Frontiers in Synaptic Neuroscience

Spontaneous synaptic transmission has been established as essential for the maintenance of synaptic weights during action potential-induced transmission. However, spontaneous transmission also changes during synaptic plasticity and has been shown to, in part, mediate changes in synaptic weights. Despite decades of research, a coherent framework for understanding the complex molecular processes that support presynaptic spontaneous transmission during maintenance and plasticity has remained elusive. We show here that presynapses modulate spontaneous transmission frequency during the early time-course of plasticity following entropic force theory. We use live primary hippocampal cultures as a model system and induce plasticity using an established Long-Term Potentiation (LTP) protocol. We then use a combination of electron microscopy, fluorescence microscopy, and computational modeling to show how spontaneous release frequency dynamically changes during early plasticity. We use our entropic force theory to show how the dynamically changing synaptic vesicle pool structure mediates spontaneous release changes. Lastly, we show how these changes are altered in the presence of P301L tau leading to degeneration. The results from this study provide new insights that not only help understand normal synaptic function but also aid in understanding neurodegeneration.

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.

Dendritic spines are the principal postsynaptic sites of excitatory transmission. For over a century, their shape has been sorted into discrete categories such as filopodia, thin, long thin, stubby, mushroom, and branched, largely by Golgi-Cox impregnation and, more recently, confocal microscopy. However, both approaches share a fundamental limitation. The histological sectioning and single-viewpoint imaging that these methods rely on cannot control the orientation of a spine relative to the observer. Because a spine is a three-dimensional object, the projection seen depends on how its parent dendrite lies within the section. Here, using the publicly available H01 petavoxel reconstruction of human temporal cortex imaged by serial-section electron microscopy (EM), we show that spine-shape classification depends strongly on viewing angle. A total of 445 spines on layer 4 basal dendrites of five pyramidal neurons were classified from an initial viewpoint (Angle 1), then reclassified after rotation in Neuroglancer (Angle 2). Only 20.9% kept their category, so chance-corrected agreement was negligible (Cohens kappa = 0.027). These observations provide direct evidence that the rigid Golgi-Cox and confocal taxonomies conflate true spine morphology with the arbitrary angle of view. Our results, therefore, support recasting spine shape as a three-dimensional continuum, measurable in petavoxel reconstructions such as H01 through free rotation in Neuroglancer. Significance statementThe classification of dendritic spines into discrete shape classes underpins a vast literature on synaptic plasticity, development, and disease. Yet it rests on two-dimensional images whose viewing angle is not controlled. By rotating the same human spines in a nanoscale EM reconstruction, this study shows that four out of five spines change category with viewpoint alone. The finding exposes a systematic bias in Golgi-Cox and confocal classifications. It argues that spine morphology should be treated as a measurable three-dimensional continuum rather than a set of fixed labels.

Dopamine signaling through dopamine 1 receptors (D1R) and dopamine 2 receptors (D2R) regulates hippocampal synaptic plasticity underlying learning and memory, yet their subcellular localization within the hippocampus is unknown. Here we performed electron microscopic immunocytochemistry to elucidate the distribution of D1R and D2R in subregions of the mouse hippocampus. In CA1 and CA3 stratum radiatum (SR), D1R- and D2R-immunoreactivity was found primarily on pyramidal cell dendritic spines and unmyelinated axons, and to a lesser extent in axon terminals and glia. In both regions, D1R-labeled terminals formed predominantly asymmetric (excitatory-type) synapses on dendritic spines, whereas D2R-labeled terminals formed mainly symmetric (inhibitory-type) synapses on pyramidal cell dendritic shafts. In the dentate gyrus (DG) hilus, D1R-labeling was almost exclusively found in unmyelinated axons and glia. D2R immunoreactivity in the hilus similarly was present in unmyelinated axons and glia but was also detected in dendritic spines originating from mossy cells and in terminals forming symmetric synapses. These findings indicate that dopamine receptors are positioned to influence excitatory and inhibitory signaling in the murine hippocampus. As D1R and D2R exert opposing effects on neuronal signaling, their localization on pyramidal neuron compartments provides a structural substrate for bidirectional modulation of synaptic plasticity and pyramidal cell activity. In addition, the presence of D2Rs on inhibitory terminals contacting pyramidal neurons and hilar interneurons suggests a role in regulating inhibitory circuitry within the hippocampus.

