD-67-positive interneurons is clearly reduced in every layer of the KA-lesioned hippocampus (B₁–B₄), compared with the intact control hippocampus (A₁–A₄).Asterisks in B₁ denote the degenerated CA3 cell layer. DG, Dentate gyrus;DH, dentate hilus; GCL, granule cell layer; ML, molecular layer; SL, stratum lucidum; SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum. Scale bars:A₁, B₁, 400 μm;A₂–A₄,B₂–B₄, 200 μm.

Fig. 3.

A₁, Nissl-stained section of the hippocampal formation from a KA-lesioned rat, which received mixed hippocampal cell transplant (T) into the lesioned CA3 region at 45 d after KA administration. A large number of surviving neurons are seen in the graft area (outlined byinterrupted lines in A₁).A₂, Magnified view of transplant region inA₁ showing clusters of larger CA3 pyramidal-like neurons. A₃ Mostly dispersed smaller neurons from a CA1 cell graft. B₁,C₁, D₁, Examples of GAD-67-immunostained sections of the hippocampal formation from kainic acid-lesioned rats, which received fetal cell grafts at 45 d after lesion. B₁, Lesioned hippocampus that received mixed hippocampal cell transplant (T);C₁, lesioned hippocampus that received CA3 cell transplant (T); D₁, lesioned hippocampus that received CA1 cell transplant (T). Note that the transplant (outlined byinterrupted lines) is predominantly located just below the degenerated CA3 cell layer (asterisks) in all of these examples. B₂, C₂,D₂, Magnified views of CA1 regions fromB₁, C₁, andD₁. Note that hippocampus receiving either mixed hippocampal or CA3 cell grafts (B₁,B₂, C₁,C₂) exhibit greater density of GAD-67 interneurons than both lesion-only hippocampus (Fig.2B₁–B₄) and hippocampus receiving CA1 cell graft (D₁,D₂). GAD-67 interneuron density inB₁ and C₁ are also comparable with the control hippocampus (Fig.2A₁). DG, Dentate gyrus;GCL, granule cell layer; ML, molecular layer; SLM, stratum lacunosum moleculare;SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum. Scale bars:A₁, B₁,C₁, D₁, 400 μm;A₂, A₃, 100 μm;B₂, C₂,D₂, = 200 μm.

Fig. 4.

Comparison of the distribution of GAD-67-positive interneurons in the dentate gyrus (A₁–A₅) and the CA1 subfield (B₁–B₅) of the intact control hippocampus (A₁,B₁), the KA-lesioned hippocampus (A₂, B₂), the KA-lesioned hippocampus receiving mixed hippocampal cell graft (A₃, B₃), the KA-lesioned hippocampus receiving CA3 cell graft (A₄,B₄), and the KA-lesioned hippocampus receiving CA1 cell graft (A₅, B₅). Note that compared with the intact hippocampus (A₁, B₁), GAD-67-positive interneuron density appears decreased in both dentate gyrus and CA1 subfield of lesion-only hippocampus (A₂,B₁) and the hippocampus receiving CA1 cell graft (A₅, B₅). In contrast, both of these regions in the lesioned hippocampus receiving either mixed hippocampal or CA3 cell grafts (A₃,A₄, B₃,B₄) exhibit interneuron density that is closer to the intact hippocampus. In dentate gyrus, recovery is apparent in the dentate hilus, including basket cells at the junction of hilus and granule cell layer, whereas in the CA1 subfield, recovery is conspicuous in strata radiatum and pyramidale. DH, Dentate hilus; GCL, granule cell layer;ML, molecular layer; SO, stratum oriens;SP, stratum pyramidale; SR, stratum radiatum. Scale bar, 100 μm.

Fig. 5.

Comparison of the distribution of GAD-67-positive interneurons in the CA3 subfield (A₁–A₅) of the control hippocampus (A₁), the KA-lesioned hippocampus (A₂), the KA-lesioned hippocampus receiving mixed hippocampal cell graft (A₁), the KA-lesioned hippocampus receiving CA3 cell graft (A₄), and the KA-lesioned hippocampus receiving CA1 cell graft (A₅). Compared with the intact hippocampus (A₁) a clear decrease in GAD-67-positive interneuron density is obvious in the CA3 subfield of both lesion-only hippocampus (A₂) and the lesioned hippocampus receiving CA1 cell graft (A₅). In contrast, the CA3 region (particularly stratum radiatum) in hippocampus receiving either mixed hippocampal or CA3 cell grafts (A₃, A₄) exhibits interneuron density that is closer to intact hippocampus. Theinterrupted line in A₅ denotes transplant–host interface; asterisks denote the degenerated CA3 cell layer. SL, Stratum lucidum;SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum. Scale bar, 100 μm.

Fig. 6.

