Anatomical Projections Research Articles

Four concurrent feedforward and feedback networks with different roles in the visual cortical hierarchy.

Visual stimuli evoke fast-evolving activity patterns that are distributed across multiple cortical areas. These areas are hierarchically structured, as indicated by their anatomical projections, but how large-scale feedforward and feedback streams are functionally organized in this system remains an important missing clue to understanding cortical processing. By analyzing visual evoked responses in laminar recordings from 6 cortical areas in awake mice, we uncovered a dominant feedforward network with scale-free interactions in the time domain. In addition, we established the simultaneous presence of a gamma band feedforward and 2 low frequency feedback networks, each with a distinct laminar functional connectivity profile, frequency spectrum, temporal dynamics, and functional hierarchy. We could identify distinct roles for each of these 4 processing streams, by leveraging stimulus contrast effects, analyzing receptive field (RF) convergency along functional interactions, and determining relationships to spiking activity. Our results support a dynamic dual counterstream view of hierarchical processing and provide new insight into how separate functional streams can simultaneously and dynamically support visual processes.

Function-specific projections from V2 to V4 in macaques

Previous studies have revealed modular projections from area V2 to area V4 in macaques. Specifically, V2 neurons in cytochrome oxidase (CO)-rich thin and CO-sparse pale stripes project to distinct regions in V4. However, how these modular projections relate to the functional subcompartments of V4 remains unclear. In this study, we injected retrograde fluorescent tracers into V4 regions with different functional properties (color, orientation, and direction) that were identified with intrinsic signal optical imaging (ISOI). We examined the labeled neurons in area V2 and their locations with respect to the CO patterns. Covariation was observed between the functional properties of the V4 injection sites and the numbers of labeled neurons in particular CO stripes. This covariation indicates that the color domains in V4 mainly received inputs from thin stripes in V2, whereas V4 orientation domains received inputs from pale stripes. Although motion-sensitive domains are present in both V2 and V4, our results did not reveal a functional specific feedforward projection between them. These results confirmed previous findings of modular projections from V2 to V4 and provided functional specificity for these anatomical projections. Together, these findings indicate that color and form remain separate in ventral mid-level visual processing.

Brain rhythms define distinct interaction networks with differential dependence on anatomy

SummaryCognitive functions are subserved by rhythmic neuronal synchronization across widely distributed brain areas. In 105 area pairs, we investigated functional connectivity (FC) through coherence, power correlation, and Granger causality (GC) in the theta, beta, high-beta, and gamma rhythms. Between rhythms, spatial FC patterns were largely independent. Thus, the rhythms defined distinct interaction networks. Importantly, networks of coherence and GC were not explained by the spatial distributions of the strengths of the rhythms. Those networks, particularly the GC networks, contained clear modules, with typically one dominant rhythm per module. To understand how this distinctiveness and modularity arises on a common anatomical backbone, we correlated, across 91 area pairs, the metrics of functional interaction with those of anatomical projection strength. Anatomy was primarily related to coherence and GC, with the largest effect sizes for GC. The correlation differed markedly between rhythms, being less pronounced for the beta and strongest for the gamma rhythm.

Angular and linear measurements of adult flexible flatfoot via weight-bearing CT scans and 3D bone reconstruction tools

Acquired adult flatfoot is a frequent deformity which implies multiple, complex and combined 3D modifications of the foot skeletal structure. The difficult thorough evaluation of the degree of severity pre-op and the corresponding assessment post-op can now be overcome by cone-beam (CBCT) technology, which can provide access to the 3D skeletal structure in weight-bearing. This study aims to report flatfoot deformities originally in 3D and in weight-bearing, with measurements taken using two different bone segmentation techniques. 21 such patients, with indication for surgical corrections, underwent CBCT (Carestream, US) while standing on one leg. From these scans, 3D models of each bone of the foot were reconstructed by using two different state-of-the-art segmentation tools: a semi-automatic (Mimics Innovation Suite, Materialise, Belgium), and an automatic (Bonelogic Ortho Foot and Ankle, Disior, Finland). From both reconstructed models, Principal Component Analysis was used to define anatomical reference frames, and original foot and ankle angles and other parameters were calculated mostly based on the longitudinal axis of the bones, in anatomical plane projections and in 3D. Both bone model reconstructions revealed a considerable valgus of the calcareous, plantarflexion and internal rotation of the talus, and typical Meary’s angles in the lateral and transverse plane projections. The mean difference from these angles between semi-automatic and automatic segmentations was larger than 3.5 degrees for only 3 of the 32 measurements, and a large number of these differences were not statistically significant. CBCT and the present techniques for bone shape reconstruction finally provide a novel and valuable 3D assessment of complex foot deformities in weight-bearing, eliminating previous limitations associated to unloaded feet and bidimensional measures. Corresponding measurements on the bone models from the two segmentation tools compared well. Other more representative measurements can be defined in the future using CBCT and these techniques.