Glutamate synaptic vesicles co-release protons, producing a brief acidification of the synaptic cleft that could modulate AMPA receptor (AMPARs) operation. To evaluate the extent of receptor acidification, we develop a diffusion-reaction model that couples vesicle-evoked proton and glutamate transients to AMPAR dynamics. Our simulations reveal that the rapid diffusion of protons and glutamate within the flat-cylindrical synaptic cleft leads to a mixture of protonated, singly glutamate-bound and doubly glutamate-bound AMPARs. We studied four postsynaptic AMPAR distributions - uniform disk, sub-disk, Gaussian cluster, and point-like cluster - and showed a [~] 50% increase in the number of acidified receptors when AMPARs are clustered on the postsynaptic cleft compared to a uniform arrangement. We further explored the impact of pH revealing that at acidic conditions (pH [~] 5), approximately 80-90% of open receptors are non-acidified, whereas under strongly acidic conditions (pH [~] 3), about 80-90% of open receptors exist in the protonated form. Finally, we explored how acidification modulates AMPARs during paired-pulse stimulation, a measure of short-term synaptic depression. While the presence of protons does not markedly alter the overall trends, acidified receptor states reduce the occupancies of their neutral counterparts by roughly 10-15%, indicating a mild redistribution toward protonated receptor conformations. To conclude, our model suggests that AMPAR protonation can influence the synaptic current, and we predict that this effect is determined primarily by the dissociation kinetics of glutamate and protons from AMPAR.

Dendritic arbor morphology is shaped in part by interactions with neighboring dendrites, and its geometry strongly influences the spatial distribution and strength of synapses. These observations raise the possibility that local dendritic contacts help determine where synapses accumulate and strengthen. Previous work in cultured hippocampal neurons showed that dendrite-dendrite contact sites are non-random and associated with local synaptic clustering. Here we asked whether a different type of dendritic contact, formed between a dendrite and the soma of a neighboring neuron, behaves similarly. Using dissociated hippocampal cultures, immunofluorescence imaging, time-lapse microscopy, quantitative image analysis, stochastic spatial simulations, and minimal quantitative modeling, we identified three recurrent classes of dendrite-soma interactions (DSIs): dendrites crossing directly over a neighboring soma, growing tangentially along the soma perimeter, or contacting the proximal region where a neighboring dendrite emerges from the soma. These interactions were abundant, occurred exclusively between different neurons, and showed substantial structural persistence over several days. Their overall frequency exceeded stochastic predictions across culture densities, and two configurations - proximal and tangential contacts - were selectively enriched above random expectation, whereas soma-crossing contacts were largely consistent with stochastic overlap. DSI composition also changed over development, with proximal contacts becoming progressively more prevalent. At DSI sites, synaptophysin-positive puncta were significantly denser and more intense than on non-interacting dendritic segments, consistent with local enrichment and strengthening of presynaptic specializations. Minimal modeling further indicated that biased formation together with developmental stabilization explains the observed organization better than stochastic geometry alone. These findings identify DSIs as non-random structural motifs in cultured hippocampal networks and suggest that dendrite contact geometry can contribute to synaptic distribution and strengthening.

