Ultrastructural localization of glutamate delta receptor 1 in the lateral habenula and central amygdala of mice and monkeys Open Access
Choi, Diane (Spring 2025)
Abstract
The delta family of ionotropic glutamate receptors (GluDs) are atypical in that they do not bind glutamate nor do they display typical ligand-induced ionotropic activity. Instead, GluDs form trans-synaptic complexes that are crucial for the formation and maintenance of synapses. GluD1 has widespread brain expression, and accumulating evidence demonstrates that depending on the synapse type, activity, and location, GluD1 can exhibit diverse functions beyond synaptogenesis. A better understanding of GluD1’s potential role in a specific brain region requires a detailed map of its subcellular and subsynaptic localization. Thus, this dissertation utilizes immuno-electron microscopy methods to investigate the ultrastructural localization of GluD1 in two previously unexplored regions: the lateral habenula (LHb) and the central amygdala (CeA).
In the LHb, GluD1 immunoreactivity was primarily expressed in dendritic profiles, with lower expression in somata, spines, and glia. We observed strong GluD1 expression in the core of symmetric GABAergic synapses, suggesting GluD1 may be associated with inhibitory transmission in the LHb. Less frequently, GluD1 was also found at the edges of asymmetric, putative glutamatergic, synapses. A combination of anterograde tracing and immunogold labeling studies showed that a significant proportion of lateral hypothalamus and entopeduncular nucleus afferents express postsynaptic GluD1 localization in the rat LHb.
Through the spino-parabrachio-amygdaloid pain pathway, the lateral capsular subdivisions of the CeA (CeLC) receives direct inputs from calcitonin gene-related peptide (CGRP)-containing parabrachial (PB) neurons. Our immunolabeling studies demonstrate that CGRP+ terminals form symmetric and asymmetric synapses with dendrites, symmetric synapses with soma, and asymmetric synapses with spines in the CeLC. Most CGRP+ terminals forming symmetric synapses expressed vGluT2 immunoreactivity and none were immunoreactive for GABA. GluD1 was expressed in the core of symmetric axo-dendritic and axo-somatic synapses and peri-synaptic to asymmetric synapses formed by PB-CGRP+ terminals. These results show that the CGRP+PB-CeLC projection mediates its effects through a heterogenous population of terminals that display strong synaptic relationships with GluD1.
Altogether, these findings provide a comprehensive analysis of the subsynaptic localization of GluD1 in the rodent and primate LHb and CeA, establishing a strong foundation for future functional, behavioral, and pharmacological studies investigating the role of GluD1 in the mammalian brain.
Table of Contents
List of Figures and Tables [1]
List of Abbreviations [3]
Chapter 1: Introduction [8]
1.1 Glutamate delta receptors: paradoxes of the ionotropic glutamate receptor superfamily [9]
1.1.1 Known ligands of GluDs [10]
1.1.2 Interactions between GluDs and other receptors subtypes [12]
1.1.3 The trans-synaptic complex [13]
1.2 Expression and roles of GluDs in the brain [16]
1.2.1 GluD2 in the cerebellum [16]
1.2.2 GluD1 at excitatory synapses [17]
1.2.3 GluD1 at inhibitory synapses [18]
1.2.4 Non-synaptogenic functions of GluD1 [20]
1.3 GluDs in brain diseases [22]
1.4 Knowledge gap [25]
1.5 The lateral habenula [28]
1.5.1 Afferents projections to the LHb [28]
1.5.2 Efferent projections of the LHb [32]
1.5.3 Lateral habenula-associated behaviors and disorders [33]
1.6 The central amygdala [35]
Chapter 2: Ultrastructural localization of glutamate delta receptor 1 in the rodent and primate lateral habenula [39]
2.1 Abstract [40]
2.2 Introduction [41]
2.3 Materials & Methods [43]
2.3.1 Animals [43]
2.3.2 Stereotaxic surgery and viral delivery [43]
2.3.3 Tissue preparation [44]
2.3.4 Antibodies [44]
2.3.5 Light Microscopy (LM) Immunohistochemistry [45]
2.3.6 Electron Microscopy (EM) Immunohistochemistry [46]
2.3.7 Data Analysis [48]
2.4 Results [54]
2.4.1 GluD1 is primarily expressed in dendritic profiles in the LHb [54]
2.4.2 Immunogold localization of GluD1 in the LHb [58]
2.4.3 GluD1 is associated with inhibitory GABAergic terminals in the LHb [63]
2.4.4 GluD1 expression at EPN vs LH afferents [66]
2.5 Discussion [73]
2.5.1 Subcellular and subsynaptic localization of GluD1 in the LHb [74]
2.5.2 GluD1 is involved in inhibitory transmission in the LHb [75]
2.5.3 GluD1 is associated with both LH and EPN afferents to the LHb [76]
2.5.4 GluD1 and LHb-associated disorders [78]
Concluding Remarks [78]
Chapter 3: Subsynaptic expression of calcitonin gene-related peptide-containing projections from the parabrachial region in the central amygdala of mice and monkeys [80]
3.1 Abstract [81]
3.2 Introduction [82]
3.3 Materials & Methods [83]
3.3.1 Animals [83]
3.3.2 Antibodies [84]
3.3.3 Light Microscopy (LM) Immunohistochemistry [85]
3.3.4 Electron Microscopy (EM) Immunohistochemistry [86]
3.3.5 Data Analysis [88]
3.4 Results [92]
3.4.1 Ultrastructural features of CGRP+ terminals in the CeLC [92]
3.4.2 Postsynaptic targets of CGRP+ terminals in the CeLC [96]
3.4.3 CGRP+ terminals form symmetric or asymmetric synapses in the CeLC [96]
3.4.4 CGRP+ terminals largely express vGluT2 in the CeLC [97]
3.4.5 GluD1 expression at synapses formed by CGRP+ terminals in the CeLC [101]
3.4.6 Lack of GABA expression in CGRP+ terminals [104]
3.5 Discussion [108]
3.5.1 Transmitter content of CGRP+ terminals forming symmetric synapses [109]
3.5.2 Could CGRP be the primary transmitter released at some PB-CeLC synapses? [112]
3.5.3 Interactions between CGRP and GluD1 in the CeLC [113]
3.6 Concluding Remarks [114]
Chapter 4: Discussion [116]
4.1 Summary of findings [117]
4.2 Limitations [119]
4.3 Future Directions [121]
4.3.1 Anatomical studies [121]
4.3.2 Electrophysiology studies [123]
4.3.3 Behavioral studies [124]
4.4 Speculations: how GluD1 fits into the PSD architecture [125]
4.5 GluD1 is an attractive pharmacological target [128]
4.6 Conclusion [129]
References [131]
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