Apical Dendrites Research Articles

Frequency-dependent entrainment of spontaneous Ca transients in the dendritic tufts of CA1 pyramidal cells in rat hippocampal slice preparations by weak AC electric field

Neurons in the central nervous systems are exposed to endogenous oscillating electric fields and their activities are likely to be modified by those fields. We had previously investigated the effects of AC electric field by using a newly developed method to monitor local Ca transients in the dendrites of a neuronal population in acute rat hippocampal slices and reported that spontaneously occurring Ca transients in the tufts of the apical dendrites of CA1 pyramidal neurons become entrained to subthreshold AC electric fields. To further our understanding of the impact of AC fields on dendritic activities, in the present study we examined three questions: how does the extent of entrainment depend on the frequency of the applied field, how does the mean phase of the dendritic activities during field application depend on the frequency of the field, and whether the entrainment can be seen in the absence of synaptic transmission. We have found that, the extent of entrainment is significantly greater at a low frequency band (1–4 Hz) compared to a high frequency band (8–16 Hz), 0.688 ± 0.027 at 2 Hz compared to 0.087 ± 0.016 at 16 Hz in case of 7 mV/mm field strength, that the entrainment can be observed when synaptic transmission is pharmacologically blocked, and that the mean phase of the Ca transients during field stimulation at a low frequency band (1–4 Hz) stays constant. These results indicate that the electric fields with physiologically feasible frequencies and intensities can entrain activities of the dendrites in a frequency-dependent manner independent of synaptic transmission. AC electric fields during oscillatory brain activities might play a role in synchronizing neural activities by modulating dendritic activities.

Somato-dendritic morphology and axon origin site specify von Economo neurons as a subclass of modified pyramidal neurons in the human anterior cingulate cortex.

Von Economo neurons (VENs) are modified pyramidal neurons characterized by an extremely elongated rod-shaped soma. They are abundant in layer V of the anterior cingulate cortex (ACC) and fronto-insular cortex (FI) of the human brain, and have long been described as a human-specific neuron type. Recently, VENs have been reported in the ACC of apes and the FI of macaque monkeys. The first description of the somato-dendritic morphology of VENs in the FI by Cajal in 1899 (Textura del Sistema Nervioso del Hombre y de los Vertebrados, Tomo II. Madrid: Nicolas Moya) strongly suggested that they were a unique neuron subtype with specific morphological features. It is surprising that a clarification of this extremely important observation has not yet been attempted, especially as possible misidentification of other oval or fusiform cells as VENs has become relevant in many recently published studies. Here, we analyzed sections of Brodmann area 24 (ACC) stained with rapid Golgi and Golgi-Cox in five adult human specimens, and confirmed Cajal's observations. In addition, we established a comprehensive morphological description of VENs. VENs have a distinct somato-dendritic morphology that allows their clear distinction from other modified pyramidal neurons. We established that VENs have a perpendicularly oriented, stick-shaped core part consisting of the cell body and two thick extensions - an apical and basal stem. The perpendicular length of the core part was 150-250μm and the thickness was 10-21μm. The core part was characterized by a lack of clear demarcation between the cell body and the two extensions. Numerous thin, spiny and horizontally oriented side dendrites arose from the cell body. The basal extension of the core part typically ended by giving numerous smaller dendrites with a brush-like branching pattern. The apical extension had a topology typical for apical dendrites of pyramidal neurons. The dendrites arising from the core parthad a high dendritic spine density. The most distinct feature of VENs was the distant origin site of the axon, which arose from the ending of the basal extension, often having a common origin with a dendrite. Quantitative analysis found that VENs could be divided into two groups based on total dendritic length - small VENs with a peak total dendritic length of 1500-2500μm and large VENs with a peak total dendritic length of 5000-6000μm. Comparative morphological analysis of VENs and other oval and fusiform modified pyramidal neurons showed that on Nissl sections small VENs might be difficult to identify, and that oval and fusiform neurons could be misidentified as VENs. Our analysis of Golgi slides of Brodmann area 9 from a total of 32 adult human subjects revealed only one cell resembling VEN morphology. Thus, our data show that the numerous recent reports on the presence of VENs in non-primates in other layers and regions of the cortex need further confirmation by showing the dendritic and axonal morphology of these cells. In conclusion, our study provides a foundation for further comprehensive morphological and functional studies on VENs between different species.

