Molecular Diversity of Neuron Types in the Salamander Amygdala and Implications for Amygdalar Evolution

Abstract

The amygdala is a complex brain structure in the vertebrate telencephalon, essential for regulating social behaviors, emotions, and (social) cognition. In contrast to the vast majority of neuron types described in the many nuclei of the mammalian amygdala, little is known about the neuronal diversity in non-mammals, making reconstruction of its evolution particularly difficult. Here, we characterize glutamatergic neuron types in the amygdala of the urodele amphibian Pleurodeles waltl. Our single-cell RNA sequencing data indicate the existence of at least ten distinct types and subtypes of glutamatergic neurons in the salamander amygdala. These neuron types are molecularly distinct from neurons in the ventral pallium (VP), suggesting that the pallial amygdala and the VP are two separate areas in the telencephalon. In situ hybridization for marker genes indicates that amygdalar glutamatergic neuron types are located in three major subdivisions: the lateral amygdala, the medial amygdala, and a newly defined area demarcated by high expression of the transcription factor Sim1. The gene expression profiles of these neuron types suggest similarities with specific neurons in the sauropsid and mammalian amygdala. In particular, we identify Sim1+ and Sim1+ Otp+ expressing neuron types, potentially homologous to the mammalian nucleus of the lateral olfactory tract (NLOT) and to hypothalamic-derived neurons of the medial amygdala, respectively. Taken together, our results reveal a surprising diversity of glutamatergic neuron types in the amygdala of salamanders, despite the anatomical simplicity of their brain. These results offer new insights on the cellular and anatomical complexity of the amygdala in tetrapod ancestors.

Keywords: Amphibian; Amygdala; Brain evolution; Neuron types.

Fig. 1.. Boundaries of the P. waltl amygdala territories.

(a) UMAP plot of 10,125 single-cell transcriptomes of telencephalic glutamatergic neurons, derived from the neuronal dataset presented in Woych et al. 2022 (highlighted in the boxed area). Clusters are annotated by the inferred pallial regions. (b) UMAP plots showing single cells colored according to the expression of Slc17a7 (cortical pallium and medial amygdala), Slc17a6 (glutamatergic amygdala and parts of the ventral and medial pallium), Sox6 (ventral pallium) and Penk (parts of the glutamatergic amygdala and medial and dorsal pallium). (c) Dot plot showing the expression of key marker genes defining ventral pallium and glutamatergic amygdala. (d) Left: schematic of the top view of the salamander telencephalon (gray) with dashed lines indicating the optical sectioning planes for images on the right. Right: optical coronal sections after whole-mount single HCR ISH, clearing and light sheet imaging for Slc17a6 (top) and Gad1 (bottom). Arrows in d5’–d7’ indicate the lateral cellular prominence. Scale bars represent 200 um. (e) Optical coronal sections after whole-mount double HCR ISH, clearing and light sheet imaging for Sox6 and Penk. (f-g) Left: schematic of a coronal section with ventral pallium and lateral amygdala highlighted. Right: Double HCR ISH of Slc17a6 and Gad1, or Sox6 and Penk indicating the borders of the lateral amygdala with the ventral pallium dorsally and the striatum ventrally. Scale bars represent 50 um. Abbreviations: aOB, accessory olfactory bulb; BST, bed nucleus of the stria terminalis; CeA, central amygdala; DP, dorsal pallium; LA, lateral amygdala; LPp, posterior lateral pallium; MeA, medial amygdala; mOB, main olfactory bulb; MP, medial pallium; MT, mitral/tufted cells; NAc, nucleus accumbens; NLOT-like, nucleus of the olfactory tract-like; OBG, olfactory bulb GABAergic; POA, preoptic area; POE, postolfactory eminence; pThe, prethalamic eminence; Sep, septum; Str, striatum; TEGLU, telencephalic glutamatergic; VPa, anterior ventral pallium; VPp, posterior ventral pallium.

Fig. 2.. Neuronal heterogeneity in the P. waltl pallial amygdala.

(a) UMAP plot of telencephalic glutamatergic neurons with cells from the amygdala highlighted in yellow. The magnified UMAP on the bottom shows the annotation of the different neuron types in the glutamatergic amygdala. (b) UMAP plots showing single cells colored according to the expression of Lhx9, Penk, Tac1, Meis1 and Sim1, illustrating the cellular diversity of the P. waltl amygdala. (c) Dot plot showing the expression of key marker genes defining distinct neuron types in the glutamatergic amygdala. (d) From left to right: schematic of a coronal section through the P. waltl telencephalon, expression of Penk, Lhx9, Meis1, Tac1 and Fezf2 in the lateral amygdala, and of Sim1 and Syt2 in the NLOT-like area. Boxes in upper panels indicate magnified regions in lower panels. Arrows in lower panels indicate the lateral cellular prominence. Scale bars represent 200 um in upper panels and 100 um in lower panels. Abbreviations: BST, bed nucleus of the stria terminalis; CeA, central amygdala; DP, dorsal pallium; lpA, lateral pallial amygdala; LA, lateral amygdala; LPp, posterior lateral pallium; MP, medial pallium; NLOT-like, nucleus of the olfactory tract-like; TEGLU, telencephalic glutamatergic; VPp, posterior ventral pallium.

Fig. 3.. The hypothalamus-amygdalar corridor in P. waltl.

(a) Dot plot showing differentially expressed genes in NLOT-like cells and the MeA. (b) Left: schematic of the side view of the salamander telencephalon (gray) with dashed lines indicating the optical sectioning plane for images on the right. Right: optical horizontal sections after whole-mount HCR ISH, clearing and light sheet imaging for Sim1 and Otp. Pink and teal arrowheads indicate the medial amygdala and the NLOT-like area, respectively. Dashed line in b3 indicates the sectioning plane for D. Scale bars represent 200 um. (c) 3D rendering (Imaris viewer) of Otp expression in the P. waltl brain. Pink arrow indicates a continuum of Otp+ cells from the periventricular hypothalamus to the medial amygdala. Scale bar represents 500 um. (d) From left to right: schematic of a coronal section at the level of the caudal telencephalon, expression of Gad1, presence of FOXG1 protein, and expression of Sim1 and Otp. Pink arrowheads indicate the medial amygdala. Scale bars represent 200 um. Abbreviations: LA, lateral amygdala; H, hypothalamus; MeA, medial amygdala; NLOT-like, nucleus of the olfactory tract-like; POA, preoptic area; pThe, prethalamic eminence; TEGLU, telencephalic glutamatergic.