Serotonin as a Regulator of Leptin-Mediated Food Intake Control Within a Novel Neuronal Circuit Between the Hypothalamus and Raphe Nuclei

The onset and exacerbation of obesity involves the overproduction of the adipocyte-derived hormone leptin, a key mediator of homeostatic appetite regulation and a signal for satiety. Although leptin’s hypothalamic regulation of food intake has been extensively investigated, its role in tandem with the anorectic neurotransmitter serotonin (5-HT) has been less characterized. 5-HT is synthesized in the dorsal raphe nucleus (DRN) where anatomical projections to many hypothalamic nuclei have previously been identified. Preliminary studies in our lab have: (1) identified serotonergic neurons responsive to leptin in the DRN that project to the arcuate nucleus (ARC) of the hypothalamus and (2) demonstrated leptin injected into the DRN significantly decreases food intake. The objective of the current study was to identify the role of 5-HT in leptin’s regulation of food intake first within the DRN, then between the DRN and the ARC. Adult male Sprague Dawley rats underwent stereotaxic surgery for guide cannula implantation in the DRN. After recovery, animals were administered 100 µg of p-chlorophenylalanine (PCPA), an inhibitor of 5-HT synthesis, in the DRN each day for four days. On the fourth day, leptin was also administered in the DRN (5 µg/rat) and food intake was measured over a 24-hour time course. ANOVA analysis revealed a significant difference in 24-hour food intake [F (3, 18) = 3.972; P = 0.0246] and post-hoc analysis showed that animals treated with leptin significantly decreased food intake (17.2 ± 2.0 g) compared to control rats (25.4 ± 0.9 g), whereas PCPA-treated rats did not differ from the control rats, suggesting that depletion of 5-HT attenuated leptin’s ability to regulate food intake within the DRN. To examine the role of 5-HT on leptin’s hypothalamic action, a subsequent experiment was conducted by implanting an additional cannula into the ARC for the administration of leptin or vehicle on the fourth day of treatment. ANOVA analysis revealed a significant difference in 24-hour food intake [F (3, 16) = 5.998; P = 0.0061] and post-hoc analysis showed that only rats treated with leptin in the ARC significantly decreased food intake (14.0 ± 1.5 g) compared to controls (21.8 ± 0.5 g). 5-HT depletion was assessed post-mortem using immunohistochemistry and was later quantified. Collectively, these results demonstrate that leptin’s ability to regulate food intake is dependent on 5-HT, regardless of the area of regulation (i.e. DRN or the hypothalamus).

Phox2b‐expressing neurons of the retrotrapezoid nucleus regulate post‐inspiration in conscious mice