Ischemic axonal injury up-regulates MARK4 in cortical neurons and primes tau phosphorylation and aggregation

Ischemic injury to white matter tracts is increasingly recognized to play a key role in age-related cognitive decline, vascular dementia, and Alzheimer’s disease. Knowledge of the effects of ischemic axonal injury on cortical neurons is limited yet critical to identifying molecular pathways that link neurodegeneration and ischemia. Using a mouse model of subcortical white matter ischemic injury coupled with retrograde neuronal tracing, we employed magnetic affinity cell sorting with fluorescence-activated cell sorting to capture layer-specific cortical neurons and performed RNA-sequencing. With this approach, we identified a role for microtubule reorganization within stroke-injured neurons acting through the regulation of tau. We find that subcortical stroke-injured Layer 5 cortical neurons up-regulate the microtubule affinity-regulating kinase, Mark4, in response to axonal injury. Stroke-induced up-regulation of Mark4 is associated with selective remodeling of the apical dendrite after stroke and the phosphorylation of tau in vivo. In a cell-based tau biosensor assay, Mark4 promotes the aggregation of human tau in vitro. Increased expression of Mark4 after ischemic axonal injury in deep layer cortical neurons provides new evidence for synergism between axonal and neurodegenerative pathologies by priming of tau phosphorylation and aggregation.

Maintenance of Fluorescence During Paraffin Embedding of Fluorescent Protein-Labeled Specimens.

Paraffin embedding is widely used in microscopic imaging for preparing biological specimens. However, owing to significant fluorescence quenching during the embedding process, it is not compatible with fluorescent-labeling techniques, such as transgenic and viral labeling using green fluorescent protein (GFP). Here, we investigate the quenching mechanism and optimize the embedding process to improve the preservation of fluorescence intensity. The results show that dehydration is the main reason for fluorescence quenching during paraffin embedding, caused by the full denaturation of GFP molecules in ethyl alcohol. To evaluate fluorescent and morphological preservation, we modified the embedding process using tertiary butanol (TBA) instead of ethyl alcohol. Fluorescence intensity following TBA dehydration increased 12.08-fold of that observed in the traditional method. We obtained uniform fluorescence maintenance throughout the whole mouse brain, while the continuous apical dendrites, spines, and axon terminals were shown evenly within the cortex, hippocampus, and the amygdala. Moreover, we embedded a whole rat brain labeled with AAV in the prelimbic cortex (Prl). With the axon terminals in different areas, such as the caudate putamen, thalamus, and pyramidal tract, the results showed a continuous tract of Prl neurons throughout the whole brain. This method was also suitable for tdTomota labeled samples. These findings indicate that this modified embedding method could be compatible with GFP and provides a potential turning point for applications in the fluorescent labeling of samples.

Chronic social defeat stress induces sustained synaptic structural changes in the prefrontal cortex and amygdala

Previous studies show that chronic stress induces synaptic structural alterations in brain regions involved in emotional processing such as the prefrontal cortex (PFC) and the basolateral amygdala (BLA). Yet, these studies are based mainly in animal exposure to unpredictable stressors or to restraint stress. On the other hand, studies using the chronic social defeat stress (CSDS), a relevant model of depression based on social conflict, are lacking. Here we aim to study the acute (24 h after CSDS) and long-term (one month after CSDS) effects of CSDS on dendritic and synaptic structures in the PFC and BLA of C57BL/6 mice. Specifically, BLA and PFC dendritic spine densities as well as BLA arborisation were analysed. Subsequently, we investigate in these regions the synaptic response to a friendly (interaction with a same strain mouse) or a fearful (interaction with a dominant strain mouse) social stimulus.Spine densities of the apical dendrites from the PFC pyramidal neurons were decreased by CSDS in the long-term (one month after CSDS). In addition, CSDS increased BLA stellate neurons spine density in the short-term (24 h after CSDS) and dendritic arborisation in the long-term. Moreover, long-term CSDS mice exposed to a fearful stimulus experienced a marked social avoidance and showed a significant increase in the expression of the immature form of the brain derived neurotrophic factor (proBDNF) in the amygdala.Taken together these results suggest the existence of persistent neuronal adaptations in the PFC and BLA in socially defeated mice. Specifically, spine density retraction in the PFC and increased BLA dendritic arborisation could represent an adaptive structural change allowing rapid expression of synaptic markers in response to fearful experiences.

