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.

Striatal-Enriched Protein Tyrosine Phosphatase (STEP) constrains synaptic potentiation by dephosphorylating postsynaptic substrates, but its presynaptic role has remained unclear. Here, we identify a previously unrecognized function of STEP in regulating axonal differentiation and synapse assembly. Genetic and pharmacological manipulation of STEP in vivo and in vitro show that STEP limits presynaptic maturation by restricting synaptic vesicle protein clustering along developing hippocampal axons. Using a reconstituted circuit-on-a-chip we show that loss of presynaptic STEP is sufficient to significantly increase the number of axodendritic synapses. Functional imaging further revealed that the increased synaptic puncta observed in STEP KO neurons actively undergo depolarization-evoked vesicle exocytosis, representing bona fide functional synapses. Multielectrode array recordings reveal that STEP deletion increases neuronal excitability, and network synchrony, hallmarks of enhanced presynaptic efficacy. Mechanistically, these effects reflect sustained phosphorylation of STEP promoting presynaptic assembly and release competence. Importantly, inhibiting STEP also rescues presynaptic differentiation defects in Fmr1 KO neurons, implicating aberrant STEP signaling in Fragile X-associated synaptic pathology. Thus, STEP serves as a phosphatase gatekeeper that restrains presynaptic differentiation and neurotransmission, and its inhibition may offer a therapeutic strategy to correct synaptic deficits in Fragile X Syndrome.

Glutamate spillover from excitatory synapses modulates neighboring inhibitory synapses, yet the ultrastructural organization of the major glutamate transporter GLT-1 at these sites remains poorly defined. Using quantitative pre-embedding electron microscopy in rat and human cortex, we found that GLT-1-positive astrocytic leaflets (ALs) were frequently juxtaposed to morphologically identified symmetric synapses, with similar prevalence across axo-somatic, proximal axo-dendritic, and distal axo-dendritic subtypes. Because inhibitory synapses are embedded in a dense excitatory neuropil, we applied distance-based phenotyping relative to the nearest asymmetric synapse to define symmetric-associated GLT-1+ ALs. Within this population, distal axo-dendritic symmetric synapses showed shorter AL-to-synaptic-edge distances and were embedded in a tighter local excitatory microenvironment. Post-embedding immunogold further showed that GLT-1 was enriched at the plasma membranes of ALs and localized extrasynaptically relative to symmetric synapses. Consistently, symmetric-associated membrane GLT-1 and closely spaced GLT-1/α2 couples (with an interdistance ≤ 50 nm) were preferentially localized within 1000 nm of distal symmetric synapses compared to proximal. Similar organizational features of membrane GLT-1/α2 couples were observed in human cortex. These findings identify a subtype-dependent extrasynaptic astrocytic GLT-1 organization at cortical inhibitory synapses and provide a morphological framework for glutamate-dependent modulation of inhibitory signaling.

Across species, simple olfactory operations primarily utilize long-range excitatory interconnections. More complex tasks, such as fine odor discrimination and plasticity, rely on local inhibitory circuits. In the olfactory bulb, previous studies focused on measuring activity patterns following broad activation of sensory inputs and highlighted the central role canonical dendrodendritic circuits play in sculpting principal cell firing patterns. The primary goal of this study is to define the inhibitory local circuits activated by focal stimulation of a single olfactory bulb glomerulus in olfactory bulb slices prepared from rats of either sex. This approach enables dissection of circuit mechanisms normally obscured by the multiglomerular activation patterns natural odorants elicit. Using glomerular stimulation, we identified two distinct local circuits that contribute to sensory-evoked inhibition and operate at different time periods. We further show these circuits are differentially affected by adenosine A1 receptor (A1R) activation. We utilize multiple experimental approaches to show early-phase mitral cell inhibition arises through excitatory synapses formed by axon collaterals of tufted cells onto the proximal dendrites of GABAergic granule cells. Blockade of NMDARs greatly attenuates dendrodendritic inhibition but fails to block the large initial phase of mitral cell inhibition. In contrast, activation of A1Rs attenuates both early-phase mitral cell inhibition and the underlying axo-dendritic excitation of granule cells. We conclude that two independent bulbar excitatory circuits converge on granule cells to generate temporally distinct phases of sensory-evoked inhibition, with A1R activation revealing a novel tufted cell-mediated pathway that contributes to early-phase responses.

