Yoland Smith

The ventral motor and the centromedian (CM) nuclei receive prominent GABAergic inputs from the basal ganglia, massive glutamatergic projections from motor cortices and significant GABAergic afferents from the reticular thalamic nucleus. There is strong evidence that disrupted processing of information through these connections may contribute to the pathophysiology of the basal ganglia-thalamocortical loop in Parkinson’s disease (PD). To further assess potential ultrastructural changes in synaptic connectivity and mitochondrial integrity that may contribute to these network dysfunctions, we used a 3D electron microscopic approach to determine whether the pattern of synaptic innervation and morphological integrity of dendritic mitochondria are altered in the basal ganglia-receiving parvocellular ventral anterior nucleus (VApc) and CM neurons of MPTP-treated parkinsonian monkeys. Three main conclusions can be drawn from our findings: (1) Although the overall pattern of synaptic innervation of VApc and CM neurons is not altered in parkinsonian monkeys, the volume of putative corticothalamic terminals is significantly increased in both nuclei, (2) the prevalence of corticothalamic terminals in contact with distal dendrites is several orders of magnitude higher in VApc than CM in both control and parkinsonian monkeys, (3) the complexity and ultrastructural integrity of dendritic mitochondria is altered in CM, but not in the VApc, of parkinsonian monkeys. These findings lay the foundation for future studies of changes in cortical neuromodulation of VApc and CM neurons in parkinsonism and suggest that mitochondrial defects may contribute to the degeneration of CM neurons in PD. Significance Statement: Parkinson’s disease is associated with complex functional changes in the neuronal communication between the basal ganglia, thalamus, and cerebral cortex. In this study, Masilamoni et al. used a high-resolution 3D electron microscopy approach to demonstrate morphological changes of corticothalamic terminals in both the ventral motor and centromedian (CM) thalamic nuclei of parkinsonian monkeys. They also provide evidence for decreased complexity and pathology of dendritic mitochondria in CM. These results suggest that morphological changes in the corticothalamic system may contribute to the pathophysiology of the cortico-thalamo-cortical loop in parkinsonism and that mitochondrial pathology may be one of the mechanisms that underlie CM neuronal loss in PD.

The right central amygdala (CeA) is involved in the processing of emotional‐affective dimensions of pain. Through the spino‐parabrachio‐amygdaloid pain pathway, the CeA receives nociceptive information from calcitonin gene‐related peptide (CGRP) neurons in the parabrachial nucleus. Recent evidence indicates that the glutamate delta 1 (GluD1) receptor, an atypical ionotropic glutamate receptor that largely functions as a synaptogenic molecule involved in the formation and maintenance of synapses, regulates this projection in mice. Despite its strong cellular expression, little is known about the subsynaptic localization of GluD1, and its potential interaction with CGRP terminals, in CeA neurons. To address this issue and further characterize the ultrastructure and synaptic connectivity of CGRP terminals across species, we used single and double immuno‐electron microscopy techniques in mice and monkeys. For all ultrastructural parameters examined, no species difference was found. In both species, CGRP‐positive (CGRP+) terminals formed symmetric or asymmetric synapses with dendrites, symmetric synapses with soma, and less commonly, asymmetric synapses with spines. Almost 90% of CGRP+ terminals forming asymmetric or symmetric synapses expressed vGluT2 immunoreactivity confirming the glutamatergic and peptidergic nature of this projection. Confocal microscopic analyses confirmed the perisomatic association between CGRP+ and GluD1+ puncta in mice and monkeys. At the ultrastructural level, GluD1 was expressed in the core of symmetric axo‐dendritic and axo‐somatic synapses and perisynaptic to asymmetric synapses formed by CGPR+ terminals. These findings demonstrate that the CGRP+ PB‐CeA projection mediates its effects through a heterogeneous population of terminals that display strong synaptic relationships with GluD1 in rodents and primates.