Reduced cognitive deficits after FLASH irradiation of whole mouse brain are associated with less hippocampal dendritic spine loss and neuroinflammation

AimTo evaluate the impact of ultra-rapid FLASH mouse whole brain irradiation on hippocampal dendritic spines and neuroinflammation, factors associated with cognitive impairment after brain irradiation. MethodsWe administered 30 Gy whole brain irradiation to C57BL6/J mice in sub-second (FLASH) vs. 240 s conventional delivery time keeping all other parameters constant, using a custom configured clinical linac. Ten weeks post-irradiation, we evaluated spatial and non-spatial object recognition using novel object location and object recognition testing. We measured dendritic spine density by tracing Golgi-stained hippocampal neurons and evaluated neuroinflammation by CD68 immunostaining, a marker of activated microglia, and expression of 10 pro-inflammatory cytokines using a multiplex immunoassay. ResultsAt ten weeks post-irradiation, compared to unirradiated controls, conventional delivery time irradiation significantly impaired novel object location and recognition tasks whereas the same dose given in FLASH delivery did not. Conventional delivery time, but not FLASH, was associated with significant loss of dendritic spine density in hippocampal apical dendrites, with a similar non-significant trend in basal dendrites. Conventional delivery time was associated with significantly increased CD68-positive microglia compared to controls whereas FLASH was not. Conventional delivery time was associated with significant increases in 5 of 10 pro-inflammatory cytokines in the hippocampus (and non-significant increases in another 3), whereas FLASH was associated with smaller increases in only 3. ConclusionReduced cognitive impairment and associated neurodegeneration were observed with FLASH compared to conventional delivery time irradiation, potentially through decreased induction of neuroinflammation, suggesting a promising approach to increasing therapeutic index in radiation therapy of brain tumors.

Vasopressin Cells in the Rodent Olfactory Bulb Resemble Non-Bursting Superficial Tufted Cells and Are Primarily Inhibited upon Olfactory Nerve Stimulation.

The intrinsic vasopressin system of the olfactory bulb is involved in social odor processing and consists of glutamatergic vasopressin cells (VPCs) located at the medial border of the glomerular layer. To characterize VPCs in detail, we combined various electrophysiological, neuroanatomical, and two-photon Ca2+ imaging techniques in acute bulb slices from juvenile transgenic rats with eGFP-labeled VPCs. VPCs showed regular non-bursting firing patterns, and displayed slower membrane time constants and higher input resistances versus other glutamatergic tufted cell types. VPC axons spread deeply into the external plexiform and superficial granule cell layer (GCL). Axonal projections fell into two subclasses, with either denser local columnar collaterals or longer-ranging single projections running laterally within the internal plexiform layer and deeper within the granule cell layer. VPCs always featured lateral dendrites and a tortuous apical dendrite that innervated a single glomerulus with a homogenously branching tuft. These tufts lacked Ca2+ transients in response to single somatically-evoked action potentials and showed a moderate Ca2+ increase upon prolonged action potential trains.Notably, electrical olfactory nerve stimulation did not result in synaptic excitation of VPCs, but triggered substantial GABAA receptor-mediated IPSPs that masked excitatory barrages with yet longer latency. Exogenous vasopressin application reduced those IPSPs, as well as olfactory nerve-evoked EPSPs recorded from external tufted cells. In summary, VPCs can be classified as non-bursting, vertical superficial tufted cells. Moreover, our findings imply that sensory input alone cannot trigger excitation of VPCs, arguing for specific additional pathways for excitation or disinhibition in social contexts.

Sonic hedgehog signaling in astrocytes mediates cell type-specific synaptic organization.

Astrocytes have emerged as integral partners with neurons in regulating synapse formation and function, but the mechanisms that mediate these interactions are not well understood. Here, we show that Sonic hedgehog (Shh) signaling in mature astrocytes is required for establishing structural organization and remodeling of cortical synapses in a cell type-specific manner. In the postnatal cortex, Shh signaling is active in a subpopulation of mature astrocytes localized primarily in deep cortical layers. Selective disruption of Shh signaling in astrocytes produces a dramatic increase in synapse number specifically on layer V apical dendrites that emerges during adolescence and persists into adulthood. Dynamic turnover of dendritic spines is impaired in mutant mice and is accompanied by an increase in neuronal excitability and a reduction of the glial-specific, inward-rectifying K+ channel Kir4.1. These data identify a critical role for Shh signaling in astrocyte-mediated modulation of neuronal activity required for sculpting synapses.