Endosomal sorting complexes required for transport (ESCRT) regulate membrane protein trafficking through coordinated cargo selection and endosomal processing, yet their contribution to neurotransmitter receptor sorting remains to be defined. Here, we examined how modulation of distinct complex components influences the surface expression of excitatory and inhibitory neurotransmitter receptors. Using surface biotinylation and imaging approaches in heterologous cells and primary neurons, we altered tumour susceptibility gene 101 (TSG101), a core complex I component, and vacuolar protein sorting-associated protein 4A (VPS4a), an ATPase required for complex III disassembly. Reduction of tumour susceptibility gene 101 increased receptor association with early endosomes and enhanced receptor surface localisation, whereas disruption of VPS4A promoted receptor accumulation within late endosomal compartments and impaired degradative progression. Inhibitory receptor subtypes displayed variable sensitivity. Together, these findings demonstrate that endosomal sorting complex components regulate receptor surface expression through stage-specific trafficking mechanisms associated with altered receptor recycling and degradative processing. Graphical abstractDistinct ESCRT components regulate neurotransmitter receptor trafficking through stage-specific control of the endosomal pathway. ESCRT-I disruption promotes early endosomal retention and recycling, whereas ESCRT-III impairment causes late endosomal accumulation and reduced degradation, together increasing receptor surface expression (created using Biorender). O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/732891v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@1fe66b9org.highwire.dtl.DTLVardef@10a29d7org.highwire.dtl.DTLVardef@4109c4org.highwire.dtl.DTLVardef@1e84f19_HPS_FORMAT_FIGEXP M_FIG C_FIG

The neurotransmitter dopamine (DA) is central to synaptic regulation that support diverse behavioral functions, including both learning and forgetting. This multi-functional role of DA is due to receptor specific signaling in specific subcellular environments that remain uncharacterized. Here we utilized proximity labelling proteomics in human cells to characterize the proximal environments of two Drosophila D1-like DA receptors (Dop1R1 and Dop1R2) in basal and DA activation environments. While DA drives both receptors to recruit Beta-Arrestin 2, Dop1R1 alone showed ligand driven recruitment of G-protein Receptor Kinase 2/3, proximity to clathrin mediated endocytosis, and WASH complex mediated endosomal trafficking. Additionally, we show evidence that Dop1R1 and Dop1R2 reside in distinct domains at the cell surface. In vivo disruption of Drosophila orthologs of Dop1R proximal proteins revealed three trafficking proteins, Sec24AB, Krz, and CG13887, that regulate R1-mediated learning, starvation induced attraction to odors, and DA-mediated cAMP responses in memory circuits. In addition to revealing DA receptor trafficking proteins that support learning, our comparative characterization of the cellular environments D1-like receptors offers insights into how DA differentially regulates diverse behavioral and synaptic functions. For TOC only O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/728438v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@656e56org.highwire.dtl.DTLVardef@12f0084org.highwire.dtl.DTLVardef@cb05cdorg.highwire.dtl.DTLVardef@e9d623_HPS_FORMAT_FIGEXP M_FIG C_FIG

P/Q-type calcium channel (Cav2.1) is the major channel that mediates Ca2+ influx during action potentials (APs) and evokes neurotransmitter release from presynaptic terminals. Repetitive activity induces its Ca2+-dependent facilitation (CDF) via binding of calmodulin (CaM) superfamily proteins to the IQ-like motif, specifically isoleucine (I) and methionine (M) sites, on the cytoplasmic c-terminus of Cav2.1. However, whether and how CDF contributes to short-term synaptic plasticity remains elusive. By recordings from the calyx of Held terminal in IQ-like motif point mutation knock-in mice (Cav2.1 IM-AA KI), we found that activity-dependent CDF is completely abolished, resulting in lower quantal output and shorter release time course as well as profound reductions in the magnitute of short-term facilitation and depression (STF and STD) in different Ca2+ concentrations. Prolonging deactivation of Ca2+ channels by broadening spike width normalizes quantal output and release time course in Cav2.1 IM-AA synapses, but does not fully rescue STF/STD. These results indicate that CDF of Cav2.1 channels governs the polarity and magnitude of short-term synaptic plasticity in fast-spiking central synapses.