The GluD1 receptor has many unusual features including expression at both excitatory and inhibitory synapses and a ligand binding domain capable of binding both D-serine and GABA. We have previously demonstrated that striatal GluD1 is critical for the regulation of behavioral flexibility, a phenotype dependent on the cholinergic system. Here, we found that GluD1 is enriched postsynaptically at cholinergic synapses in the mouse and monkey dorsal striatum. Further, loss of GluD1 reduces the abundance of cholinergic terminals, excitatory responses at cholinergic synapses as well as muscarinic receptor-induced plasticity. In addition, optogenetic stimulation of cholinergic interneurons or puff-application of ACh, in the presence of cholinergic and AMPA/GABA _A receptor blockers, produced current responses in medium spiny neurons (MSNs) that were sensitive to the GluD1-channel blocker NASPM. These responses were absent in GluD1 KO and overexpression of GluD1 on KO background rescued Ach puff-induced currents suggesting potential conductance via GluD1. Finally, using GluD1-Cbln1 interaction assay as an indirect method to evaluate ligand binding interaction, we found that ACh can bind GluD1 and induce conformational changes. A similar ACh-induced conformational change was observed for GluD2 in the cell binding assay. Importantly, molecular dynamics simulations and mutagenesis analysis demonstrated that ACh binding orientation in the GluD1 ligand binding domain is different from D-serine and GABA. Overall, our results identified an unprecedented feature of GluD1 in the regulation of cholinergic synapses.

The glutamate delta 1 receptor (GluD1) represents a unique subtype of ionotropic glutamate receptors that is strongly expressed in the mammalian striatum. Disruptions of the GRID1 gene, which encodes GluD1, have been associated with neuropsychiatric disorders, including schizophrenia and autism spectrum disorder; however, the role of GluD1 in the brain remains poorly understood. Previous studies in mice have demonstrated that the knockout of striatal GluD1 led to fear-conditioning deficits and depressive-like behaviors. Furthermore, these mice exhibited reduced excitatory input to the striatum due to a loss of thalamostriatal innervation, whereas corticostriatal innervation was unaffected. In this study, we examined whether changes in synapse morphology contribute to the observed functional deficits. We found that the ablation of GluD1 does not affect synaptic targeting patterns of corticostriatal and thalamostriatal terminals, using transmission electron microscopy. We further utilized three-dimensional reconstruction to obtain quantitative data on synapse ultrastructure and found no significant changes in corticostriatal and thalamostriatal synaptic components, including the presynaptic terminal volume, postsynaptic density area and morphology, and postsynaptic dendritic spine volume. These findings support a model in which GluD1 regulates input-specific circuit organization and synaptic connectivity rather than the structural morphology of individual synapses.

The glutamate delta 1 receptor (GluD1) remained largely unexplored since its cloning three decades ago because it lacked typical ligand‐gated ion channel activity. In the last decade, much progress has been made in identifying its potential function. This research has been greatly enhanced by the development of specific tools to determine receptor expression and distribution and genetic mouse models to explore region specific roles in regulating circuits and behavior. Major strides have also been taken in understanding the structure–function of the receptor. These studies demonstrate that GluD1 has many distinctive characteristics including synaptogenic activity at both excitatory and inhibitory synapses, the ability of the ligand‐binding domain to bind not only D‐serine but also GABA, its unique structural arrangement among the ionotropic glutamate receptor family in relation to domain swapping and the ability to induce tonic currents in the native system. Studies have also identified its role in regulating the postsynaptic content of AMPA and NMDA receptors and synaptic plasticity. Finally, human genetic studies revealed the relationship of GluD1 with neuropsychiatric disorders, including schizoaffective disorders and intellectual disability, which is consistent with the phenotypes observed in mice upon GluD1 ablation. The role of GluD1 is also becoming evident in neurological disorders, particularly chronic pain. Thus, GluD1 has quickly emerged as a receptor with multifaceted roles in physiology and pathology.

The lateral and capsular divisions of the central amygdala (CeLC) is a key region involved in the processing of emotional-affective dimensions of pain. In the spino-parabrachio-amygdaloid pain pathway, the CeLC receives nociceptive information from calcitonin gene-related peptide (CGRP) neurons in the parabrachial nucleus. Recent evidence indicates that glutamate delta receptor 1 (GluD1) regulates this projection in mice. GluD1 is an atypical ionotropic glutamate receptor that largely functions as a synaptogenic molecule involved in the formation and maintenance of synapses. Despite its strong cellular expression, little is known about the subsynaptic localization of GluD1 and its potential interaction with CGRP terminals in CeLC neurons. To address this issue, we used single and double immuno-electron microscopy techniques in rodents and monkeys. In both species, CGRP-positive (CGRP+) terminals formed symmetric and asymmetric synapses with dendrites, symmetric synapses with soma, and less commonly, asymmetric synapses with spines. Approximately 80% of CGRP+ terminals forming clear symmetric synapses expressed vGluT2 immunoreactivity and none were immunoreactive for GABA, confirming the glutamatergic nature of this projection and suggesting that some PB-CGRP terminals may modulate transmission to the CeLC in a peptidergic manner. GluD1 was expressed in the core of symmetric axo-dendritic and axo-somatic synapses and peri-synaptic to asymmetric synapses. These findings show that the ascending CGRP+PB-CeLC projection mediates its effects through a heterogenous population of terminals that display strong synaptic relationships with GluD1 in both mice and monkeys.