Close Homolog of L1 Regulates Dendritic Spine Density in the Mouse Cerebral Cortex Through Semaphorin 3B.

Dendritic spines in the developing mammalian neocortex are initially overproduced and then eliminated during adolescence to achieve appropriate levels of excitation in mature networks. We show here that the L1 family cell adhesion molecule Close Homolog of L1 (CHL1) and secreted repellent ligand Semaphorin 3B (Sema3B) function together to induce dendritic spine pruning in developing cortical pyramidal neurons. Loss of CHL1 in null mutant mice in both genders resulted in increased spine density and a greater proportion of immature spines on apical dendrites in the prefrontal and visual cortex. Electron microscopy showed that excitatory spine synapses with postsynaptic densities were increased in the CHL1-null cortex, and electrophysiological recording in prefrontal slices from mutant mice revealed deficiencies in excitatory synaptic transmission. Mechanistically, Sema3B protein induced elimination of spines on apical dendrites of cortical neurons cultured from wild-type but not CHL1-null embryos. Sema3B was secreted by the cortical neuron cultures, and its levels increased when cells were treated with the GABA antagonist gabazine. In vivo CHL1 was coexpressed with Sema3B in pyramidal neuron subpopulations and formed a complex with Sema3B receptor subunits Neuropilin-2 and PlexinA4. CHL1 and NrCAM, a closely related L1 adhesion molecule, localized primarily to distinct spines and promoted spine elimination to Sema3B or Sema3F, respectively. These results support a new concept in which selective spine elimination is achieved through different secreted semaphorins and L1 family adhesion molecules to sculpt functional neural circuits during postnatal maturation.SIGNIFICANCE STATEMENT Dendritic spines in the mammalian neocortex are initially overproduced and then pruned in adolescent life through unclear mechanisms to sculpt maturing cortical circuits. Here, we show that spine and excitatory synapse density of pyramidal neurons in the developing neocortex is regulated by the L1 adhesion molecule, Close Homolog of L1 (CHL1). CHL1 mediated spine pruning in response to the secreted repellent ligand Semaphorin 3B and associated with receptor subunits Neuropilin-2 and PlexinA4. CHL1 and related L1 adhesion molecule NrCAM localized to distinct spines, and promoted spine elimination to Semaphorin 3B and -3F, respectively. These results support a new concept in which selective elimination of individual spines and nascent synapses can be achieved through the action of distinct secreted semaphorins and L1 adhesion molecules.

Widespread and Highly Correlated Somato-dendritic Activity in Cortical Layer 5 Neurons

Dendritic integration can expand the information-processing capabilities of neurons. However, the recruitment of active dendritic processing invivo and its relationship to somatic activity remain poorly understood. Here, we use two-photon GCaMP6f imaging to simultaneously monitor dendritic and somatic compartments in the awake primary visual cortex. Activity in layer 5 pyramidal neuron somata and distal apical trunk dendrites shows surprisingly high functional correlation. This strong coupling persists across neural activity levels and is unchanged by visual stimuli and locomotion. Exvivo combined somato-dendritic patch-clamp and GCaMP6f recordings indicate that dendritic signals specifically reflect local electrogenesis triggered by dendritic inputs or high-frequency bursts of somatic action potentials. In contrast to the view that dendrites are only sparsely recruited under highly specific conditions invivo, our results provide evidence that active dendritic integration is a widespread and intrinsic feature of cortical computation.

Differences in Sodium Channel Densities in the Apical Dendrites of Pyramidal Cells of the Electrosensory Lateral Line Lobe.