Synaptojanin 1 is a brain enriched phosphoinositide phosphatase implicated in endocytosis at the synapse. A mutation (R258Q) that selectively impairs its Sac1 phosphatase domain causes early onset familial Parkinsonism. Neurons of mice with this mutation display synaptic vesicle traffic defects across the brain, but selective dystrophic changes in a subset of dopaminergic axons in the dorsolateral striatum. Using correlative light microscopy-FIB-SEM of mutant mouse striata to visualize in 3D these abnormal structures we show that they represent clusters of focal axonal dilations harboring massive, onion-like DAT enriched plasma membrane infoldings, generally localized next to cell bodies of neighboring cells, often engulfing evaginations of such cells. This dysmorphia was associated with a deficit in dopamine release in the same striatal region. Given the involvement of Synj1 in endocytic mechanisms, these structures may reflect an imbalance between exocytosis and endocytosis. Their occurrence only in a subset of axons suggest a vulnerability threshold of these axons beyond which the expansion of the plasma membrane is not counteracted by compensatory mechanisms.

Optical recording from large numbers of neurons is an indispensable technique for studying neuronal ensembles. We use optical sectioning through pinhole illumination to reduce the background fluorescence (F0) and increase the optical signal ({Delta}F/F0) in ex vivo brain slices densely labeled with GCaMP6f, allowing an ordinary fluorescence microscope to capture calcium transients from over 300 individual CA1 neurons - a marked increase compared to ordinary wide field fluorescence illumination. Multiple layers of overlapping neurons can be identified by their locations and the shape in space of their {Delta}F/F0 images. A single pinhole mask was placed at the field stop of a wide field illuminator, and the image of the pinhole was projected onto the tissue by a 20X NA 0.95 water immersion objective (Olympus). This created an illuminated disk with a diameter of [~]200 m and optical sections of hippocampal CA1 pyramidal layer tissue [~]100 m thick. This illumination blocked a large fraction of the F0, which in turn increased the {Delta}F/F0 5-10-fold compared to that of wide field illumination. When putative pyramidal neurons fire sparsely in the brain slice, up to 300 partially superimposed neurons can be identified by their shape and spatial location in the thick ([~]480 m) ex vivo slice in the CA1 area surrounding the pinhole image. The signal-to-noise ratio was adequate even at a low excitation light level of [~]20k photoelectrons per pixel well on the camera, allowing for 3,000 seconds of total recording time without significant bleaching. This pinhole "half confocal" method has created a useful way to sample calcium transient signals in thick tissue with a large population of neurons densely labeled with GCaMP-6f.

Plasma Membrane Calcium ATPase (PMCA) is essential for maintaining intracellular calcium homeostasis. Previously, we used constitutive PMCA downregulation in Drosophila melanogaster dopaminergic neurons as a model to increase intracellular calcium and mimic early neuronal alterations associated with Parkinsons disease. Here, we examined the mechanisms underlying the effects mediated by the conditional, adult-specific downregulation of PMCA in dopaminergic neurons in Drosophila melanogaster, both in vivo and in primary neuronal cultures. Adult-specific conditional silencing of PMCA in dopaminergic neurons reduced lifespan but to a lesser extent than the constitutive model and impaired locomotor performance. At the cellular level, PMCA-downregulated dopaminergic neurons exhibited elevated basal calcium, indicating disrupted calcium regulation. This was associated with a progressive increase in presynaptic vesicles and extracellular dopamine levels, suggesting enhanced neurotransmitter release. Notably, the synaptic active zone structure was preserved, indicating primarily functional rather than structural alterations. In primary neuronal cultures, PMCA downregulation reduced dopaminergic neuron survival and induced transient increases in neurite branching. Together, these findings show that PMCA downregulation leads to calcium dysregulation and presynaptic dysfunction without overt neurodegeneration in vivo, while promoting premature neuronal death in culture, indicating increased vulnerability and supporting a pre-degenerative state in which synaptic alterations precede neuronal loss.