The retrotrapezoid nucleus (RTN) contains Phox2b-expressing glutamatergic neurons that regulate all aspects of breathing in a CO2-depende manner. In the present study, we hypothesized that neurons in this nucleus would also be involved in the post-inspiratory phase of the respiratory cycle. Here we use a combination of phamacogenetic and genetic modification mice (Phox2b-cre+/-) to explore how these neurons control breathing in conscious and anesthetized adult mice. Phox2b-expressing neurons in the RTN region were targeted with a adeno associate vector to induce the expression of an artificial G(q) protein-coupled receptor termed designer receptors exclusively activated by designer drugs (DREADD-Gq). The transduced neurons were activated by systemic administration of otherwise inert ligand clozapine-n-oxide (CNO). Breathing measurements were recorded in conscious freely moving mce before and after RTN stimulation. Under baseline conditions, administration of CNO (90 µg/mL - 0.1 mL/animal) increased the respiratory rate (fR: 207.7 ± 25.3 vs. vehicle: 152.3 ± 5.8 bpm), tidal volume (VT: 5.0 ± 0.64, vs. vehicle: 4.1 ± 1.5 mL/kg) and ventilation (VE: 1103.0 ± 234 vs. vehicle: 613.6 ± 40.2 mL/min/kg). The CNO injection was also able to elicit an increase in the post-inspiratory phase (0.1077 ± 0.023, vs. vehicle: 0.076 ± 0.008) and in the total expiratory phase (0.0992 ± 0.037, vs vehicle: 0.0476 ± 0.018) in Phox2b-Cre+/- mice. In urethane-anesthetized mice, CNO injection was also able to elicit an increase in intercostal muscle activity (IntEMG: 61 ± 1.3, vs. vehicle: 8 ± 1.4 mV) and an increase in the post-inspiratory of vagal activity (cVN: 62 ± 1.8, vs. vehicle: 29 ± 1.5 mV) and post-inspiratory peak of the vagus nerve (cVN peak: 142 ± 4.2, vs. vehicle: 64 ± 1.4 V). Importantly, pharmacogenetic stimulation of the RTN-Phox2b neurons and a saturating CO₂ concentration produced similar effects on breathing. Therefore, consistent with their anatomical projections, RTN-Phox2b neurons regulate lung ventilation by controlling all aspects of breathing, i.e breathing frequency, inspiration, post-inspiration and active expiration.

Noradrenergic Locus Coeruleus Neurons Send Monosynaptic Excitatory Drive to Opioid‐sensitive Kölliker‐Fuse Neurons in Mice

The opioid epidemic remains a major public health crisis, with constantly increasing overdoses. Fatal outcomes from opioid overdose primarily result from respiratory failure, mediated by mu-opioid-receptors in respiratory control regions of the brain. Additionally, sedation mediated by the inhibition of arousal/wakefulness centers may further depress breathing. However, the neural substrates and underlying mechanisms of opioid-inhibition of arousal centers, and their impact on respiratory modulation are poorly understood. The pontine Kölliker-Fuse nucleus (KF) is highly susceptible to inhibition by opioids, significantly contributing to opioid-induced rate and pattern changes. Additionally, prior work has identified anatomical projections between the KF and the main noradrenergic arousal center of the brain, the locus coeruleus (LC), representing a potential link between arousal/sedation and respiratory control. Here, using retrograde viral tracer injected into the KF, combined with immunolabeling for the noradrenergic marker, tyrosine hydroxylase (TH), we show that on average ~30% of noradrenergic LC neurons send projections to the KF. To explore the functional role and neurochemical identity of LC to KF projections, we expressed channelrhodopsin-2 in the LC of TH-Cre hemizygous mice. Whole-cell patch clamp recordings combined with optogenetic stimulation of LC neurons, and immunohistochemistry confirmed selective, functional channelrhodopsin-2 expression in noradrenergic LC cells. Further, optically stimulated LC axon terminals in coronal KF slices induced light-evoked excitatory postsynaptic potentials in KF neurons. Experiments utilizing pharmacological isolation of LC to KF synapses indicate that the projections between the LC and KF are monosynaptic and glutamatergic. We also found that a subset of KF neurons receiving monosynaptic drive from the LC is sensitive to opioids, as application of a mu-opioid receptor agonist resulted in a hyperpolarizing outward K+ current. This work provides exciting novel evidence for an opioid-sensitive, excitatory pathway between the LC and the pontine respiratory group.

Characterization of Tyrosine Hydroxylase‐Positive Neurons Projections from the Paraventricular Nucleus of Hypothalamus to Hindbrain Autonomic Regulatory Centers