Mesenchymal stromal cells (MSCs) have been proposed as a promising therapeutic tool for traumatic disorders of the nervous system, due to their neuro-regenerative properties. These outcomes have been extensively connected to paracrine mechanisms. Because synaptic reintegration of injured axons into existing circuitry is required for functional recovery, we investigated the synaptogenic potential of MSC secretome. Using neuronal primary cultures, hippocampal organotypic slices, and human cortical brain organoids, we found that MSC secretome induces presynaptic differentiation. Moreover, the number of axodendritic synapses also increases after treatment with MSC secretome. This increase in synapses correlates with an enhancement in synaptic activity, revealing that newly formed synapses are functional. Finally, we unraveled the mechanism underlying this effect and identified thrombospondin-1 (TSP1) as the major synaptogenic factor in MSC secretome. Together, our findings demonstrate that MSC secretome displays synaptogenic properties, pointing to a potential role as a cell-free therapy to restore lost synaptic connectivity.

Understanding the evolution of the bilaterian brain requires a detailed exploration of the precise nature of cellular and subcellular differences between related species. We undertook an electron micrographic reconstruction of the brain of the predatory nematode Pristionchus pacificus and compared the results with the brain of Caenorhabditis elegans, which diverged at least 100 million years ago. We revealed changes in neuronal cell death, neuronal cell position, axodendritic projection patterns, and synaptic connectivity of homologous neurons that display no obvious changes in overall neurite morphology and projection patterns. These multiscale patterns of evolutionary changes show no bias to specific brain regions or neuron types.

Olfactory bulb glomeruli have a complex organization that includes axodendritic synapses between olfactory sensory neurons (OSNs) and excitatory mitral cells (MCs) and tufted cells (TCs), as well as dendrodendritic synapses between MCs/TCs and GABAergic periglomerular cells (PGCs). MCs also receive excitatory signals from one subclass of TCs, the external tufted cells (eTCs). While these signals are driven by glutamate released from eTC dendrites, they appear not to reflect direct eTC > MC synaptic connections but rather "spill-over" of glutamate released at eTC > PGC synapses acting on nearby MC dendrites. Here, we used serial section electron microscopy images of rat olfactory bulb glomeruli with biocytin-labeled MC and eTC dendrites to evaluate potential ultrastructural underpinnings of "extrasynaptic" signaling. We compared the environment around eTC > PGC synapses with that of MC > PGC synapses using several quantitative measures and, as a further point of comparison, also evaluated axodendritic OSN > MC and OSN > eTC synapses. Across the four synapse types, one unique feature of eTC > PGC synapses was their much closer distance to the nearest dendrites of excitatory cells (including MCs), averaging ∼160 nm. In contrast, astroglial processes were positioned quite far away from eTC > PGC synapses, with a mean distance of ∼500 nm. These distance values would suggest that glutamate released from eTC > PGC synapses may access nearby excitatory dendrites without interference from glial glutamate transporters, thus providing an ultrastructural basis for extrasynaptic signaling. Our battery of ultrastructural measures, which included brick analyses, also supported a long-standing model for the organization of glomeruli in which OSN and dendrodendritic synapses are segregated into discrete compartments.

Kainic acid (KA) model of epilepsy is a reliable tool to study temporal lobe epilepsy (TLE), the most common type of partial epilepsy in adults. Substantial body of data suggest that the KA-induced status epilepticus (SE) leads to several molecular and structural changes in the hippocampus, including sclerosis, sprouting of mossy fiber, reorganization of inter-neuronal networks, alterations in neuropeptide signaling, gliosis, and synaptic transmission dysregulation. However, no details on the ultrastructural changes, especially in relationship to synapses are available. This information is important in providing a comprehensive understanding of subtle changes that occur in this debilitating disease. Thus, in this study, applying electron-microscopic morphometric analysis, we evaluated the ultrastructural effects of KA on the CA1 region of the hippocampus, an area intimately involved in SE. The total number of synaptic vesicles (SVs), the number of docking SVs, the length of synapse active zone (AZ) and the number and area of presynaptic and postsynaptic mitochondria in axo-dendritic (excitatory) synapses were measured at 24 h, and 8 and 21 days after KA administration. Results indicate a decrease in the total number and docking of SVs, an increase in the length of AZ and the number and area of presynaptic and postsynaptic mitochondria, which were more prominent at 8 days after KA injection. The findings suggest a time-dependent ultrastructural changes in CA1 region of the hippocampus in an animal model of focal epilepsy.

