Long-Range Projections of Cortical GABAergic Neurons to the Midline Dorsal Thalamic Nuclei in GAD67-GFP Mice

Background: Cortical γ-aminobutyric acidergic (GABAergic) neurons are characterized primarily as local inhibitory interneurons that modulate cortical pyramidal neuronal activity. However, emerging evidence has demonstrated that some of them may project to subcortical structures, such as the midline dorsal thalamic nuclei (MDTN), which play a pivotal role in sensory information transmission and emotional regulation. The present study aimed to investigate whether cortical GABAergic neurons project to the MDTN.

Methods: To address this question, this study combined retrograde tracing with immunofluorescent histochemical staining in GAD67-green fluorescence protein (GAD67-GFP) mice.

Results: Cholera toxin B subunit (CTB) retrograde-labeled (CTB⁺), GAD67-GFP-immunoreactive (GAD⁺), and GAD and CTB double-labeled (GAD⁺+CTB⁺) neurons were identified across many cortical regions. CTB⁺ neurons were mainly observed in the motor cortices, cingulate cortex (Cg), prelimbic cortex (PrL), and insular cortex (IC) with sparse distributions in the sensory cortices, orbitofrontal cortex (OFC), piriform cortex (Pir) and claustrum (CL). GAD⁺ neurons were distributed throughout all cortical layers. In the sensory, motor, and granular insular cortices, the highest density was observed in layers II/III or V, with a relatively sparse distribution in layers I and IV. These layers were also widely distributed in Other cortical regions such as the OFC, Cg, PrL, and Pir. GAD⁺+CTB⁺ neurons were mainly concentrated in layers V/VI of the motor, sensory, and IC cortices, with sparse distributions in the OFC, PrL, and Cg. These neurons spanned a rostrocaudal range of +2.34 mm to -0.46 mm from the bregma. Quantitative analysis showed that GAD⁺+CTB⁺ neurons accounted for 0.25%-0.55% of GAD⁺ neurons and 2.52%-4.93% of CTB⁺ neurons, respectively.

Conclusions: The present results confirmed the existence of long-range GABAergic projections from the cortex to the MDTN and provide a morphological basis for the functional study of corticothalamic regulation through GABAergic projections.

GABAergic neurons; cerebral cortex; midline thalamic nuclei; neuronal pathway tracing.