Hypothalamus is a key brain region for cardiovascular, behavioral, and metabolic regulation that contains complex type of excitatory or inhibitory neurons. Tyrosine hydroxylase (TH)-positive neurons in the hypothalamus play an important role in cardiometabolic regulation; however, the anatomical projections of TH neurons in the paraventricular nucleus hypothalamus (PVN) to key parasympathetic and sympathetic premotor nuclei in preautonomic regions of the brain and their connections to the liver remains undefined. This study aims to characterize the projections from THPVN neurons to ventral preautonomic regulating centers and their connection to liver neuronal projections. To achieve our goal, Cre recombinase-dependent TIT2L-eGFP reporter mice were microinjected with adeno-associated virus encoding the Cre recombinase driven by a rat TH promoter (AAV2-TH-Cre, 6.8 x 108 vg/site) bilaterally into the PVN using a stereotaxic apparatus. A retrograde pseudorabies virus with red fluorescence reporter (PRV-614-RFP) was injected into the liver (3 sites, 3ul/site) to identify liver-related neurons in the brainstem preautonotmic centers. One month after injection, mice were transcardially perfused and brain tissues were processed for confocal imaging. The AAV2-TH-Cre induced a robust expression of GFP mostly in the PVN neuronal cell bodies but not in other brain regions. In the brainstem preautonomic centers, we found dense projections in the nucleus tractus solitarius (NTS) and some projections to the dorsal motor nucleus of the vagus (DMV) and rostral ventrolateral medulla (RVLM). Interestingly, there was also dense projects into the median eminence (ME) that regulates neural hormones release. Importantly, the PRV-RFP retrograded from liver was found throughout the DMV and ventrolateral NTS . Particularly in the NTS, there was dense synaptic contact of the THPVN neuronal projections with the liver related neurons (PRV-RFP+ cell). In addition, we also observed that some PRV-RFP+ cells colocalized with TH-positive neurons in the PVN. In sum, the THPVN neurons project anatomically to the autonomic and neurosecretory regions of brain and may modulate the cardiometabolic activity and liver metabolism through these projections.

Aesthetic Enhancement of the Vermilion Using Dermofat Graft in Patients With Cleft Lip Deformity.

There is an increasing patient expectation for better esthetics, manifesting through anatomical harmony, projection, and volume of the upper lip relative to the lower lip, in patients with cleft deformity. The aim of this study is to investigate the outcome of vermilion augmentation using autologous dermofat graft (DFG) to enhance the lip using both quantitative and qualitative assessment. Patients with secondary cleft lip deformity who received the surgical treatment in our institution from 2015 to 2018 were recruited. Panel assessment was performed on standardized preoperative and postoperative digital photographs. A patient questionnaire was used for the reported outcome. Image processing and analyses were applied to measure the lip change. Statistical analyses were performed. A total of 91 patients were included. The mean age at operation was 22.7 years, and postoperative follow-up was 3.6 years. There were no complications in the study group. The panel assessment showed significant improvement (P < .00001) on upper lip vermilion in both frontal and lateral profile views. Ninety-five percent of patients reported improvement of the upper lip projection and volume. Quantitative image analysis showed an increase in the upper lip vermilion ratio in 97% of the cases in frontal views and improvement of the vermilion projection in 87% of the cases in the superimposed lateral views. The use of DFG is an effective and reproducible method for vermilion augmentation and aesthetic enhancement in patients presenting with upper lip insufficiency relative to the lower lip.

Anatomical projections to the dorsal tegmental nucleus and abducens nucleus arise from separate cell populations in the nucleus prepositus hypoglossi, but overlapping cell populations in the medial vestibular nucleus.

Specialized circuitry in the brain processes spatial information to provide a sense of direction used for navigation. The dorsal tegmental nucleus (DTN) is a core component of this circuitry and utilizes vestibular inputs to generate neural activity encoding the animal's directional heading. Projections arising from the nucleus prepositus hypoglossi (NPH) and the medial vestibular nucleus (MVe) are thought to transmit critical vestibular signals to the DTN and other brain areas, including the abducens nucleus (ABN), a component of eye movement circuitry. Here, we utilized a dual retrograde tracer approach in rats to investigate whether overlapping or distinct populations of neurons project from the NPH or MVe to the DTN and ABN. We report that individual MVe neurons project to both the DTN and ABN. In contrast, we observed individual NPH neurons that project to either the DTN or ABN, but rarely to both structures simultaneously. We also examined labeling patterns in other structures located in the brainstem and posterior cortex and observed (1) complex patterns of interhemispheric connectivity between the left and right DTN, (2) projections from the supragenual nucleus, interpeduncular nucleus, and retrosplenial cortex to the DTN, (3) projections from the lateral superior olive to the ABN, and (4) a unique population of cerebrospinal fluid-contacting neurons in the dorsal raphe nucleus. Collectively, our experiments provide valuable new information that extends our understanding of the anatomical organization of the brain's spatial processing circuitry.