In the mammalian cochlea, sensory hair cells are crucial for the transduction of acoustic stimuli into electrical signals, which are then relayed to the central auditory pathway via spiral ganglion neuron (SGN) afferent dendrites. The SGN output is directly modulated by inhibitory cholinergic axodendritic synapses from the efferent fibers originating in the superior olivary complex. When the adult cochlea is subjected to noxious stimuli or aging, the efferent system undergoes major rewiring, such that it reestablishes direct axosomatic contacts with the inner hair cells (IHCs), which occur only transiently during prehearing stages of development. The trigger, origin, and degree of efferent plasticity in the cochlea remains largely unknown. Using functional and morphological approaches, we demonstrate that efferent plasticity in the adult cochlea occurs as a direct consequence of mechanoelectrical transducer current dysfunction. We also show that, different from prehearing stages of development, the lateral olivocochlear - but not the medial olivocochlear - efferent fibers are those that form the axosomatic synapses with the IHCs. The study also demonstrates that in vivo restoration of IHC function using AAV-Myo7a rescue reestablishes the synaptic profile of adult IHCs and improves hearing, highlighting the potential of using gene-replacement therapy for progressive hearing loss.

Several studies suggest that hearing loss results in changes in the balance between inhibition and excitation in the inferior colliculus (IC). The IC is an integral nucleus within the auditory brainstem. The majority of ascending pathways from the lateral lemniscus (LL), superior olivary complex (SOC), and cochlear nucleus (CN) synapse in the IC before projecting to the thalamus and cortex. Many of these ascending projections provide inhibitory innervation to neurons within the IC. However, the nature and the distribution of this inhibitory input have only been partially elucidated in the rat. The inhibitory neurotransmitter, gamma aminobutyric acid (GABA), from the ventral nucleus of the lateral lemniscus (VNLL), provides the primary inhibitory input to the IC of the rat with GABA from other lemniscal and SOC nuclei providing lesser, but prominent innervation. There is evidence that hearing related conditions can result in dysfunction of IC neurons. These changes may be mediated in part by changes in GABA inputs to IC neurons. We have previously used gene micro-arrays in a study of deafness-related changes in gene expression in the IC and found significant changes in GAD as well as the GABA transporters and GABA receptors (Holt 2005). This is consistent with reports of age and trauma related changes in GABA (Bledsoe et al., 1995; Mossop et al., 2000; Salvi et al., 2000). Ototoxic lesions of the cochlea produced a permanent threshold shift. The number, intensity, and density of GABA positive axon terminals in the IC were compared in normal hearing and deafened rats. While the number of GABA immunolabeled puncta was only minimally different between groups, the intensity of labeling was significantly reduced. The ultrastructural localization and distribution of labeling was also examined. In deafened animals, the number of immuno gold particles was reduced by 78 % in axodendritic and 82 % in axosomatic GABAergic puncta. The affected puncta were primarily associated with small IC neurons. These results suggest that reduced inhibition to IC neurons contribute to the increased neuronal excitability observed in the IC following noise or drug induced hearing loss. Whether these deafness diminished inhibitory inputs originate from intrinsic or extrinsic CNIC sources awaits further study.

The aim of this study is to investigate whether the dorsal claustrum receives afferent input from the intralaminar thalamic nuclei - centromedian nucleus, central lateral nucleus and paracentral nucleus. The intralaminar thalamic nuclei of eight cats were electrolytically lesioned. We obtained samples from the dorsal claustrum for electron microscopic analysis from the second to the seventh post-procedural day. Two types of degenerated synaptic boutons were observed: electron-dense which formed the majority of boutons, and electron-lucent comprising the remaining samples. Between the second and seventh post-procedural day, we observed a steady increase in the number of electron-dense boutons which were diffusely distributed throughout the dorsal claustrum. Electron-dense degenerated boutons formed asymmetrical contacts with dendritic spines as well as with small and medium-sized dendrites. In contrast, electron-lucent degenerated boutons were observed in earlier post-procedural periods and formed symmetrical axodendritic contacts.

