Appropriate termination of regenerative processes is critical for producing the correct number of cells in tissues. Here we provide evidence for an end-product inhibition of dopamine neuron regeneration that is mediated by dopamine. Ablation of midbrain dopamine neurons leads to complete regeneration in salamanders. Regeneration involves extensive neurogenesis and requires activation of quiescent ependymoglia cells, which express dopamine receptors. Pharmacological compensation for dopamine loss by L-dopa inhibits ependymoglia proliferation and regeneration in a dopamine receptor-signaling-dependent manner, specifically after ablation of dopamine neurons. Systemic administration of the dopamine receptor antagonist haloperidol alone causes ependymoglia proliferation and the appearance of excessive number of neurons. Our data show that stem cell quiescence is under dopamine control and provide a model for termination once normal homeostasis is restored. The findings establish a role for dopamine in the reversible suppression of neurogenesis in the midbrain and have implications for regenerative strategies in Parkinson's disease.

After chemical ablation of midbrain dopamine neurons by 6-OHDA ( Figures 1 A–1D ;), regeneration in salamanders leads to complete histological restoration and to full recovery of motor behavior (). To date, the salamander provides the sole available animal model that allows discovery of principles of robust midbrain dopamine neurogenesis in a Parkinson's disease-like condition. Dopaminergic regeneration depends on cellular proliferation and is characterized by cell cycle reentry of ependymoglia stem cells () lining the ventricle in the normally quiescent midbrain (). Dopamine neurons, which express the evolutionarily conserved markers tyrosine hydroxylase (TH) and Nurr1, are born gradually during regeneration and reach normal numbers within 4 weeks after ablation (). No new dopamine neurons are detected in the midbrain of control animals (); conversely no excessive neurons are formed after full recovery ( Figure S1 A available online;).

n = 3–16 for each group. Error bars represent SEM. Ve indicates the third ventricle. Scale bars represent 50 μm. See also Figure S1 and Movie S1

(K) The GABA A receptor agonist muscimol does not inhibit regeneration of TH + neurons in the midbrain.

(H and I) L-dopa administration between days 9 and 15 inhibits the regeneration of TH + (H) and Nurr1 + (I) neurons.

(E–G) L-dopa administration between days 2 and 8 inhibits the regeneration of TH + (E) and Nurr1 + (F) neurons as well as locomotor recovery (G).

(A–D) Ablation of midbrain DA neurons revealed by the loss of TH- and Nurr1-expressing neurons (B, D) compared to control brains (A, C).

Pharmacological Compensation for Dopamine Loss Specifically Inhibits the Regeneration of Dopamine Neurons in the Midbrain

Figure 1 Pharmacological Compensation for Dopamine Loss Specifically Inhibits the Regeneration of Dopamine Neurons in the Midbrain

Efficient regeneration by activation of neurogenesis in homeostatically quiescent regions of the adult vertebrate brain.

A clonal analysis of neural progenitors during axolotl spinal cord regeneration reveals evidence for both spatially restricted and multipotent progenitors.

Clonal cell cultures from adult spinal cord of the amphibian urodele Pleurodeles waltl to study the identity and potentialities of cells during tail regeneration.

Local stem and progenitor cells are targets for promoting regeneration (), for which it is critical to understand how they sense the extent of cell loss in relation to the normal homeostatic turnover. An insufficient regenerative response or a rampant regeneration process could result either in an incomplete or superfluous number of cells. Therefore, two critical steps to regulate are the initiation and appropriate termination of the generation of new cells. However, very little is known about the mechanisms that control the duration of replacement after cell loss, particularly in otherwise essentially quiescent tissues. We addressed these questions by studying the regeneration of dopamine neurons in the midbrain of an adult salamander, the red spotted newt. Newts are powerful models that possess the most extensive regenerative capacities among adult vertebrates () and their dopaminergic system has been well characterized ().

Results

Harms et al., 2007 Harms C.

Albrecht K.

Harms U.

Seidel K.

Hauck L.

Baldinger T.

Hübner D.

Kronenberg G.

An J.

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et al. Phosphatidylinositol 3-Akt-kinase-dependent phosphorylation of p21(Waf1/Cip1) as a novel mechanism of neuroprotection by glucocorticoids. Rinner et al., 1997 Rinner W.A.

Pifl C.

Lassmann H.

Hörtnagl H. Induction of apoptosis in vitro and in vivo by the cholinergic neurotoxin ethylcholine aziridinium. Next, we ablated cholinergic neurons in the midbrain by injecting ethylcholine aziridinium (AF64A) (). Cholinergic neurons were identified by the expression of choline acetyltransferase (ChAT) ( Figures S1 B–S1D). Regeneration of cholinergic neurons was gradual and took 7 weeks ( Figures S1 E–S1G). In contrast to the regeneration of dopamine neurons after 6-OHDA injection, L-dopa administration between days 9 and 15 after AF64A injection did not influence the regeneration of cholinergic neurons ( Figure 1 J).