Defective AMPA-mediated synaptic transmission and morphology in human neurons with hemizygous SHANK3 deletion engrafted in mouse prefrontal cortex

Genetic abnormalities in synaptic proteins are common in individuals with autism; however, our understanding of the cellular and molecular mechanisms disrupted by these abnormalities is limited. SHANK3 is a postsynaptic scaffolding protein of excitatory synapses that has been found mutated or deleted in most patients with 22q13 deletion syndrome and about 2% of individuals with idiopathic autism and intellectual disability. Here, we generated CRISPR/Cas9-engineered human pluripotent stem cells (PSCs) with complete hemizygous SHANK3 deletion (SHANK3+/–), which is the most common genetic abnormality in patients, and investigated the synaptic and morphological properties of SHANK3-deficient PSC-derived cortical neurons engrafted in the mouse prefrontal cortex. We show that human PSC-derived neurons integrate into the mouse cortex by acquiring appropriate cortical layer identities and by receiving and sending anatomical projections from/to multiple different brain regions. We also demonstrate that SHANK3-deficient human neurons have reduced AMPA-, but not NMDA- or GABA-mediated synaptic transmission and exhibit impaired dendritic arbors and spines, as compared to isogenic control neurons co-engrafted in the same brain region. Together, this study reveals specific synaptic and morphological deficits caused by SHANK3 hemizygosity in human cortical neurons at different developmental stages under physiological conditions and validates the use of co-engrafted control and mutant human neurons as a new platform for studying connectivity deficits in genetic neurodevelopmental disorders associated with autism.

Animal Models of Tinnitus Treatment: Cochlear and Brain Stimulation.

Neuromodulation, via stimulation of a variety of peripheral and central structures, is used to suppress tinnitus. However, investigative limitations in humans due to ethical reasons have made it difficult to decipher the mechanisms underlying treatment-induced tinnitus relief, so a number of animal models have arisen to address these unknowns. This chapter reviews animal models of cochlear and brain stimulation and assesses their modulatory effects on behavioral evidence of tinnitus and its related neural correlates. When a structure is stimulated, localized modulation, often presenting as downregulation of spontaneous neuronal spike firing rate, bursting and neurosynchrony, occurs within the brain area. Through anatomical projections and transmitter pathways, the interventions activate both auditory- and non-auditory structures by taking bottom-up ascending and top-down descending modes to influence their target brain structures. Furthermore, it is the brain oscillations that cochlear or brain stimulation evoke and connect the prefrontal cortex, striatal systems, and other limbic structures to refresh neural networks and relieve auditory, attentive, conscious, as well as emotional reactive aspects of tinnitus. This oscillatory neural network connectivity is achieved via the thalamocorticothalamic circuitry including the lemniscal and non-lemniscal auditory brain structures. Beyond existing technologies, the review also reveals opportunities for developing advanced animal models using new modalities to achieve precision neuromodulation and tinnitus abatement, such as optogenetic cochlear and/or brain stimulation.

Three-Dimensional Determination of the Fusion Zone between the Distal Maxilla and the Pterygoid Plate of the Sphenoid Bone and Considerations for Implant Treatment Procedure

During pre-operation planning, an implantologist has to decide about the location of a dental implant based on the available bone, anatomical structures and future prosthetics. The aim of this study was to provide an overview of the configurations of the junction zone of the pterygoid process, maxillary tuberosity and pyramidal process among the population and to determine the usefulness of 3D model visualization in regard to precision of anatomical structure projections for clinical planning. A total of 72 cases were analyzed for seven measurements (lateral, medial, rostral, caudal, area, line-1 longitudinal, line-2 transverse) on both body sides—right (R) and left (L). In 57 cases, age and sex of the patient were given. In 15 cases this information was missing. Among the group of 57 cases with complete data, there were 30 females (F) and 27 males (M). A total of 57 models of upper jaws including the adjacent pterygoid process of the sphenoid bone were taken for investigation. The results of the comparison between the right and left side showed no differences (p &gt; 0.05) in values of the measured parameters. The results of the comparison between males and females showed a statistically significant difference when assessing the line-2 transverse (p &lt; 0.05)—in the male group the average was 8.22 mm, in the female group the average was lower (7.83 mm). No statistically significant differences in values of the measured parameters for females and males were found for the left side nor for the right side. In all examined specimens there was enough bone surface in the fusion zone to allow for the stable placement of one tuberopterygoid implant.