It is well documented that propionic acid (PPA) produces behavioral, morphological, molecular and immune responses in rats that are characteristic of autism spectrum disorder in humans. However, whether PPA affects the ultrastructure and synaptic architecture of regions of autistic brain has not been adequately addressed. Earlier we show that single intraperitoneal (IP) injection of PPA (175 mg/kg) produces superficial changes in the spatial memory and learning of adolescent male Wistar rats. However, in neurons, synapses and glial cells of hippocampal CA1 area and medial prefrontal cortex transient (mainly) or enduring alterations were detected. In this study, we used electron microscopic morphometric analysis to test the effect of PPA on different structural parameters of axodendritic synapses of the hippocampus and prefrontal cortex. The animals were treated with a single IP injection of PPA (175 mg/kg). The length and width of synaptic active zone, the area of presynaptic and postsynaptic mitochondria, the distance between presynaptic mitochondria and the synapse active zone, the distance between postsynaptic mitochondria and postsynaptic density and the depth and opening diameter of neuronal porosome complex were evaluated. Our results show that synaptic mitochondria of the hippocampus and prefrontal cortex are the most vulnerable to PPA treatment: in both regions, the area of postsynaptic mitochondria were increased. In general, our results show that even small dose of PPA, which produces only superficial effects on spatial memory and learning is able to alter the synapse architecture in brain regions involved in cognition and autism pathogenesis. Therefore, the microbiome may be involved in the control of neurotransmission in these regions.

DYT1 dystonia is associated with decreased striatal dopamine release. In this study, we examined the possibility that ultrastructural changes of nigrostriatal dopamine terminals could contribute to this neurochemical imbalance using a serial block face/scanning electron microscope (SBF/SEM) and three-dimensional reconstruction to analyse striatal tyrosine hydroxylase-immunoreactive (TH-IR) terminals and their synapses in a DYT1(ΔE) knockin (DYT1-KI) mouse model of DYT1 dystonia. Furthermore, to study possible changes in vesicle packaging capacity of dopamine, we used transmission electron microscopy to assess the synaptic vesicle size in striatal dopamine terminals. Quantitative comparative analysis of 80 fully reconstructed TH-IR terminals in the WT and DYT1-KI mice indicate (1) no significant difference in the volume of TH-IR terminals; (2) no major change in the proportion of axo-spinous versus axo-dendritic synapses; (3) no significant change in the post-synaptic density (PSD) area of axo-dendritic synapses, while the PSDs of axo-spinous synapses were significantly smaller in DYT1-KI mice; (4) no significant change in the contact area between TH-IR terminals and dendritic shafts or spines, while the ratio of PSD area/contact area decreased significantly for both axo-dendritic and axo-spinous synapses in DYT1-KI mice; (5) no significant difference in the mitochondria volume; and (6) no significant difference in the synaptic vesicle area between the two groups. Altogether, these findings suggest that abnormal morphometric changes of nigrostriatal dopamine terminals and their post-synaptic targets are unlikely to be a major source of reduced striatal dopamine release in DYT1 dystonia.

The cellular mechanisms of neuroplastic changes in the structure of motoneurons and neuropils of the oculomotor (III) nuclei in mice after a 30-day space flight and 7 days after landing were studied. The results showed that microgravity caused degenerative phenomena in neurons: a decrease in the number of terminal dendritic branches was found both after flight and after readaptation to Earth's gravity. In mice after the flight, the number of axodendritic synapses was less than in the control, and their number was not restored after the readaptation. The number of mitochondria in the motoneurons of animals after the flight also decreased and after the readaptation reached only the control value. In addition, a significant number of dark motorneurons were found in mice after readaptation, which indicates that degeneration was caused not only by microgravity, but also by a reaction to the landing of the biosatellite. On the contrary, in the trochlear nucleus, as we showed earlier (Mikheeva et al. in Brain Res 15(1795):148077. https://doi.org/10.1016/j.brainres.2022.148077 , 2022), after readaptation, the dendrites and synaptic contacts were restored, and mitogenesis is significantly enhanced. It has been suggested that morphological changes in the oculomotor nucleus may be the main cause of microgravity-induced nystagmus.