Histogram comparing GAD-67-positive interneuron density per cubic millimeter volume of tissue in both the entire and different layers of the dentate gyrus among the control intact hippocampus (n = 8), the lesioned hippocampus at 6 months after lesion, the lesioned hippocampus receiving mixed hippocampal cell graft, the lesioned hippocampus receiving CA3 cell graft, and the lesioned hippocampus receiving CA1 cell graft (n = 6 in each group). ANOVA with the Student—Newman–Keuls multiple comparisons post hoctest reveals significant differences between groups (p < 0.0001). Compared with the intact control hippocampus, interneuron density in lesion-only hippocampus and the lesioned hippocampus receiving CA1 cell graft is significantly reduced for the entire dentate gyrus (p < 0.001) as well as for all three layers of the dentate gyrus (p < 0.05). In contrast, interneuron density in dentate gyrus of lesioned hippocampus receiving grafts of either mixed hippocampal or CA3 cells is comparable with that in the intact control hippocampus (p > 0.05) and significantly greater than both lesion-only hippocampus and lesioned hippocampus receiving CA1 cell grafts (p < 0.05). The only exception is the granule cell layer, where the recovery is partial (i.e., significantly greater than lesion-only hippocampus but significantly less than control hippocampus; p< 0.05). Values are means ± SE.

Fig. 7.

Histogram comparing the density of GAD-67 interneurons per cubic millimeter volume of tissue in both the entire and different strata of the CA1 subfield among the intact control hippocampus (n = 8), the lesioned hippocampus at 6 months after lesion, the lesioned hippocampus receiving mixed hippocampal cell graft, the lesioned hippocampus receiving CA3 cell graft, and the lesioned hippocampus receiving CA1 cell graft (n = 6 in each group). ANOVA reveals significant differences between groups (p < 0.0001). Note that compared with the intact control hippocampus, interneuron density in lesion-only hippocampus and the hippocampus receiving CA1 cell graft is significantly reduced for the entire CA1 subfield (p < 0.001) and also for all layers of the CA1 subfield (p < 0.05). However, the interneuron density in the CA1 subfield of the lesioned hippocampus receiving grafts of either mixed hippocampal or CA3 cells is comparable with that in the control intact hippocampus (p > 0.05) and significantly greater than both lesion-only hippocampus and hippocampus receiving CA1 cell graft (p < 0.05). The only exception is the stratum pyramidale of the lesioned hippocampus receiving mixed hippocampal transplants, where recovery of GAD-67 interneuron density is partial (i.e., significantly comparable with control intact hippocampus but not significantly greater than lesion-only hippocampus;p > 0.05). Values are means ± SE.

Fig. 8.

Histogram showing GAD-67 interneuron density per cubic millimeter volume of tissue in both the entire and different strata of the CA3 subfield. Comparison among the control intact hippocampus (n = 8), the lesioned hippocampus at 6 months after lesion, the lesioned hippocampus receiving mixed hippocampal cell graft, the lesioned hippocampus receiving CA3 cell graft, and the lesioned hippocampus receiving CA1 cell graft (n = 6 in each group) using ANOVA reveals significant differences (p < 0.0001). The GAD interneuron density in lesion-only hippocampus and the hippocampus receiving CA1 cell graft is significantly reduced for the entire CA3 subfield (p < 0.001) and for all different strata of the CA3 subfield (p < 0.05) compared with the control intact hippocampus. Interneuron density in the entire CA3 region of the lesioned hippocampus receiving grafts of either mixed hippocampal or CA3 cells is significantly less than that of the intact hippocampus (p < 0.05) but significantly greater than both lesion-only hippocampus and hippocampus receiving CA1 cell graft (p < 0.05), suggesting partial recovery of GAD-67 interneuron density with mixed hippocampal or CA3 cell grafting. Among different layers, only stratum radiatum exhibited complete recovery with mixed hippocampal or CA3 cell grafting; density was comparable with intact control hippocampus (p > 0.05) and significantly greater than lesion-only hippocampus (p < 0.01). GAD-67 interneuron density in strata oriens and pyramidale remained less than intact control hippocampus (p < 0.05) and comparable with lesion-only hippocampus and hippocampus receiving CA1 cell grafts (p > 0.05). Values are means ± SE.

Fig. 9.

Histogram comparing GAD-67 interneuron density per cubic millimeter volume of tissue in the entire septal hippocampus among the control intact hippocampus (n = 8), the lesioned hippocampus at 6 months after lesion, the lesioned hippocampus receiving mixed hippocampal cell graft, the lesioned hippocampus receiving CA3 cell graft, and the lesioned hippocampus receiving CA1 cell graft (n = 6 in each group). ANOVA analysis with the Student—Newman–Keuls multiple comparisons post hoc test reveals significant differences among groups (p < 0.0001). Note that the density of GAD interneurons in lesion-only hippocampus and hippocampus receiving CA1 cell grafts is significantly reduced compared with control intact hippocampus (p < 0.01). In contrast, GAD interneuron density in lesioned hippocampus receiving grafts of either mixed hippocampal or CA3 cells shows complete recovery. Density is highly comparable with that in control intact hippocampus (p > 0.05) and significantly greater than in both lesion-only hippocampus (p < 0.001) and hippocampus receiving CA1 cell grafts (p< 0.01). Values are means ± SE.

Fig. 10.

Histogram showing comparison of the mean diameter of soma of GAD-67-positive interneurons among intact hippocampus, lesion-only hippocampus, and lesioned hippocampus receiving different cell grafts. Note that the soma size of GAD-67 interneurons is greater in the dentate gyrus and the CA3 subfield of lesion-only hippocampus, and this increase was shown to be significant (p < 0.05). However, the size is comparable with that of the control intact hippocampus in the lesioned hippocampus receiving fetal grafts (mixed hippocampal, CA3, or CA1 cell grafts). Values are means ± SE.