This study directly builds on our earlier work demonstrating that cannabidiol (CBD, 10 mg/kg daily) protects vocal quality and accelerates recovery of learned song patterns after HVC microlesions in adult male zebra finches [16]. Vocal recovery was associated with robust CBD suppression of microlesion-induced neuroinflammation (reduced IL6, IL1B, and TNFA), oxidative stress (reduced SOD2 and DHE fluorescence), microglial activation (reduced TMEM119), and circuit-wide synaptic loss (preserved VGLUT2/PSD95 colocalization), as well as activation of NRF2 nuclear translocation with upregulation of plasticity-associated transcripts (BDNF, ARC/ARG3.1, MSK1, Tripson et al., 2023). We designed the present experiments to identify cellular substrates underlying these protective effects. We now show that astrocytes are a primary, though not exclusive, target of CBD-mediated neuroprotection.

These findings complement recent independent evidence that CBD interacts with the TrkB adaptor FRS2 to stabilize receptor signaling and inhibit JAK2/STAT3/SOCS1-driven inflammatory cascades [35]. In that work, FRS2-dependent TrkB activation was associated with reduced astrogliosis (GFAP), cytokine production, and synaptic loss: outcomes that align closely with the astrocyte-preserving effects, lowered reactivity markers (C3, S100A10, aromatase), and synaptic protection we observe across song-control nuclei after acute TBI. The present astrocyte-centric data therefore extend the known neuroprotective profile of cannabidiol to a vocal-learning model of focal injury, reinforcing the potential for glial-targeted mechanisms in circuit resilience.

CBD preserved astrocyte viability across regions of both the anterior forebrain pathway and vocal motor circuits (HVC, RA, and Area X; see Fig. 1), attenuated injury-induced lysosomal stress and reactivity programs, and enhanced mediators of astrocyte metabolic and antioxidant capacity. These glial-associated actions provide a mechanistic link between previously reported anti-inflammatory, anti-oxidative, and synaptoprotective outcomes and the accelerated behavioral recovery of song, a complex sensorimotor skill.

A central finding is that HVC microlesions trigger substantial astrocyte apoptosis, accounting for a large fraction of cleaved caspase-3–positive cells in the three song nuclei investigated (HVC, RA, and Area X; Fig. 3). CBD markedly reduces this astrocyte loss while preserving or expanding astrocyte populations in a region-dependent manner (Fig. 3D,E). This astrocyte-specific protection is particularly relevant to secondary injury processes, as the primary electrolytic damage remains constant across treatment groups while CBD limits the subsequent apoptotic, lysosomal, and reactivity cascades that would otherwise amplify circuit-wide dysfunction (see Alalawi et al., 2019; Tripson et al., 2023, for propagation of inflammatory and synaptic responses). This general, circuit-wide astrocytic resilience contrasts with more localized neuronal preservation (confined largely to the directly-lesioned HVC; Fig. 4) and aligns with evidence that astrocyte viability is a critical determinant of circuit recovery after focal CNS injury [38–40]. Astrocytes maintain neuronal survival and synaptic stability through metabolic coupling (e.g., lactate shuttling, glutamate homeostasis, redox buffering) and structural support [41,42]. Astrocytic aerobic glycolysis generates energy- and survival-promoting lactate that is shuttled to neurons via selective transporters, providing oxidative substrate while preserving neuronal redox balance and supporting synaptic plasticity [43]. Astrocytes also clear excess glutamate to protect against excitotoxicity and maintain synaptic homeostasis [44] and serve as the primary CNS source of glutathione for redox buffering and neuroprotection [45].

The endocannabinoid system (ECS) regulates many of these functions, and astrocytes express both canonical (CB₁, inducibly CB₂) and non-canonical ECS-linked receptors (TRPV1/4, PPARG; [25,46]). Although CBD is not a direct CB₁/CB₂ agonist, it modulates astrocytic signaling via PPARG and TRPV channels [47,48] consistent with the broad circuit-wide preservation of astrocyte populations observed here. By stabilizing astrocytes, CBD may prevent secondary propagation of injury signals that would otherwise amplify neuronal vulnerability and synaptic destabilization: deficits reversed in our prior study [20].

Injury also imposes significant lysosomal stress on astrocytes, as evidenced by enlarged LAMP1-immunopositive lysosomes and upregulation of LAMP1 and LC3 mRNA (Fig. 5). In the case of enlarged lysosomes, these were within GS-expressing astrocytes. In the case of LC3 upregulation, this may have also included extra-astrocytic expression (e.g. from microglia and or neurons). CBD normalized lysosomal morphology and suppressed these transcriptional responses, indicating reduced proteostatic stress rather than global lysosomal suppression. Lysosomal/autophagic pathways are tightly coupled to cellular redox balance and mitochondrial quality control [49]. Their dysregulation exacerbates oxidative stress and inflammation [50].