Abnormal deposition of the microtubule-associated protein tau has long been associated with neurodegenerative diseases. While spine loss and neuronal death are hallmarks of tauopathy, how pathological tau affects synaptic activity in vivo and whether functional properties of individual synapses dictate the survival fate of dendritic spines remain elusive. Here we examined the visual response properties of dendrites and spines of layer 2/3 primary visual cortical neurons, using longitudinal two-photon calcium imaging in P301S mouse model of tauopathy. We found that neuronal outputs in tau mutant mice were hyperactive and poorly tuned whereas dendritic spine responses were also poorly tuned but hypoactive. Moreover, we found that spines that were stably retained across two imaging sessions were larger in size and more sharply tuned but less active compared to those that turned over in controls. Such structure-to-function relationship was not observed in mutants. Our findings illustrate how the preferential maintenance of well-tuned inputs in healthy neural circuitry may be affected by tauopathy, resulting in neurons with poorly tuned visual responses.

The mechanisms that underlie glutamatergic synapse organization and function in GABAergic inhibitory interneurons (INs) are not well described, despite evidence that impaired glutamatergic excitation of INs is implicated in psychiatric disorders such as schizophrenia and anxiety. Glutamatergic synapses received by INs have unique basal transmission properties and exhibit distinct synaptic plasticity compared to those received by excitatory neurons, likely due to cell-type specific differences in postsynaptic density (PSD) composition and maintenance mechanisms. In the present study, we show that the interneuron-specific protein Btbd11 regulates excitatory synapse transmission in hippocampal interneurons through promotion of phase separation and support of postsynaptic nanoarchitecture. Btbd11 forms a phase separated protein complex with Psd-95 and TARP{gamma}2 and impacts the stability of TARP{gamma}2 and GluA1 within glutamatergic IN synapses in an expression- and phase separation-dependent manner. Using super resolution imaging, we show that Btbd11 displays nanoscale clustering properties within IN synapses that correlate with Psd-95 nanostructure. Furthermore, genetic deletion of Btbd11 decreases PSD protein expression, reduces synapse size, and disrupts Psd-95 nanocluster organization. These effects manifest as a drastic reduction in glutamatergic synaptic transmission onto INs when Btbd11 is deleted. Together, these data provide insights into a novel cell type-specific synaptic regulatory mechanism in an understudied synapse population.

Cav1.2 is an L-type voltage-gated Ca2+ channel (VGCC) that supports Ca2+ influx in response to membrane depolarization. Ca2+ entering via Cav1.2 alters gene expression, activates Ca2+-dependent enzymes and has been implicated in synaptic plasticity. ORL-1 is a Gi/o-coupled G protein-coupled receptor (GPCR) that is expressed in the peripheral and central nervous systems. Both Cav1.2 and ORL-1 are expressed in the hippocampus, where they have been implicated in learning and memory. It is well-documented that ORL-1 interacts with another VGCC, Cav2.2. However, less is known about potential interactions between Cav1.2 and ORL-1. Here, we examine the interplay between Cav1.2 (Cav1c, Cav2{delta}-1, Cav{beta}1) and ORL-1 co-expressed in tsA-201 cells by using biochemical, electrophysiological and confocal imaging analysis. Co-immunoprecipitations revealed that ORL-1 independently interacts with Cav1c and Cav2{delta}-1 subunits of the Cav1.2 channel complex. Electrophysiological recordings revealed that co-expression with ORL-1 reduced Cav1.2 peak current density without altering its biophysical properties. Acute perfusion with the ORL-1 receptor agonist nociceptin (1 M) did not alter Cav1.2 current density. Confocal imaging experiments revealed that ORL-1 significantly decreases Cav1.2 plasma membrane expression by disrupting forward trafficking. Interestingly, ORL-1 did not affect Cav1.2 endocytosis. Overall, our results demonstrate a previously unrecognized interaction between ORL-1 and Cav1.2 that alters Cav1.2 membrane expression without affecting biophysical properties.