A receptor agonist muscimol between days 9 and 15 after ablation. In contrast to L-dopa, muscimol had no effect on the regeneration of TH+ neurons ( Next we treated animals with high doses of the γ-aminobutyric acid (GABA)receptor agonist muscimol between days 9 and 15 after ablation. In contrast to L-dopa, muscimol had no effect on the regeneration of THneurons ( Figure 1 K; Figure S2 B).

These results together show that pharmacological compensation for dopamine loss specifically inhibits dopamine neuron regeneration.

+ ependymoglia cell/midbrain ( Berg et al., 2010 Berg D.A.

Kirkham M.

Beljajeva A.

Knapp D.

Habermann B.

Ryge J.

Tanaka E.M.

Simon A. Efficient regeneration by activation of neurogenesis in homeostatically quiescent regions of the adult vertebrate brain. +/GFAP+ cells ( Figure 4 Dopamine Receptor Antagonism Undermines Ependymoglia Quiescence and Causes the Appearance of Excessive Numbers of Neurons Show full caption (A) Haloperidol administration evokes cell cycle reentry by normally quiescent midbrain ependymoglia cells. (B and C) Haloperidol administration leads to the appearance of excessive numbers of Nurr1+ (B) and TH+ (C) neurons. n = 3–5 for each group. Error bars represent SEM. (D–G) Haloperidol administration results in newly formed Nurr1+ cells shown by BrdU+/Nurr1+ cells. Arrows point to Nurr1−/BrdU− cells, filled arrowheads point to Nurr1+/BrdU- cells, arrowhead points to a BrdU+/Nurr1+ cell (E–G is the area indicated by the rectangle in D). (H–K) Specific labeling of ependymoglia cells in vivo by electroporation. Expression of YFP is restricted to ependymoglia cells 24 hr after electroporation. (L and M) Haloperidol administration results in the transition of ependymoglia to TH+ cells. Arrowhead points to an ependymoglia cell expressing YFP; arrow points to a TH+ cell expressing YFP. (N) Schematic model illustrating that loss of dopamine after ablation of its producing neurons lifts the proliferation block, allowing cells to enter a regenerative program. Conversely, the regenerative process ends when normal dopaminergic homeostasis has been restored, and dopamine signaling is required for the reversible suppression of neurogenesis in the adult newt midbrain. Ve indicates the third ventricle, DA (dopamine). Scale bars represent 50 μm. See also Figure S3 Given the strong receptor signaling-dependent inhibitory effect of dopamine on ependymoglia proliferation, we tested whether antagonizing dopamine receptors alone is sufficient to evoke cell cycle reentry by quiescent ependymoglia cells. Nonablated animals received daily intraperitoneal injections of haloperidol for 4 days (2 mg/kg/injection), for 8 days (1 mg/kg/injection), or for 15 days (2 mg/kg/injection). While almost all cells in the adult newt midbrain are quiescent with the exception of 3 ± 2 PCNAependymoglia cell/midbrain ( Figure 4 A ;), haloperidol treatment caused their cell cycle reentry. Treatment with haloperidol resulted in significant increase in the number of PCNA/GFAPcells ( Figure 4 A). Although the number of ependymoglia cells entering the cell cycle after a 4 day treatment with 2 mg/kg haloperidol corresponds to only 50% of the maximum number of cycling ependymoglia cells after ablation of the dopamine neurons, the results establish that dopamine receptor signaling is critical in preventing ependymoglia proliferation.

1 and D 2 dopamine receptor signaling, we treated animals with the selective D 1 receptor antagonist SCH23390 ( O'Boyle and Waddington, 1987 O'Boyle K.M.

Waddington J.L. [3H]SCH 23390 binding to human putamen D-1 dopamine receptors: Stereochemical and structure-affinity relationships among 1-phenyl-1H-3-benzazepine derivatives as a guide to D-1 receptor topography. 1 and D 2 receptors showed overlapping expression pattern in the midbrain ( Berg et al., 2010 Berg D.A.

Kirkham M.

Beljajeva A.

Knapp D.

Habermann B.

Ryge J.

Tanaka E.M.

Simon A. Efficient regeneration by activation of neurogenesis in homeostatically quiescent regions of the adult vertebrate brain. To distinguish between Dand Ddopamine receptor signaling, we treated animals with the selective Dreceptor antagonist SCH23390 (). Despite the fact that that Dand Dreceptors showed overlapping expression pattern in the midbrain ( Figures S3 D–S3G), treatment with SCH23390 had no effect on cell cycle reentry in the midbrain (data not shown). In contrast, SCH23390 reduced mitotic activity in a constitutively active proliferation hotspot, which is located between the dorsal and lateral pallium in the forebrain (data not shown;).