Distinct structural and functional connectivity of genetically segregated thalamoreticular subnetworks

Abstract

The thalamic reticular nucleus (TRN), the major inhibitory source of the thalamus, plays essential roles in sensory processing, attention, and cognition. However, our understanding of how TRN circuitry contributes to these diverse functions remains limited, largely due to the lack of genetic tools for selectively targeting TRN neurons with discrete structural and physiological properties. Here, we develop Cre mouse lines targeting two genetically segregated populations of TRN neurons that engage first-order (FO) and higher-order (HO) thalamic nuclei, respectively. In addition to substantially distinct electrophysiological properties, these TRN subnetworks are further distinguished by biases in top-down cortical and bottom-up thalamic inputs, along with significant differences in brain-wide synaptic convergence. Furthermore, we demonstrate that dysfunction of each subnetwork results in distinct cortical electroencephalogram (EEG) and sensory processing deficits commonly observed in neuropsychiatric disorders, underscoring the potential involvement of TRN subnetworks in the pathophysiology of these conditions.

Keywords: CP: Neuroscience; EEG; TRN subnetwork synaptic connectivity; electrophysiology; neurodevelopmental disorders; neuropsychiatric disorders; thalamic reticular nucleus, TRN; thalamocortical circuits.

Figure 1.. Generation of Spp1-Cre and Ecel1-Cre mice permits genetic access to two TRN subnetworks|

(A) CRISPR-Cas9 strategy for generating Cre lines. (B) Schematic of the AAV injection strategy for labeling TRN neurons. (C) Shell-like distribution of Ecel1+ neurons in the TRN. Scale bar: 500 μm. (D) Ecel1+ axonal projections extending outside of the FO somatosensory nuclei to the HO thalamus (Po, posteromedial thalamic nucleus; VL, ventrolateral; VM, ventromedial; CM, centromedial thalamus). Scale bar: 500 μm. (E) Core-like distribution of Spp1+ neurons in the TRN. Scale bar: 500 μm. (F) Spp1+ axonal projections in FO somatosensory thalamus. VPL, ventroposterior lateral; VPM, ventroposterior medial. Scale bar: 500 μm. (G) FISH images depicting overlap of Spp1 and Ecel1 transcripts in GFP-expressing neurons from Ecel1-Cre mice. Scale bar: 250 μm. An asterisk indicates presence of Spp1 mRNA in posterior thalamic glia. (H) FISH images depicting overlap of Spp1 and Ecel1 transcripts in GFP-expressing neurons from Spp1-Cre mice. Scale bar: 250 μm. (I) Magnified image of the inset from (G). Scale bar: 50 μm. (J) Magnified image of the inset from (H). Scale bar: 50 μm. (K) Quantification of GFP+ neurons from each Cre line expressing one or both transcripts. Bar graphs are depicted as mean + SEM (average from 3 slices permouse collected between −0.70 and −1.34 mm from the bregma; n = 3 mice per Cre line).

Figure 2.. FO and HO TRN subnetwork neurons have distinct electrophysiological properties

(A) Schematic of the AAV injection strategy for labeling FO and HO TRN subnetwork neurons. Scale bars: 1.5 mm. mCherry+ cells were identified for whole-cell patch clamping. (B–E) Passive membrane properties. RMP, resting membrane potential; τ, time constant. Two-tailed unpaired t tests (*p < 0.05, **p < 0.01, ****p < 0.0001). (F) Representative traces of rebound bursting in Spp1+ and Ecel1+ neurons; top traces depict the first burst following membrane hyperpolarization. (G) Number of rebound bursts generated across membrane potentials. (H) Maximum number of rebound bursts; two-tailed unpaired t test (****p < 0.0001). (I) Intraburst action potential (AP) frequency from the first rebound burst; two-tailed unpaired t test (****p < 0.0001). (J) Afterhyperpolarization (AHP) amplitude following the first burst. (K) Hyperpolarization slope of the AHP following the first burst (depicted as absolute value); two-tailed unpaired t test (***p < 0.001). (L) Linear regression and correlation of maximum number of bursts as a function of cell capacitance (****p < 0.0001). (M) Representative trace of an Spp1+ runaway bursting neuron. (N) Percentage of Spp1+ neurons expressing the runaway bursting phenotype. (O) Representative traces of tonic AP firing from Spp1+ and Ecel1+ neurons at 400 pA current injection. (P) Number of APs elicited by depolarization steps. (Q) Instantaneous AP frequency per depolarizing step. (R) Maximum number of APs elicited by suprathreshold depolarization for Spp1+ and Ecel1+ neurons; two-tailed unpaired t test (****p < 0.0001). (S and T) AP waveform half-width and amplitude (*p < 0.05, ****p < 0.0001). (U) AP spike frequency adaptation ratio; two-tailed unpaired t test (**p < 0.01). (V) AP spike train amplitude ratio; two-tailed unpaired t test (****p < 0.0001). Bar graphs, bursting curves, and tonic firing curves are presented as mean ± SEM. The circle graph is depicted as parts of whole (n = 26 cells and 5 mice per group).