Heterogeneity of neural properties within a given neural class is ubiquitous in the nervous system and permits different sub-classes of neurons to specialize for specific purposes. This principle has been thoroughly investigated in the hindbrain of the weakly electric fish A. leptorhynchus in the primary electrosensory area, the Electrosensory Lateral Line lobe (ELL). The pyramidal cells (PCs) that receive inputs from tuberous electroreceptors are organized in three maps in distinct segments of the ELL. The properties of these cells vary greatly across maps due to differences in connectivity, receptor expression, and ion channel composition. These cells are a seminal example of bursting neurons and their bursting dynamic relies on the presence of voltage-gated Na+ channels in the extensive apical dendrites of the superficial PCs. Other ion channels can affect burst generation and their expression varies across ELL neurons and segments. For example, SK channels cause hyperpolarizing after-potentials decreasing the likelihood of bursting, yet bursting propensity is similar across segments. We question whether the depolarizing mechanism that generates the bursts presents quantitative differences across segments that could counterbalance other differences having the opposite effect. Although their presence and role are established, the distribution and density of the apical dendrites’ Na+ channels have not been quantified and compared across ELL maps. Therefore, we test the hypothesis that Na+ channel density varies across segment by quantifying their distribution in the apical dendrites of immunolabeled ELL sections. We found the Na+ channels to be two-fold denser in the lateral segment (LS) than in the centro-medial segment (CMS), the centro-lateral segment (CLS) being intermediate. Our results imply that this differential expression of voltage-gated Na+ channels could counterbalance or interact with other aspects of neuronal physiology that vary across segments (e.g., SK channels). We argue that burst coding of sensory signals, and the way the network regulates bursting, should be influenced by these variations in Na+ channel density.

Amplifying the redistribution of somato-dendritic inhibition by the interplay of three interneuron types.

GABAergic interneurons play an important role in shaping the activity of excitatory pyramidal cells (PCs). How the various inhibitory cell types contribute to neuronal information processing, however, is not resolved. Here, we propose a functional role for a widespread network motif consisting of parvalbumin- (PV), somatostatin- (SOM) and vasoactive intestinal peptide (VIP)-expressing interneurons. Following the idea that PV and SOM interneurons control the distribution of somatic and dendritic inhibition onto PCs, we suggest that mutual inhibition between VIP and SOM cells translates weak inputs to VIP interneurons into large changes of somato-dendritic inhibition of PCs. Using a computational model, we show that the neuronal and synaptic properties of the circuit support this hypothesis. Moreover, we demonstrate that the SOM-VIP motif allows transient inputs to persistently switch the circuit between two processing modes, in which top-down inputs onto apical dendrites of PCs are either integrated or cancelled.

Rational Engineering of XCaMPs, a Multicolor GECI Suite for In Vivo Imaging of Complex Brain Circuit Dynamics

To decipher dynamic brain information processing, current genetically encoded calcium indicators (GECIs) are limited in single action potential (AP) detection speed, combinatorial spectral compatibility, and two-photon imaging depth. To address this, here, we rationally engineered a next-generation quadricolor GECI suite, XCaMPs. Single AP detection was achieved within 3-10ms of spike onset, enabling measurements of fast-spike trains in parvalbumin (PV)-positive interneurons in the barrel cortex invivo and recording three distinct (two inhibitory and one excitatory) ensembles during pre-motion activity in freely moving mice. Invivo paired recording of pre- and postsynaptic firing revealed spatiotemporal constraints of dendritic inhibition in layer 1 invivo, between axons of somatostatin (SST)-positive interneurons and apical tufts dendrites of excitatory pyramidal neurons. Finally, non-invasive, subcortical imaging using red XCaMP-R uncovered somatosensation-evoked persistent activity in hippocampal CA1 neurons. Thus, the XCaMPs offer a critical enhancement of solution space in studies of complex neuronal circuit dynamics. VIDEO ABSTRACT.

Estrogen-Dependent Functional Spine Dynamics in Neocortical Pyramidal Neurons of the Mouse.

Surgical ovariectomy has been shown to reduce spine density in hippocampal CA1 pyramidal cells of rodents, and this reduction is reversed by 17β-estradiol (E2) treatment in a model of human estrogen replacement therapy. Here, we report reduction of spine density in apical dendrites of layer 5 pyramidal neurons of several neocortical regions that is reversed by subsequent E2 treatment in ovariectomized (OVX) female Thy1M-EGFP mice. We also found that OVX-associated reduction of spine density in somatosensory cortex was accompanied by a reduction in miniature EPSC (mEPSC) frequency (but not mIPSC frequency), indicating a change in functional synapses. OVX-associated spine loss in somatosensory cortex was also rescued by an agonist of the G-protein-linked estrogen receptor (GPER) but not by agonists of the classic estrogen receptors ERα/ERβ, whereas the opposite selectivity was found in area CA1. Acute treatment of neocortical slices with E2 also rescued the OVX-associated reduction in mEPSC frequency, which could be mimicked by a GPER agonist and abolished by a GPER antagonist. Time-lapse in vivo two-photon imaging showed that OVX-associated reduction in spine density is achieved by both an increase in spine loss rate and a decrease in spine gain rate and that subsequent rescue by E2 reversed both of these processes. Crucially, the spines added after E2 rescue were no more likely to reappear at or nearby the sites of pre-OVX spines than those in control mice treated with vehicle. Thus, a model of estrogen replacement therapy, although restoring spine density and dynamics, does not entirely restore functional connectivity.SIGNIFICANCE STATEMENT Estrogen replacement therapy following menopause or surgical removal of the ovaries is a widespread medical practice, yet little is known about the consequences of such treatment for cells in the brain. Here, we show that estrogen replacement reverses some of the effects of surgical removal of the ovaries on the structure and function of brain cells in the mouse. Yet, importantly, the fine wiring of the brain is not returned to the presurgery state by estrogen treatment, suggesting lasting functional consequences.

