Molecular Definitions of the Zebrafish Amygdaloid Complex. Precommissural (A–E), supra- and postcommissural (F–Q) telencephalon, and delineation of the dorsal pallium in its periventricular {(S+T, U1/left) and central (U1, right, U2)} portions. Pallium: Analyzing the distributions of GABA (A–C,H,K), parvalbumin (E,F,J,N), and GFP in the transgenic line Tg(vGlut2a:GFP)(A–C,H,I,K,M) allowed to discern the dorsomedial pallial zone, the putative core region of the pallial amygdala, from the hippocampal division (medial pallium/MP) and the teleostean dorsal pallium (DP) (A–C). DP lacks both vGlut2a-driven GFP and parvalbumin-positive neurons but shows parvalbumin positive fibers (F,J,H,U2). The strongly GFP-positive DM lacks parvalbumin expression (F,J). The mostly vGlut2a:GFP-free hippocampal division (MP) exhibits both parvalbumin-positive fibers and neurons in addition to GABA-neurons (C,F,J). Only at posteriormost sections, we found vGlut2a:GFP positive neurons in the MP (H). At anteriormost sections (A,B), the DP reaches the dorsalmost periventricular zone, which holds proliferative stem cells. More posteriorly (C,F,J), the DP is shown in its secondarily centralized position overgrown by both the pAmy and hippocampal division (medial pallium/MP). In addition, we found many large GABAergic interneurons in DP (A–C), as well as sparsely distributed GABAergic interneurons in the pAmy and MP (B,C). Subpallium: Dense population of GABAergic neurons define the subpallium (A–C,H,K). Within the subpallium, isl1-driven GFP labels striatopallidal and septal divisions as well as the BSTm (D1,D2,F,G). In contrast, GABAergic territories that lack isl1-driven GFP define the majority of subpallial amygdaloid territories. The anterior (precommissural) dorsalmost. GABAergic population (CeAa) together with the migrated GABAergic nuclei (CeAl = former Vc) predominantly form the zebrafish central amygdala [CeA in (A–C)]. The ventricular-close GABAergic territory is juxtaposed and contiguous with the former supracommissural (Vs) and postcommissural (Vp) nuclei of medial amygdala (MeA; former Vs/Vp). In addition, based on the lack of isl1-driven GFP in both anterior and posterior TH-positive dopaminergic and their previous reported projection to the hypothalamus, we consider as the anterior and posterior bed nucleus of the stria termalis (BSTa/BSTpd). The medial bed nucleus of the stria terminalis (BSTm) is the only isl1:GFP positive medial nucleus [BSTm in (F,G)]. We also found sparse dopaminergic neurons within the posteromedial amygdala (MeAp) that project into part of the nLOT (B2). Supra- and postcommissural extended medial amygdaloid nuclei (MeAc, MeAd, MeAp, BSTm, BSTpd) are defined by the presence of numerous calretinin-positive neurons (F,I,M) similar to the mammalian situation. In contrast, the CeAd that comprises numerous laterally displaced GABAergic neurons shows many parvalbumin-positive fibers and few parvalbumin neurons, but lacks calretinin neurons. The MeAp was formerly assigned to the teleostean subpallium, because there are GABAergic neurons in its vicinity (K). However, the otp-a (L) and calretinin (I,M) positive neurons that define the MeAp are likely glutamatergic and originate from the thalamic eminence according to our results. Discerning PMPa, IOP, and nLOT Comparing secondary olfactory projections in the transgenic line Tg(lhx2a:GAP-YFP)(J,N) with parvalbumin allowed us to identify primary olfactory pallial territories and their topological relationships. The distribution of secondary olfactory projections in comparison to parvalbumin and calretinin fibers identifies two olfactory integrative structures: (1) the parvalbumin-positive integrative olfactory pallium (IOP) (I,J,M,N) formerly misinterpreted as part of the medial pallium (“Dl/Dlp”) in zebrafish; (2) the largely parvalbumin-free (migrated) amygdaloid nucleus of the lateral olfactory tract (nLOT). The nLOT is a predominately glutamatergic nucleus that consists of both vGlut2a:GFP positive (anterior; C) and negative (posterior) domains as well as GAD67-positive [arrows in (O,P)] and gad1b-driven dsRed expressing [arrow in (Q)] GABAergic zones resembling the mammalian nLOT that consists of alternating GABAergic and glutamatergic layers. Previously, the nLOT has been mislabeled as the posterior division of the dorsal telencephalon (“Dp”) and mistakenly considered the piriform cortex homolog. The identification of the nLOT together with the piriform cortex homolog (IOP) solves their topological relationships within the complexly everted telencephalon (R indicates orientation of sections A–N). (F) Schematic figure shows orientation of section in the sagittal view. New hallmarks of the complexly everted zebrafish telencephalon – the rostral (superficial) versus posterior centralized parts of the dorsal pallium (DP). (S–U2) Analyzing distribution of parvalbumin versus vGlut2a-driven GFP shows that the dorsal pallium reaches the periventricular zone and includes its germinative layer of origin. Note, that DM and medial pallium (MP = dorsolateral (Dl) zone of the pallium) overgrow the dorsal pallium at posterior sections at the point of convergence (circled area). (V) Schematics of a zebrafish telencephalon from dorsal perspective illustrating levels of cross sections.
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