Figure 3.. Brain-wide input mapping to FO and HO TRN subnetworks

(A) Schematic of the rabies tracing strategy in Cre mice. (B) Representative images of rabies virus targeting to the TRN, with arrows depicting the presence of starter cells. Scale bars: 300 μm. Inset scale bars: 100 μm. (C) Brain-wide input map of afferents extending to the FO and HO TRN subnetworks; multiple unpaired t tests with Holm-Šidák correction for multiple comparisons (****p < 0.0001). (D) Representative images of presynaptically labeled cells from cortical regions. Scale bars: 400 μm. (E) Representative images of presynaptically labeled cells from thalamic nuclei. Scale bars: 400 μm. (F) Representative images of retrogradely labeled L5 and L6 neurons in S1 from TRN rabies tracing. Scale bars: 250 μm. (G) Normalized L5/L6 input difference across the rostrocaudal axis. (H) Average normalized L5/L6 input difference across the cortex; two-tailed unpaired t test (*p < 0.05). Normalized difference = (# of L5 neurons − # of L6 neurons) / (# of L5 neurons + # of L6 neurons). (I) Average total synaptic convergence onto Spp1+ and Ecel1+ neurons across major brain regions; two-tailed unpaired t tests (*p < 0.05, **p < 0.01). The xy plot is depicted as mean ± SEM, and bar graphs are depicted as mean + SEM. n = 6 mice per group. See also Table S1 for corresponding brain region acronyms.

Figure 4.. FO and HO TRN subnetwork neurons display top-down corticothalamic and bottom-up thalamocortical input biases

(A–D) Percentage of presynaptic input cells and synaptic convergence from the sensory cortex; multiple unpaired t tests with Holm-Šidák correction for multiple comparisons (***p < 0.001). (E–H) Percentage of presynaptic input cells and synaptic convergence from the thalamus; multiple unpaired t tests with Holm-Šidák correction for multiple comparisons (***p < 0.001, ****p < 0.0001). (I) Synaptic convergence ratio of secondary over primary somatosensory and motor cortex inputs; two-tailed unpaired t test (*p < 0.05). (J) Synaptic convergence ratio of HO over FO thalamic inputs; two-tailed unpaired t test (****p < 0.0001). (K) Representative images of presynaptically labeled cells from the brain stem. Scale bars: 300 μm. (L) Percentage of presynaptic input cells and synaptic convergence from brain stem nuclei; multiple unpaired t tests with Holm-Šidák correction for multiple comparisons (***p < 0.001). Bar graphs are depicted as mean + SEM (n = 6 mice per group). See also Table S1 for corresponding brain region acronyms.

Figure 5.. Dysfunction of FO and HO TRN subnetworks reproduces distinct cortical EEG abnormalities

(A) Schematic of the injection strategy for expressing the inhibitory DREADD hM4D(Gi)-mCherry or mCherry control fluorophore in the TRN of each Cre line. (B) Example of DREADD or control mCherry expression in the FO Spp1- or the HO Ecel1-TRN neurons. Scale bars: 500 μm. (C) Schematic of the strategy for polysomnography and frontocortical EEG recording in awake mice. (D) Spectral power from spontaneous EEG in control or Spp1+ neuron-inhibited mice and total EEG power of control and Spp1+ neuron-inhibited mice; two-tailed unpaired t test (**p < 0.01). (E) ASSR-evoked gamma power of an auditory stimulus in control or Spp1+ neuron-inhibited mice; two-tailed unpaired t test (*p < 0.05). (F) Frontal mismatch negativity (MMN) of control or Spp1+ neuron-inhibited mice. (G) Spectral power from spontaneous EEG in control or Ecel1+ neuron-inhibited mice and total EEG power of control and Ecel1+ neuron-inhibited mice. (H) ASSR-evoked gamma power of an auditory stimulus in control or Ecel1+ neuron-inhibited mice. (I) Frontal MMN of control and Ecel1+ neuron-inhibited mice; two-tailed unpaired t test (*p < 0.05). The xy plots are depicted as mean ± SEM, and bar graphs are depicted as mean + SEM (n = 10 Spp1-Inhib and 9 control mice; n = 9 Ecel1-Inhib and 8 control mice).