Endogenous Cholinergic Signaling Modulates Sound-Evoked Responses of the Medial Nucleus of the Trapezoid Body.

The medial nucleus of trapezoid body (MNTB) is a major source of inhibition in auditory brainstem circuitry. The MNTB projects well-timed inhibitory output to principal sound-localization nuclei in the superior olive (SOC) as well as other computationally important centers. Acoustic information is conveyed to MNTB neurons through a single calyx of Held excitatory synapse arising from the cochlear nucleus. The encoding efficacy of this large synapse depends on its activity rate, which is primarily determined by sound intensity and stimulus frequency. However, MNTB activity rate is additionally influenced by inhibition and possibly neuromodulatory inputs, albeit their functional role is unclear. Happe and Morley (2004) discovered prominent expression of α7 nAChRs in rat SOC, suggesting possible engagement of ACh-mediated modulation of neural activity in the MNTB. However, the existence and nature of this putative modulation have never been physiologically demonstrated. We probed nicotinic cholinergic influences on acoustic responses of MNTB neurons from adult gerbils (Meriones unguiculatus) of either sex. We recorded tone-evoked MNTB single-neuron activity in vivo using extracellular single-unit recording. Piggyback multibarrel electrodes enabled pharmacological manipulation of nAChRs by reversibly applying antagonists to two receptor types, α7 and α4β2. We observed that tone-evoked responses are dependent on ACh modulation by both nAChR subtypes. Spontaneous activity was not affected by antagonist application. Functionally, we demonstrate that ACh contributes to sustaining high discharge rates and enhances signal encoding efficacy. Additionally, we report anatomic evidence revealing novel cholinergic projections to MNTB arising from pontine and superior olivary nuclei.SIGNIFICANCE STATEMENT This study is the first to physiologically probe how acetylcholine, a pervasive neuromodulator in the brain, influences the encoding of acoustic information by the medial nucleus of trapezoid body, the most prominent source of inhibition in brainstem sound-localization circuitry. We demonstrate that this cholinergic input enhances neural discrimination of tones from noise stimuli, which may contribute to processing important acoustic signals, such as speech. Additionally, we describe novel anatomic projections providing cholinergic input to the MNTB. Together, these findings shed new light on the contribution of neuromodulation to fundamental computational processes in auditory brainstem circuitry and to a more holistic understanding of modulatory influences in sensory processing.

White Matter Hyperintensity Burden Is Associated With Hippocampal Subfield Volume in Stroke.

White matter hyperintensities of presumed vascular origin (WMH) are a prevalent form of cerebral small-vessel disease and an important risk factor for post-stroke cognitive dysfunction. Despite this prevalence, it is not well understood how WMH contributes to post-stroke cognitive dysfunction. Preliminary findings suggest that increasing WMH volume is associated with total hippocampal volume in chronic stroke patients. The hippocampus, however, is a complex structure with distinct subfields that have varying roles in the function of the hippocampal circuitry and unique anatomical projections to different brain regions. For these reasons, an investigation into the relationship between WMH and hippocampal subfield volume may further delineate how WMH predispose to post-stroke cognitive dysfunction. In a prospective study of acute ischemic stroke patients with moderate/severe WMH burden, we assessed the relationship between quantitative WMH burden and hippocampal subfield volumes. Patients underwent a 3T MRI brain within 2–5 days of stroke onset. Total WMH volume was calculated in a semi-automated manner. Mean cortical thickness and hippocampal volumes were measured in the contralesional hemisphere. Total and subfield hippocampal volumes were measured using an automated, high-resolution, ex vivo computational atlas. Linear regression analyses were performed for predictors of total and subfield hippocampal volumes. Forty patients with acute ischemic stroke and moderate/severe white matter hyperintensity burden were included in this analysis. Median WMH volume was 9.0 cm3. Adjusting for intracranial volume and stroke laterality, age (β = −3.7, P < 0.001), hypertension (β = −44.7, P = 0.04), WMH volume (β = −0.89, P = 0.049), and mean cortical thickness (β = 286.2, P = 0.006) were associated with total hippocampal volume. In multivariable analysis, age (β = −3.3, P < 0.001) and cortical thickness (β = 205.2, P = 0.028) remained independently associated with total hippocampal volume. In linear regression for predictors of hippocampal subfield volume, increasing WMH volume was associated with decreased hippocampal-amygdala transition area volume (β = −0.04, P = 0.001). These finding suggest that in ischemic stroke patients, increased WMH burden is associated with selective hippocampal subfield degeneration in the hippocampal-amygdala transition area.