Glutamate delta receptor 1 (GluD1) is a unique synaptogenic molecule expressed at excitatory and inhibitory synapses. The lateral habenula (LHb), a subcortical structure that regulates negative reward prediction error and major monoaminergic systems, is enriched in GluD1. LHb dysfunction has been implicated in psychiatric disorders such as depression and schizophrenia, both of which are associated with GRID1, the gene that encodes GluD1. Thus, disruption in GluD1 synaptic signaling may contribute to LHb dysfunction and the pathophysiology of LHb‐associated disorders. Despite its strong cellular expression, little is known about the subsynaptic and subcellular localization of GluD1 in LHb neurons. Given that GluD1 is involved in the development and/or regulation of glutamatergic and GABAergic synapses in various brain regions, a detailed map of GluD1 synaptic localization is essential to elucidate its role in the LHb. To address this issue, we used immunoelectron microscopy methods in rodents and monkeys. In both species, GluD1 immunoreactivity was primarily expressed in dendritic profiles, with lower expression in somata, spines, and glial elements. Pre‐ and post‐embedding immunogold experiments revealed strong GluD1 expression in the core of symmetric GABAergic synapses. Albeit less frequent, GluD1 was also found at the edges (i.e., perisynaptic) of asymmetric, putative glutamatergic synapses. Through the combination of anterograde tracing with immunogold labeling in rats, we showed that axon terminals from the entopeduncular nucleus and the lateral hypothalamus express postsynaptic GluD1 immunolabeling in the LHb. Our findings suggest that GluD1 may play a critical role in modulating GABAergic transmission in the rodent and primate LHb.

Regulator of G protein signalling 14 (RGS14) is a multifunctional signalling protein that serves as a natural suppressor of synaptic plasticity in the mouse brain. Our previous studies showed that RGS14 is highly expressed in postsynaptic dendrites and spines of pyramidal neurons in hippocampal area CA2 of the developing mouse brain. However, our more recent work with monkey brain shows that RGS14 is found in multiple neuron populations throughout hippocampal area CA1 and CA2, caudate nucleus, putamen, globus pallidus, substantia nigra and amygdala. In the mouse brain, we also have observed RGS14 protein in discrete limbic regions linked to reward behaviour and addiction, including the central amygdala and the nucleus accumbens, but a comprehensive mapping of RGS14 protein expression in the adult mouse brain is lacking. Here, we report that RGS14 is more broadly expressed in mouse brain than previously known. Intense RGS14 staining is observed in specific neuron populations of the hippocampal formation, amygdala, septum, bed nucleus of stria terminalis and ventral striatum/nucleus accumbens. RGS14 is also observed in axon fibre tracts including the dorsal fornix, fimbria, stria terminalis and the ventrohippocampal commissure. Moderate RGS14 staining is observed in various other adjacent regions not previously reported. These findings show that RGS14 is expressed in brain regions that govern aspects of core cognitive functions such as sensory perception, emotion, memory, motivation and execution of actions and suggest that RGS14 may serve to suppress plasticity and filter inputs in these brain regions to set the overall tone on experience‐to‐action processes.

Astrocytes intricately weave within the neuropil, giving rise to characteristic bushy morphologies. Pioneering studies suggested that primate astrocytes are more complex due to increased branch numbers and territory size compared to rodent counterparts. However, there has been no comprehensive comparison of astrocyte morphology across species. We employed several techniques to investigate astrocyte morphology and directly compared them between mice and rhesus macaques in cortical and subcortical regions. We assessed astrocyte density, territory size, branching structure, fine morphological complexity, and interactions with neuronal synapses using a combination of techniques, including immunohistochemistry, adeno‐associated virus–mediated transduction of astrocytes, diOlistics, confocal imaging, and electron microscopy. We found significant morphological similarities between primate and rodent astrocytes, suggesting that astrocyte structure has scaled with evolution. Our findings show that primate astrocytes are larger and more numerous than those in rodents but contest the view that primate astrocytes are morphologically far more complex.