Astrocyte-derived neurons provide excitatory input to the adult striatal circuitry

Abstract

Astrocytes have emerged as a potential source for new neurons in the adult mammalian brain. In mice, adult striatal neurogenesis can be stimulated by local damage, which recruits striatal astrocytes into a neurogenic program by suppression of active Notch signaling (J. P. Magnusson et al., Science 346, 237-241 [2014]). Here, we induced adult striatal neurogenesis in the intact mouse brain by the inhibition of Notch signaling in astrocytes. We show that most striatal astrocyte-derived neurons are confined to the anterior medial striatum, do not express established striatal neuronal markers, and exhibit dendritic spines, which are atypical for striatal interneurons. In contrast to striatal neurons generated during development, which are GABAergic or cholinergic, most adult astrocyte-derived striatal neurons possess distinct electrophysiological properties, constituting the only glutamatergic striatal population. Astrocyte-derived neurons integrate into the adult striatal microcircuitry, both receiving and providing synaptic input. The glutamatergic nature of these neurons has the potential to provide excitatory input to the striatal circuitry and may represent an efficient strategy to compensate for reduced neuronal activity caused by aging or lesion-induced neuronal loss.

Keywords: astrocyte-derived neurogenesis; glutamatergic; neurons; striatum.

Fig. 1.

Astrocyte-derived neurons are preferentially located in the anterior medial striatum. (A) Schematic of the transgenic mouse line and experimental outline of the study. Adult animals (8 wk-old) homozygous for the conditional Rbpj-κ gene were given tamoxifen for 5 consecutive d to induce selective ablation of RBPj-κ and reporter expression (tdTom, tdTomato) in astrocytes and their progeny. Two weeks after tamoxifen-mediated genetic recombination, animals received EdU via the drinking water for 4 wk, and brain tissue was collected for analyses 6 wk later (12 wk after RBPj-κ deletion). (B) Conditional homozygous deletion of RBPj-κ in adult Cx30-CreER^T2–expressing striatal astrocytes leads to the production of adult-born neurons via proliferation. Arrowhead shows a recombined (tdTom⁺) astrocyte-derived neuron (NeuN⁺) that incorporated EdU. (C and D) Distribution (C) and quantification (D) of adult-born, astrocyte-derived neurons in the striatum along the anterior–posterior axis, from region 1 (R1) to R4. Each colored dot represents an adult-born, astrocyte-derived neuron. (E) Recombined striatal cells (tdTom⁺) do not express DARPP-32, ChAT, parvalbumin, calretinin, or somatostatin. Arrowheads point at striatal neurons positive for the neuron subtype marker and negative for tdTom. (F) A fraction of astrocyte-derived neurons (NeuN⁺/tdTom⁺ cells; arrowheads) expresses nNOS. The arrow marks a nonrecombined nNOS-expressing striatal interneuron. (Scale bars, 10 µm [B], 50 µm [E], and 20 µm.) (F). Data shown as mean ± SEM (n = 4 animals), *P = 0.0376, **P = 0.0074 by independent t test (external versus medial striatum) in D.

Fig. 2.

Astrocyte-derived neurons have unique electrophysiological properties distinct from the major striatal neuron subtypes. (A) Adult-born, astrocyte-derived neurons were identified in the region surrounding the anterior commissure (aca) in the caudate putamen (CPu), lateral of the lateral ventricle. (B) Neurons were selected by their expression of tdTom and identified by their distinct morphology and electrophysiological properties. (C) Current-voltage responses of astrocyte-derived neurons (ADNs), astrocytes (ast), MSNs, FSIs, Cholinergic Interneurons (ChINs), and LTS interneurons. Dotted lines mark baseline membrane potential. (D) TdTom-expressing cells were classified as neurons based on their discharge of action potentials. (E) ADNs (red) have longer membrane time constants compared to astrocytes (black). (F) Examples from three different ADNs discharging action potentials in response to ramp current injections. (G) Distinct membrane properties reveal ADNs as a unique population unlike well-known caudal neurons, most notably by their shallow action potentials, input resistance (R_in), and resting membrane potential (V_rest). *P < 0.05, **P < 0.01, and ***P < 0.001 by independent t test for all comparisons (ADNs versus astrocytes, MSN, FSI, ChIN, or LTS interneurons). (H) Patched neurons were filled with neurobiotin for post hoc staining and morphological analysis. ADNs presented highly branched neurites and dendritic spines (arrows). Scale bars represent 250 µm (A) and 25 µm (B and H).

Fig. 3.

Astrocyte-derived neurons receive GABAergic and glutamatergic input. (A and B) Spontaneous postsynaptic events observed in astrocyte-derived neurons were not affected by gabazine (n = 6 responding neurons, Z = −0.405, P = 0.686) but reduced in the presence of NBQX (n = 10 responding neurons, Z = −2.090, P = 0.037, Wilcoxon signed-rank test). Data points marked in red (B) correspond to example traces in A. (C and D) An extracellular stimulation electrode (C) was used to trigger postsynaptic responses in astrocyte-derived neurons. (D, Right) Synaptic responses in the presence of gabazine subtracted from responses under control conditions reveal a large GABA-mediated component.

Fig. 4.

Astrocyte-derived neurons are glutamatergic and functionally connect to nearby MSNs. (A–C) In mice crossed with a ChR2-tdTom reporter (A), astrocyte-derived neurons and astrocytes (B) exhibit a clear light-induced depolarization (C). (D) Postsynaptic responses in MSNs induced by a 5-ms light pulse were attenuated by gabazine (10 μM) and strongly reduced by NBQX (10 μM). In a subset of experiments, the additional application of NBQX and amino-5-phosphonopentanoate (AP5) abolished postsynaptic responses completely. (E) A 40 Hz train of 3-ms light pulses produced sharply depressing postsynaptic responses. (F) Light-induced responses were reduced in amplitude in the presence of gabazine (P = 0.03, n = 8 responding neurons) and more strongly reduced in the presence of NBQX (P = 0.007, n = 9 responding neurons). Peak delay was unaffected by gabazine (P = 0.87, n = 7 responding neurons) but increased in NBQX (P = 0.05, n = 9 responding neurons, Wilcoxon signed-rank test for all comparisons). (G) Fast postsynaptic responses to light activation were detected in mice expressing ChR2 in astrocytes and astrocyte-derived neurons (ADNs) (n = 13 mice) but not in mice expressing ChR2 exclusively in astrocytes (n = 2 mice). (H and I) Detection of Vglut1 (Slc17a7) mRNA in astrocyte-derived striatal neurons (tdTom⁺/NeuN⁺ cells, arrowheads) by RNAscope in situ hybridization combined with immunofluorescence for NeuN and tdTomato (H). Note that in the striatum, Vglut1 mRNA signals in astrocyte-derived neurons are weaker than in glutamatergic excitatory neurons of the cerebral cortex. Vglut2 (Slc17a6) mRNA signals are virtually absent in astrocyte-derived striatal neurons (I). Cell nuclei are labeled with DAPI (blue). (Scale bar, 10 µm.)