Extraction of Distinct Neuronal Cell Types from within a Genetically Continuous Population

Single-cell transcriptomics of neocortical neurons have revealed more than 100 clusters corresponding to putative cell types. For inhibitory and subcortical projection neurons (SCPNs), there is a strong concordance between clusters and anatomical descriptions of cell types. In contrast, cortico-cortical projection neurons (CCPNs) separate into surprisingly few transcriptomic clusters, despite their diverse anatomical projection types. We used projection-dependent single-cell transcriptomic analyses and monosynaptic rabies tracing to compare mouse primary visual cortex CCPNs projecting to different higher visual areas. We find that layer 2/3 CCPNs with different anatomical projections differ systematically in their gene expressions, despite forming only a single genetic cluster. Furthermore, these neurons receive feedback selectively from the same areas to which they project. These findings demonstrate that gene-expression analysis in isolation is insufficient to identify neuron types and have important implications for understanding the functional role of cortical feedback circuits.

OR16-03 Metabolic Effects Of Hypothalamic Pomc Neurons Generated Postnatally From Tanycytes On A Pomc Null Genetic Background

Hypothalamic proopiomelanocortin (POMC) neurons are an integral part of the central melanocortin system and regulate feeding and energy balance in vertebrates. Tanycytes are radial glial-like cells lining the third ventricle that contain a subpopulation of adult stem cells, which can differentiate under specific circumstances into glia and neurons, including POMC neurons. However, the capacity of these stem cell-derived neurons to fully mature and integrate into existing neural circuits of physiological relevance is unknown. This study systematically tested whether Pomc mRNA-positive cells newly generated from tanycyte precursors can differentiate into melanocortin-secreting POMC neurons, integrate into the normal anatomical projection pathways of these cells and rescue the obesity phenotype caused by the loss of Pomc expression in ArcPomcfneo/fneo mice. We generated an inducible compound genetic mouse model by crossing RaxCreERT2 with the Cre-dependent ArcPomcfneo/fneo and LSL-syp-tdTomato alleles. Rax is expressed exclusively in postnatal tanycytes, thereby limiting tamoxifen-induced recombination of the two floxed alleles by CreERT2 to tanycytes. As expected, tamoxifen treatment of the mice at age 4–5 wk recapitulated endogenous Rax expression 16 wk later as observed by red fluorescent tdTomato expression in all tanycytes. In addition, Cre recombinase-mediated deletion of the floxed-neomycin cassette from the neuronal enhancer region of the ArcPomcfneo alleles relieved their constitutive transcriptional silencing. Consequently, tamoxifen treatment consistently generated a significant number of newly generated POMC neurons from tanycytes (~10% of the POMC neurons in a WT mouse), identified by Pomc FISH and POMC/α-MSH immunofluorescence in the soma and established terminal projections to hypothalamic nuclei including the PVH and DMH involved in energy homeostasis. A subpopulation of these neurons also expressed the synaptophysin-tDTomato reporter. We performed serial body weight, food intake, body composition, oral GTT and insulin measurements with the RaxCreERT2/+, ArcPomcfneo/fneo mice and found no significant differences in any of these metabolic variables compared to untreated obese ArcPomcfneo/fneo mice. These data are consistent with previous studies from our lab suggesting that Pomc expression has to be at least ~30% of normal to mitigate the obesity phenotype in Pomc-null mice. In conclusion, we demonstrated that tanycytes are capable of generating mature Pomc-expressing neurons in the hypothalamus of adult mice. However, we propose that determining the underlying mechanisms involved in the generation of hypothalamic POMC neurons from tanycytes and interventions to increase their number, might lead to a novel approach to treat obesity. Nothing to Disclose: SG, GW, RML, MJL

GABAergic neurons of the medullary raphe regulate active expiration during hypercapnia.

