Embryonic MGE precursor cells grafted into adult rat striatum integrate and ameliorate motor symptoms in 6-OHDA-lesioned rats

Abstract

We investigated a strategy to ameliorate the motor symptoms of rats that received 6-hydroxydopamine (6-OHDA) lesions, a rodent model of Parkinson's disease, through transplantation of embryonic medial ganglionic eminence (MGE) cells into the striatum. During brain development, embryonic MGE cells migrate into the striatum and neocortex where they mature into GABAergic interneurons and play a key role in establishing the balance between excitation and inhibition. Unlike most other embryonic neurons, MGE cells retain the capacity for migration and integration when transplanted into the postnatal and adult brain. We performed MGE cell transplantation into the basal ganglia of control and 6-OHDA-lesioned rats. Transplanted MGE cells survived, differentiated into GABA(+) neurons, integrated into host circuitry, and modified motor behavior in both lesioned and control rats. Our data suggest that MGE cell transplantation into the striatum is a promising approach to investigate the potential benefits of remodeling basal ganglia circuitry in neurodegenerative diseases.

Figure 1. Transplanted MGE Cells Migrated Long Distances from the Site of Injection throughout the Striatum

(A) Striatum 4 weeks after transplantation. The transplanted GFP-MGE cells migrated up to 2.5 mm in all directions from the sites of injection but did not migrate beyond the borders of the striatum. (B) Eight weeks after transplantation, the MGE cells had a mature appearance. Most MGE cells had an oblong body shape and numerous processes that could be traced up to hundreds of μm from the soma. The GFP⁺ processes were present throughout the striatum even in areas far away from GFP-MGE cell bodies. Scale bars represent 0.5 mm in (A) and 25 μm in (B).

Figure 2. Most MGE Cells Adopted an Inter-neuronal Fate when Transplanted into the Adult Striatum

(A) Four weeks after transplantation, most MGE cells expressed the mature neuronal markers NeuN (75% ± 6%). (B–D) Most MGE cells also expressed markers related to GABA storage or synthesis such as GABA (B, 75% ± 4%), GAD (C, 60% ± 11%), and GAT (D, 50% ± 9%). (E–I) Some transplanted MGE cells also expressed interneuron subtype markers CB (E, 24% ± 7%), CR (F, 8.3% ± 0.5%), PV (G, 0.5% ± 0.5%), Substance P (H, 6% ± 2%), and Somatostatin (I, 1% ± 0.5%). (J) One quarter of the MGE cells transplanted into the striatum (25% ± 4%) expressed the oligodendrocyte marker CNPase. Arrows point to examples of MGE cells that stained positive for each marker, and insets for each panel (labeled 2 and 3) show higher power examples of immunopositive cells. Scale bar represents 30 μm in (A1) and applies to lower-power images shown in (A1)–(J1); scale bar represents 10 μm in (A2) and applies to all higher-power inset images (A2–J2 and A3–J3).

Figure 3. MGE Cells Transplanted into the Striatum Displayed the Basic Membrane Properties Characteristic of Mature Forebrain Interneurons and Showed Evidence of Synaptic Integration

(A) 67% of the MGE cells (green) expressed the synapse marker synaptophysin (red) along their processes (white arrows). (B) We obtained whole-cell patch-clamp recordings from transplanted cells up to 20 weeks after transplantation. Alexa-594 (red) was included in the fill solution of the glass microelectrode to confirm that recordings were obtained from targeted GFP-expressing MGE transplant cells. (C) Most MGE cells (37/44) fired repetitive nonaccommodating action potentials with large after hyperpolarizations as is common for MGE-derived interneurons. (D) A small percent of the transplanted MGE cells that were recorded from (n = 7/44) did not exhibit neuronal membrane properties and did not fire action potentials when stimulated with a series of depolarizing currents, consistent with glial cell identity. (E₁) Most MGE neurons were fast spiking (FS) cells and did not fire rebound action potentials after hyperpolarization. (E₂) The FS population included stuttering FS cell subtypes. (E₃) The second most common cell type was low-threshold-spiking (LTS) cells. These cells displayed much lower spiking frequencies than did FS cells and displayed two or more rebound spikes in response to hyperpolarization. (F) Transplanted cells demonstrated both AMPA and GABA-mediated spontaneous synaptic events. (G) A transplanted MGE cell filled with Texas red-dextran demonstrated the branched aspiny processes that are characteristic of striatal interneurons. Scale bars represent 3 μm in (A), 5 μm in (B), and 10 μm in (G).

Figure 4. MGE Cell Transplantation Ameliorates the Behavioral Deficits of 6-OHDA Lesions

(A) Rotation test. 6-OHDA-lesioned rats that received MGE cell transplantation (n = 18) performed fewer contralateral rotations under apomorphine stimulation than nontransplanted 6-OHDA controls (n = 15), 6-OHDA-lesioned rats that received sham injection (n = 11), or dead MGE cells (n = 10). ANOVA showed that the difference between groups was significant at 9, 11, and 14 weeks (asterisks). Postcomparison analysis showed that the MGE group rotated significantly less than the 6-OHDA-treated control groups at 9, 11, and 14 weeks. Error bars depict standard error of the mean. (B) Length of stride test. 6-OHDA-lesioned rats that received MGE cell transplantation (n = 18) had a longer stride than did nontransplanted 6-OHDA controls (n = 15) and 6-OHDA-lesioned rats that received sham injection (n = 11). Naive rats (n = 10) are included for comparison. ANOVA showed that the difference between groups was significant at 11 weeks. Postcomparison analysis showed that the MGE group had a significantly longer stride than the 6-OHDA-lesioned control groups at 11 weeks (asterisk). See also Figure S1.

Figure 5. MGE Transplanted Cells Expressed Dopamine Receptors

(A1 and B1) MGE cells (green) expressed the dopamine receptor D1 (red) and dopamine receptor D2 (red). (A2 and B2) Higher-magnification images showing that the MGE transplant cell (green, white arrowhead) expresses D1 and D2 (red) on the somatic membrane and processes. Merge panel shows colocalization. Scale bars represent 30 μm for (A1) and (B1) and 3 μm for (A2) and (B2).

Figure 6. Transplantation of MGE Cells into Control Rats that Had Not Been Lesioned with 6-OHDA Increased the Length of Stride and the Level of Activity

(A) The length of stride was significantly higher in control rats injected with MGE cells (n = 6) than in control rats (n = 16). (B) Open field tests showed an increase in activity for control animals that received MGE cell transplantation in comparison to control rats. Error bars depict standard error of the means. (C) Representative open field zone maps from two rats showing that transplantation of MGE cells into control rats increased the level of motor activity over that of control rats.

Figure 7. MGA Cells Transplanted in the Subthalamic Nucleus

(A) MGE cells transplanted into the subthalamic nucleus (STN) survived but did not migrate from the site of injection and did not mature into neurons. (B–K) None of the MGE cells that were transplanted into the STN expressed the neuronal marker NeuN (B), the inhibitory interneuron marker GABA (E) or GAD (G), or the calcium sequestering proteins CR (H) or CB (I). Most MGE cells transplanted into the STN expressed the astrocyte marker GFAP (75% ± 5%, C and J) or the oligodendrocyte marker CNPase (30% ± 9%, D and K). A small percentage of MGE cells in the STN expressed the GABA transporter GAT1 (13% ± 5%, F). Scale bars represent 25 μm in (A); 25 μm for (I) and (B)–(H); and 5 μm for (K) and (J).