Basal forebrain GABAergic innervation of olfactory bulb periglomerular interneurons.

Basal forebrain long-range projections to the olfactory bulb are important for olfactory sensitivity and odour discrimination. Using optogenetics, it was confirmed that basal forebrain afferents mediate IPSCs on granule and deep short axon cells. It was also shown that they selectively innervate specific subtypes of periglomerular (PG) cells. Three different subtypes of type 2 PG cells receive GABAergic IPSCs from the basal forebrain but not from other PG cells. Type 1 PG cells, in contrast, do not receive inputs from the basal forebrain but do receive inhibition from other PG cells. These results shed new light on the complexity and specificity of glomerular inhibitory circuits, as well as on their modulation by the basal forebrain. Olfactory bulb circuits are dominated by multiple inhibitory pathways that finely tune the activity of mitral and tufted cells, the principal neurons, and regulate odour discrimination. Granule cells mediate interglomerular lateral inhibition between mitral and tufted cells' lateral dendrites whereas diverse subtypes of periglomerular (PG) cells mediate intraglomerular lateral inhibition between their apical dendrites. Deep short axon cells form broad intrabulbar inhibitory circuits that regulate both populations of interneurons. Little is known about the extrabulbar GABAergic circuits that control the activity of these various interneurons. We examined this question using patch-clamp recordings and optogenetics in olfactory bulb slices from transgenic mice. We showed that axonal projections emanating from diverse basal forebrain GABAergic neurons densely project in all layers of the olfactory bulb. These long-range GABAergic projections provide a prominent synaptic input on granule and short axon cells in deep layers as well as on selective subtypes of PG cells. Specifically, three different subclasses of type 2 PG cells receive robust and target-specific basal forebrain inputs but have little local interactions with other PG cells. In contrast, type 1 PG cells are not innervated by basal forebrain fibres but do interact with other PG cells. Thus, attention-regulated basal forebrain inputs regulate inhibition in all layers of the olfactory bulb with a previously overlooked synaptic complexity that further defines interneuron subclasses.

Neuron‐Targeted Caveolin‐1 Gene Therapy Preserves Cognitive Function and Synaptic Plasticity in a Mouse Model of Alzheimer's Disease (AD)