Reductions in lysosomal burden and LAMP1/LC3 expression are consistent with the hypothesis that CBD improves autophagic clearance of injury-related debris, thereby limiting accumulation of damaged organelles and proteins and promoting astrocyte and circuit resilience. However, the observed decreases could alternatively reflect reduced production of cellular debris upstream of autophagy (e.g., via CBD’s established suppression of neuroinflammation, oxidative stress, microglial activation, and synaptic loss; Tripson et al., 2023).

Pharmacological assessment of autophagic flux via LC3-II accumulation in the presence/absence of lysosomal inhibitors (such as bafilomycin A1 or chloroquine) has been successfully applied in vivo in mammalian brain and other tissues, as well as in cell culture systems [51,52]. Systemic administration of these inhibitors to zebra finches, combined with western blot or immunofluorescence quantification of LC3-II in microdissected song nuclei could distinguish increased clearance from reduced debris generation. It may be helpful to develop methods to isolate and culture primary zebra finch astrocyte cultures, as in vitro approaches using lesion-mimetic stressors with and without CBD would allow more direct mechanistic dissection (e.g., flux assays with lysosomal blockade or p62/SQSTM1 turnover). Such combined in vivo and in vitro expansion would strengthen causal inference regarding CBD’s effects on astrocyte autophagic processes. The present lysosomal findings mechanistically extend our earlier observations of CBD-mediated reductions in SOD2, DHE fluorescence, and NRF2 pathway activation [20]. Activation of PPARG, a known transcriptional coordinator of antioxidant and metabolic programs in glia [53,54] offers a plausible convergent mechanism, as PPARG regulates both lysosomal function and mitochondrial turnover [55]. Future experiments using selective PPARG and/or TRPV antagonists may help to confirm pathway specificity.

CBD further enhanced astrocyte metabolic resilience by increasing expression of glutamine synthetase (GS) and glutamate-cysteine ligase modifier subunit (GCLM), the regulatory component of the rate-limiting enzyme in glutathione biosynthesis (Fig. 6). GS supports glutamate recycling and prevents excitotoxicity, while GCLM is a key cofactor for glutathione synthesis and downstream redox buffering: functions essential for maintaining synaptic homeostasis under injury stress [44,56,57]. These changes are consistent with the previously observed song circuit synaptic preservation and reduced excitotoxic vulnerability and suggest that CBD shifts astrocytes toward an oxidative stress–resistant, metabolically efficient phenotype [47,58] likely via enhanced GS/GCLM expression and NRF2-induced antioxidant responses. While NRF2 activation underpins antioxidant responses observed herein and in prior work, CBD likely engages complementary targets like PPARG for glial reactivity suppression and TLR4 inhibition for broader anti-inflammatory effects, as supported by recent mechanistic studies [59,60]. Future experiments with selective antagonists (e.g., GW9662 for PPARG) could help dissect these contributions in the zebra finch model. Although protein-level confirmation and assessment of the primary astrocytic glutamate transporter GLT1 also remains for future work, the transcriptional data (Figs 6 and 7) provide strong evidence that improved astrocytic management of glutamate/excitotoxic signaling supports the circuit stability required for sensorimotor relearning and vocal recovery.

Finally, CBD suppressed injury-induced astrocyte reactivity markers (C3, S100A10, aromatase; Fig. 7). C3 is typically associated with pro-inflammatory astrocytes, while S100A10 usually marks more homeostatic, anti-inflammatory astroglial states [27,61] and aromatase provides a general reactivity index [32,62]. Coordinated downregulation of all three transcripts, rather than selective promotion of a reparative phenotype (e.g., S100A10-expressing “A2-like” population), suggests that CBD prevents diversion of astrocytic resources into stress programs and sustains core homeostatic functions [47]. Such suppression is consistent with CBD-mediated inhibition of NFKB–driven transcription via PPARG and TRPV1 signaling [63,64] and aligns with the broad anti-inflammatory profile we documented previously. By limiting reactive astrogliosis, CBD appears to favor maintenance of the supportive astrocytic environment necessary for ongoing synaptic refinement and vocal circuit integrity.

Region-specific neuronal preservation in HVC (a site of adult neurogenesis) further suggests that CBD may enhance post-injury recruitment in addition to neuronal survival, potentially augmenting circuit repair [65,66]. Although proliferation markers (e.g., BrdU, DCX) were not assessed here, results raise the possibility that astrocyte-derived trophic support contributes to this effect [67,68].

Collectively, these findings establish astrocyte-targeted neuroprotection as a key mechanism by which CBD preserves song circuit integrity and accelerates recovery of a complex learned behavior after a focal TBI-like injury [16,20]. The zebra finch model, with discrete, well-mapped nuclei that support sensitive-period learning and lifelong sensorimotor maintenance, offers a powerful, translationally relevant platform for dissecting glial contributions to circuit resilience [69,70]. Many human sensorimotor skills (speech, language, manual dexterity) likewise depend on analogous specialized circuits vulnerable to traumatic disruption [65,71]. Our results therefore highlight the therapeutic potential of glial-directed interventions, with CBD or its downstream modulators as promising candidates for promoting functional recovery after CNS trauma [72,73].