Local protein synthesis in neurons occurs in both axons and dendrites and plays a central role in synaptic function. High-throughput-based sequencing and imaging studies have demonstrated the presence and translation of synaptically localised mRNAs. However, quantification of activity-dependent translation dynamics at synapses at the transcriptome-wide scale remains limited. Here, we apply ribosome profiling to synapse-enriched fractions (synaptoneurosomes) derived from rat cortical tissue following stimulation with the group 1 mGluR agonist DHPG. DHPG stimulation induced translation of mRNAs involved in synaptic processes, including synaptic vesicle exocytosis and axo-dendritic transport. Notably, translation of ribosomal protein mRNAs was upregulated upon mGluR activation, consistent with the expected increase in de novo protein synthesis. Together, these results demonstrate the use of ribosome profiling to capture changes in local mRNA translation from isolated preparations.

Hippocampal area CA2 has recently emerged as a critical region for social recognition memory. Furthermore, this understudied region has been implicated in psychiatric diseases and neurodegenerative diseases. There has been accumulating evidence indicating that the pyramidal neurons (PNs) in area CA2 exhibit functional specializations that correlate with somatic position in stratum pyramidale (sp). In this study, we investigated the morphological differences in dendritic architecture of CA2 PNs with a focus on the radial gradient, i.e., along the deep-superficial axis of the sp. We conducted a comprehensive morphological analysis including Sholl intersection profiles, branching order distributions, root angle distributions, and dendritic cable lengths. We found that CA2 PNs have fewer oblique dendrites and a larger number of tuft-like dendrites as compared to CA1 PNs. Furthermore, within the CA2 population, we found that many of the dendritic structural features gradually changed along the radial axis from deep to superficial somatic location, indicating a continuum of dendritic morphology rather than two sharply defined subtypes of pyramidal neurons. This morphological characterization may serve as a starting point to better understand the corresponding functional organization of CA2. The gradual difference between deeper and superficial CA2 PNs suggests a continuum of their computational capabilities beyond two binary functional classes. In briefUsing several methods, we examine the dendritic morphology of over 130 CA2 and CA1 pyramidal neurons and find that many properties such as the cable length and terminal numbers of the dendritic arbors vary as a with the location of the soma in the pyramidal layer. HighlightsO_LIWe use scholl analysis, graph theory and machine learning techniques to quantify the different dendritic morphologies of CA2 pyramidal neurons. C_LIO_LIMany properties of CA2 pyramidal neuron apical dendrites vary as a function of somatic location in the pyramidal layer. C_LIO_LIMore superficial CA2 pyramidal neurons have longer oblique apical dendrites, and shorter tuft dendrites. C_LI

Background: Norepinephrine (NE) released from locus coeruleus (LC) projections regulates astrocyte structure and function through adrenergic receptor signaling. We previously showed that increasing noradrenergic tone with the NE reuptake inhibitor desipramine increases astrocyte ramification in the molecular layer of the dentate gyrus. However, whether tonic LC-derived noradrenergic tone is required to maintain astrocyte morphological complexity in vivo, and whether {beta}-adrenergic receptor activation is the effector pathway, remained unclear. Methods: Adult male C57BL/6J mice received DSP-4 (50 mg/kg X 3 days i.p.), a selective LC neurotoxin, with or without concurrent isoproterenol that continued for 21 additional days post cessation of DSP-4 treatment (ISO; 2 mg/kg/day X 24 days), or saline (n = 4 mice per group). Animals were sacrificed 22 days after the final DSP-4 injection. Noradrenergic denervation was confirmed by dopamine {beta}-hydroxylase (DBH) immunostaining. GFAP-immunostained astrocytes in the molecular layer of the dentate gyrus were morphologically characterized using Sholl analysis. Astrocyte density was quantified by SOX9 immunostaining. Results: DSP-4 produced >83% reduction in DBH fiber coverage in the molecular layer. Sholl analysis revealed significant reductions in astrocyte branching complexity in both treatment groups, with the reductions concentrated at distances of 5-15 m from the soma. The maximum number of intersections was also significantly reduced in both groups. Unexpectedly, ISO did not rescue morphological complexity. While DSP-4 alone did not alter astrocyte density, as measured by the number of SOX9-expressing astrocytes, DSP-4+ISO increased SOX9-positive cell density, dissociating the effects of adrenergic signaling on morphology from those on cell numbers. Conclusions: LC-derived noradrenergic tone is required for the maintenance of astrocyte arbour complexity in the dentate gyrus molecular layer. {beta}-adrenergic receptor activation alone is insufficient to restore structural integrity following noradrenergic denervation, yet promotes astrocyte density independently of structural remodeling. These findings have implications for understanding how LC neurodegeneration in Alzheimer's disease and depression may compromise hippocampal astrocyte structure and function.