Introduction Alzheimer's disease (AD) is a devastating neurodegenerative condition. AD patients develop severe cognitive deficits, which are closely correlated with loss of synapses and decreased plasticity. Caveolin-1 (Cav-1), a scaffolding protein within the microdomains of membrane/lipid rafts (MLRs), organizes signaling complexes that promote functional neuronal and synaptic plasticity. Previously we showed that neuron-targeted overexpression of Cav-1 using a synapsin promoter (SynCav1) promotes neuroplasticity, increases synaptic plasticity, and improves memory in aged mice. The current study investigated the impact of SynCav-1 on markers of synaptic plasticity and cognitive function in an APPSwePS1d9 mouse model of AD. Methods AAV9-SynRFP (red fluorescent protein) or AAV9-SynCav1 was delivered into the hippocampus of wild type (WT) or AD mice at 2.5 months of age. At 9 months, open field and fear conditioning test were used to evaluate learning and memory performance. After behavior tests, animal were sacrificed for immunoblot (IB) and immunofluorence microscopy (IF). Electron microscopy (EM) was used to observe the ultrastructure morphology. Results AD-SynRFP mice exhibited significant decreased fear learning (day 1) compared to WT-SynRFP mice and AD-SynCav1 mice. In contrast, AD-SynCav1 mice showed preserved fear learning (day 1), and improved contextual and cued memory recall (day 2 & 3). Both IB and IF showed decreased Cav-1 expression in 9 month-old AD-SynRFP mice. AD-SynCav1 mice showed preserved Cav-1 levels and increased expression of neuroplasticity markers (TrkB, MAP2) compared to AD-SynRFP mice. IB analysis of MLR (buoyant fractions 4 & 5) demonstrate decreased MLR-localization of TrkB, Cav-1, CT-B, synaptophysin, TrkA, PSD95, LRP in AD-SynRFP mice. In contrast, AD-SynCav1 exhibited preserved MLR-localization of TrkB, Cav-1, CT-B, synaptophysin, TrkA, PSD95 and LRP1. IF showed preserved markers of dendritic integrity (MAP2) in AD-SynCav1 mice. EM analysis of the hippocampus revealed that AD-SynRFP mice had fewer type I excitatory synapses and decreased number of presynaptic vesicles per axonal bouton, and altered spine morphology (reduced spine length and increased spine neck diameter) in CA1 apical dendrites within the stratum radiatum, which were not exhibited by AD-SynCav1 mice. Additional analysis revealed preserved myelinated Schaffer collateral axons in AD-SynCav1 mice compared to AD-SynRFP. Conclusion SynCav1 gene delivery to the hippocampus of pre-symptomatic adult AD mice (2.5 m) preserves fear learning, improves memory, and preserves ultrastructural indicators of synaptic plasticity and myelination in mild symptomatic AD mice (9 months), therefore may serve as a novel gene therapy to restore synaptic and neuroplasticity and improve brain function in individuals afflicted with Alzheimer's disease. Support or Funding Information Veterans Affairs Merit Award from the Department of Veterans Affairs BX003671 (B. P. Head), National Institutes of Health, Bethesda, MD, U.S.A., NS073653 (B. P. Head); GM085179 (P. M. Patel). (A) AD - SynRFP at 2.5 months of age exhibited learning deficits on acquisition Day 1 training compared to both WT -SynRFP and AD -SynCav1 at 9 months of age. There was no significant difference between WT - SynRFP versus AD-SynCav1. SynCav1 delivery to AD mice significantly improved hippocampal-dependent contextual memory recall on Day 2 (B) and hippocampal-independent cued memory recall on Day 3 (C). Data are presented as mean ± SEM. Significance was assumed when p < 0.05. IF microscopy revealed that, in 9 month-old age, AD-SynRFP exhibited decreased in hippocampal Cav-1 expression compared to WT-SynRFP, while SynCav1 gene delivery preserved Cav-1 expression. Scale bar = 50 mm. IB analysis of MLR fractions (buoyant fractions 4 & 5) demonstrates decreased MLR-localization of full length TrkB, Cav-1, CT-B, synaptophysin, TrkA, LRP1 in AD-SynRFP mice; while SynCav1 treatment preserve MLR-localized synaptic proteins. CA1 distal apical dendrites in the stratum radiatum were examined for ultrastructure changes of type I asymmetric synapses and presynaptic vesicles (PSVs)/axonal bouton. SynCav1 preserves total number of synapses and PSVs in the hippocampus of 9 month old AD mice. CA1 distal apical dendrites in the stratum radiatum were examined for alterations in dendritic spine morphology, i.e., spine length (mm), spine neck diameter (mm), and spine area (mm2). SynCav1 preserves dendritic spine morphology in the hippocampus of 9 month old APPSwePS1d9 mice. G ratio analysis (axon lumen diameter (Di)/fiber (axon lumen + myelin) diameter (Do)) was used to measure changes in axonal myelin fiber content of CA3 Schaffer collaterals innervating the CA1 apical dendritic arbor. SynCav1 preserves axonal myelin fiber content of hippocampal CA3 Schaffer collaterals in 9 month old APPSwePS1d9 mice. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Clock Mechanism Influences Neurobiology and Adaptations to Heart Failure in Clock\u220619/\u220619 Mice With Implications for Circadian Medicine