The parafacial respiratory group (pFRG), located in the lateral aspect of the rostroventral lateral medulla, has been described as a conditional expiratory oscillator that emerges mainly in conditions of high metabolic challenges to increase breathing. The convergence of inhibitory and excitatory inputs to pFRG and the generation of active expiration may be more complex than previously thought. We hypothesized that the medullary raphe, a region that has long been described to be involved in breathing activity, is also responsible for the expiratory activity under hypercapnic condition. To test this hypothesis, we performed anatomical and physiological experiments in urethane-anesthetized adult male Wistar rats. Our data showed anatomical projections from serotonergic (5-HT-ergic) and GABAergic neurons of raphe magnus (RMg) and obscurus (ROb) to the pFRG region. Pharmacological inhibition of RMg or ROb with muscimol (60 pmol/30 nL) did not change the frequency or amplitude of diaphragm activity and did not generate active expiration. However, under hypercapnia (9-10% CO2), the inhibition of RMg or ROb increased the amplitude of abdominal activity, without changing the increased amplitude of diaphragm activity. Depletion of serotonergic neurons with saporin anti-SERT injections into ROb and RMg did not increase the amplitude of abdominal activity during hypercapnia. These results show that the presumably GABAergic neurons within the RMg and ROb may be the inhibitory source to modulate the activity of pFRG during hypercapnia condition.NEW & NOTEWORTHY Medullary raphe has been involved in the inspiratory response to central chemoreflex; however, these reports have never addressed the role of raphe neurons on active expiration induced by hypercapnia. Here, we showed that a subset of GABA cells within the medullary raphe directly project to the parafacial respiratory region, modulating active expiration under high levels of CO2.

Density center-based fast clustering of widefield fluorescence imaging of cortical mesoscale functional connectivity and relation to structural connectivity.

.Spontaneous resting-state neural activity or hemodynamics has been used to reveal functional connectivity in the brain. However, most of the commonly used clustering algorithms for functional parcellation are time-consuming, especially for high-resolution imaging data. We propose a density center-based fast clustering (DCBFC) method that can rapidly perform the functional parcellation of isocortex. DCBFC was validated using both simulation data and the spontaneous calcium signals from widefield fluorescence imaging of excitatory neuron-expressing transgenic mice (Vglut2-GCaMP6s). Compared to commonly used clustering methods such as k-means, hierarchical, and spectral, DCBFC showed a higher adjusted Rand index when the signal-to-noise ratio was greater than for simulated data and higher silhouette coefficient for in vivo mouse data. The resting-state functional connectivity (RSFC) patterns obtained by DCBFC were compared with the anatomic axonal projection density (PDs) maps derived from the voxel-scale model. The results showed a high spatial correlation between RSFC patterns and PDs.

Involvement of rat posterior prelimbic and cingulate area 2 in vocalization control.

Microstimulation mapping identified vocalization areas in primate anterior cingulate cortex. Rat anterior cingulate and medial prefrontal areas have also been intensely investigated, but we do not know, how these cortical areas contribute to vocalizations and no systematic mapping of stimulation‐evoked vocalizations has been performed. To address this question, we mapped microstimulation‐evoked (ultrasonic) vocalizations in rat cingulate and medial prefrontal cortex. The incidence of evoked vocalizations differed markedly between frontal cortical areas. Vocalizations were most often evoked in posterior prelimbic cortex and cingulate area 2, whereas vocalizations were rarely evoked in dorsal areas (vibrissa motor cortex, secondary motor cortex and cingulate area 1) and anterior areas (anterior prelimbic, medial‐/ventral‐orbital cortex). Vocalizations were observed at intermediate frequencies in ventro‐medial areas (infralimbic and dorsopeduncular cortex). Various complete, naturally occurring calls could be elicited. In prelimbic cortex superficial layer microstimulation evoked mainly fear calls with low efficacy, whereas deep layer microstimulation evoked mainly 50 kHz calls with high efficacy. Vocalization stimulation thresholds were substantial (70–500 μA, the maximum tested; on average ~400 μA) and latencies were long (median 175 ms). Posterior prelimbic cortex projected to numerous targets and innervated brainstem vocalization centers such as the intermediate reticular formation and the nucleus retroambiguus disynaptically via the periaqueductal gray. Anatomical position, stimulation effects and projection targets of posterior prelimbic cortex were similar to that of monkey anterior cingulate vocalization cortex. Our data suggest that posterior prelimbic cortex is more closely involved in control of vocalization initiation than in specifying acoustic details of vocalizations.