The glymphatic system mediates brain-wide cerebrospinal fluid (CSF) transport and is highly sensitive to brain state. Experimental studies show that 2-adrenergic agonist- based anesthesia enhances glymphatic CSF influx, whereas isoflurane markedly suppresses it. However, it has been suggested that the reduced tracer influx observed during isoflurane anesthesia may reflect rapid clearance of tracer from the basal cisterns rather than genuine inhibition of glymphatic transport. To address this question, we compared conventional short-duration cisterna magna tracer injections with prolonged low-rate infusion while maintaining identical total tracer dose and anesthesia duration. Across both paradigms, ketamine/dexmedetomidine anesthesia consistently produced substantially greater perivascular CSF influx than isoflurane. In contrast, tracer accumulation in blood and cervical lymph nodes remained largely unchanged between conditions. These findings demonstrate that suppression of glymphatic influx during isoflurane anesthesia is independent of tracer injection duration and support the conclusion that 2-adrenergic agonist-based anesthesia promotes glymphatic transport through mechanisms linked to sleep-like brain states.

Activity of the mesolimbic dopamine system has long been implicated in encoding primary rewards and contributing to the addictive properties of drugs of abuse. Dopamine neurons in the ventral tegmental area (VTADA) of the midbrain typically show patterns of spontaneous burst activity that align with the onset of salient events or rewarding stimuli, resulting in phasic dopamine release in the nucleus accumbens (NAc). Fiber photometry is increasingly being used as an accessible technique to quantify neural activity with high temporal resolution at sensors offering signal specificity in stable recordings over extended periods of time. It has been well established by multiple techniques that opioids increase mesolimbic dopamine activity, likely through disinhibition of VTADA neurons. Here we used fiber photometry to compare sub-second transient events from VTADA neurons with GCaMP6f and dopamine release in the lateral shell of the NAc with dLight1.3b and GRABDA2h in response to morphine treatment. In weekly sessions, one dose of morphine was administered in escalating order (2.5, 5,7.5, and 10 mg/kg, intraperitoneal). Consistent with prior literature, both GCaMP6f in VTADA neurons and dLight1.3b in NAc showed patterns of increased signal following morphine treatment. In contrast, morphine suppressed transient activity at GRABDA2h sensors. Further analyses of whole signal streams from each sensor showed a generalized increase, but reduction in variability of the GRABDA2h signal, consistent with the interpretation of sensor saturation. Such results emphasize the importance of the inclusion of appropriate controls to contextualize the interpretation of biosensor responses, particularly in response to pharmacological treatment. HIGHLIGHTSO_LIMorphine elicited increased signaling in VTADA GCaMP6f and NAc dLight1.3b, consistent with prior literature C_LIO_LIMorphine suppressed NAc GRABDA2h signaling of transient events, suggesting saturation of GRABDA2h sensor C_LIO_LISensor validation with pharmacological challenges is critical for interpretation of data C_LI