In this study we investigated the role of the circadian mechanism on cognition-relevant brain regions and neurobiological impairments associated with heart failure (HF), using murine models. We found that the circadian mechanism is an important regulator of healthy cognitive system neurobiology. Normal Clock∆19/∆19 mice had neurons with smaller apical dendrite trees in the medial prefrontal cortex (mPFC), and hippocampus, showed impaired visual-spatial memory, and exhibited lower cerebrovascular myogenic tone, versus wild types (WT). We then used the left anterior descending coronary artery ligation model to investigate adaptations in response to HF. Intriguingly, adaptations to neuron morphology, memory, and cerebrovascular tone occurred in differing magnitude and direction between Clock∆19/∆19 and WT mice, ultimately converging in HF. To investigate this dichotomous response, we performed microarrays and found genes crucial for growth and stress pathways that were altered in Clock∆19/∆19 mPFC and hippocampus. Thus these data demonstrate for the first time that (i) the circadian mechanism plays a role in neuron morphology and function; (ii) there are changes in neuron morphology and function in HF; (iii) CLOCK influences neurobiological gene adaptations to HF at a cellular level. These findings have clinical relevance as patients with HF often present with concurrent neurocognitive impairments. There is no cure for HF, and new understanding is needed to reduce morbidity and improve the quality of life for HF patients.

Complementary networks of cortical somatostatin interneurons enforce layer specific control.

The neocortex is functionally organized into layers. Layer four receives the densest bottom up sensory inputs, while layers 2/3 and 5 receive top down inputs that may convey predictive information. A subset of cortical somatostatin (SST) neurons, the Martinotti cells, gate top down input by inhibiting the apical dendrites of pyramidal cells in layers 2/3 and 5, but it is unknown whether an analogous inhibitory mechanism controls activity in layer 4. Using high precision circuit mapping, in vivo optogenetic perturbations, and single cell transcriptional profiling, we reveal complementary circuits in the mouse barrel cortex involving genetically distinct SST subtypes that specifically and reciprocally interconnect with excitatory cells in different layers: Martinotti cells connect with layers 2/3 and 5, whereas non-Martinotti cells connect with layer 4. By enforcing layer-specific inhibition, these parallel SST subnetworks could independently regulate the balance between bottom up and top down input.

Fear conditioning and extinction induce opposing changes in dendritic spine remodeling and somatic activity of layer 5 pyramidal neurons in the mouse motor cortex

Multiple brain regions including the amygdala and prefrontal cortex are crucial for modulating fear conditioning and extinction. The primary motor cortex is known to participate in the planning, control, and execution of voluntary movements. Whether and how the primary motor cortex is involved in modulating freezing responses related to fear conditioning and extinction remains unclear. Here we show that inactivation of the mouse primary motor cortex impairs both the acquisition and extinction of freezing responses induced by auditory-cued fear conditioning. Fear conditioning significantly increases the elimination of dendritic spines on apical dendrites of layer 5 pyramidal neurons in the motor cortex. These eliminated spines are further apart from each other than expected from random distribution along dendrites. On the other hand, fear extinction causes the formation of new spines that are located near the site of spines eliminated previously after fear conditioning. We further show that fear conditioning decreases and fear extinction increases somatic activities of layer 5 pyramidal neurons in the motor cortex respectively. Taken together, these findings indicate fear conditioning and extinction induce opposing changes in synaptic connections and somatic activities of layer 5 pyramidal neurons in the primary motor cortex, a cortical region important for the acquisition and extinction of auditory-cued conditioned freezing responses.

Long-range inhibitory intersection of a retrosplenial thalamocortical circuit by apical tuft-targeting CA1 neurons.

Hippocampus, granular retrosplenial cortex (RSCg), and anterior thalamic nuclei (ATN) interact to mediate diverse cognitive functions. To identify cellular mechanisms underlying hippocampo-thalamo-retrosplenial interactions, we investigated the potential circuit suggested by projections to RSCg layer 1 (L1) from GABAergic CA1 neurons and ATN. We find that CA1→RSCg projections stem from GABAergic neurons with a distinct morphology, electrophysiology, and molecular profile. Their long-range axons inhibit L5 pyramidal neurons in RSCg via potent synapses onto apical tuft dendrites in L1. These inhibitory inputs intercept L1-targeting thalamocortical excitatory inputs from ATN to co-regulate RSCg activity. Subicular axons, in contrast, excite proximal dendrites in deeper layers. Short-term plasticity differs at each connection. Chemogenetically abrogating CA1→RSCg or ATN→RSCg connections oppositely affects the encoding of contextual fear memory. Our findings establish retrosplenial-projecting CA1 neurons as a distinct class of long-range dendrite-targeting GABAergic neuron, and delineate an unusual cortical circuit specialized for integrating long-range inhibition and thalamocortical excitation.