Retrograde Tracing Research Articles

A Novel Retrograde AAV Variant for Functional Manipulation of Cortical Projection Neurons in Mice and Monkeys.

Retrograde adeno-associated viruses (AAVs) are capable of infecting the axons of projection neurons and serve as a powerful tool for the anatomical and functional characterization of neural networks. However, few retrograde AAV capsids have been shown to offer access to cortical projection neurons across different species and enable the manipulation of neural function in non-human primates (NHPs). Here, we report the development of a novel retrograde AAV capsid, AAV-DJ8R, which efficiently labeled cortical projection neurons after local administration into the striatum of mice and macaques. In addition, intrastriatally injected AAV-DJ8R mediated opsin expression in the mouse motor cortex and induced robust behavioral alterations. Moreover, AAV-DJ8R markedly increased motor cortical neuron firing upon optogenetic light stimulation after viral delivery into the macaque putamen. These data demonstrate the usefulness of AAV-DJ8R as an efficient retrograde tracer for cortical projection neurons in rodents and NHPs and indicate its suitability for use in conducting functional interrogations.

Whole-brain monosynaptic inputs to lateral periaqueductal gray glutamatergic neurons in mice.

The lateral periaqueductal gray (LPAG), which mainly contains glutamatergic neurons, plays an important role in social responses, pain, and offensive and defensive behaviors. Currently, the whole-brain monosynaptic inputs to LPAG glutamatergic neurons are unknown. This study aims to explore the structural framework of the underlying neural mechanisms of LPAG glutamatergic neurons. This study used retrograde tracing systems based on the rabies virus, Cre-LoxP technology, and immunofluorescence analysis. We found that 59 nuclei projected monosynaptic inputs to the LPAG glutamatergic neurons. In addition, seven hypothalamic nuclei, namely the lateral hypothalamic area (LH), lateral preoptic area (LPO), substantia innominata (SI), medial preoptic area, ventral pallidum, posterior hypothalamic area, and lateral globus pallidus, projected most densely to the LPAG glutamatergic neurons. Notably, we discovered through further immunofluorescence analysis that the inputs to the LPAG glutamatergic neurons were colocalized with several markers related to important neurological functions associated with physiological behaviors. The LPAG glutamatergic neurons received dense projections from the hypothalamus, especially nuclei such as LH, LPO, and SI. The input neurons were colocalized with several markers of physiological behaviors, which show the pivotal role of glutamatergic neurons in the physiological behaviors regulation by LPAG.

Expansion microscopy in honeybee brains for high-resolution neuroanatomical analyses in social insects

The diffraction limit of light microscopy poses a problem that is frequently faced in structural analyses of social insect brains. With the introduction of expansion microscopy (ExM), a tool became available to overcome this limitation by isotropic physical expansion of preserved specimens. Our analyses focus on synaptic microcircuits (microglomeruli, MG) in the mushroom body (MB) of social insects, high-order brain centers for sensory integration, learning, and memory. MG undergo significant structural reorganizations with age, sensory experience, and during long-term memory formation. However, the changes in subcellular architecture involved in this plasticity have only partially been accessed yet. Using the western honeybee Apis mellifera as an experimental model, we established ExM for the first time in a social insect species and applied it to investigate plasticity in synaptic microcircuits within MG of the MB calyces. Using combinations of antibody staining and neuronal tracing, we demonstrate that this technique enables quantitative and qualitative analyses of structural neuronal plasticity at high resolution in a social insect brain.

A Novel Mouse Model for Polysynaptic Retrograde Tracing and Rabies Pathological Research.

Retrograde tracing is an important method for dissecting neuronal connections and mapping neural circuits. Over the past decades, several virus-based retrograde tracers have been developed and have contributed to display multiple neural circuits in the brain. However, most of the previously widely used viral tools have focused on mono-transsynaptic neural tracing within the central nervous system, with very limited options for achieving polysynaptic tracing between the central and peripheral nervous systems. In this study, we generated a novel mouse line, GT mice, in which both glycoprotein (G) and ASLV-A receptor (TVA) were expressed throughout the body. Using this mouse model, in combination with the well-developed rabies virus tools (RABV-EnvA-ΔG) for monosynaptic retrograde tracing, polysynaptic retrograde tracing can be achieved. This allows functional forward mapping and long-term tracing. Furthermore, since the G-deleted rabies virus can travel upstream against the nervous system as the original strain, this mouse model can also be used for rabies pathological studies. Schematic illustrations about the application principles of GT mice in polysynaptic retrograde tracing and rabies pathological research.

The Brain/MINDS Marmoset Connectivity Resource: An open-access platform for cellular-level tracing and tractography in the primate brain.

The primate brain has unique anatomical characteristics, which translate into advanced cognitive, sensory, and motor abilities. Thus, it is important that we gain insight on its structure to provide a solid basis for models that will clarify function. Here, we report on the implementation and features of the Brain/MINDS Marmoset Connectivity Resource (BMCR), a new open-access platform that provides access to high-resolution anterograde neuronal tracer data in the marmoset brain, integrated to retrograde tracer and tractography data. Unlike other existing image explorers, the BMCR allows visualization of data from different individuals and modalities in a common reference space. This feature, allied to an unprecedented high resolution, enables analyses of features such as reciprocity, directionality, and spatial segregation of connections. The present release of the BMCR focuses on the prefrontal cortex (PFC), a uniquely developed region of the primate brain that is linked to advanced cognition, including the results of 52 anterograde and 164 retrograde tracer injections in the cortex of the marmoset. Moreover, the inclusion of tractography data from diffusion MRI allows systematic analyses of this noninvasive modality against gold-standard cellular connectivity data, enabling detection of false positives and negatives, which provide a basis for future development of tractography. This paper introduces the BMCR image preprocessing pipeline and resources, which include new tools for exploring and reviewing the data.

AAV11 enables efficient retrograde targeting of projection neurons and enhances astrocyte-directed transduction

Viral tracers that enable efficient retrograde labeling of projection neurons are powerful vehicles for structural and functional dissections of the neural circuit and for the treatment of brain diseases. Currently, some recombinant adeno-associated viruses (rAAVs) based on capsid engineering are widely used for retrograde tracing, but display undesirable brain area selectivity due to inefficient retrograde transduction in certain neural connections. Here we developed an easily editable toolkit to produce high titer AAV11 and demonstrated that it exhibits potent and stringent retrograde labeling of projection neurons in adult male wild-type or Cre transgenic mice. AAV11 can function as a powerful retrograde viral tracer complementary to AAV2-retro in multiple neural connections. In combination with fiber photometry, AAV11 can be used to monitor neuronal activities in the functional network by retrograde delivering calcium-sensitive indicator under the control of a neuron-specific promoter or the Cre-lox system. Furthermore, we showed that GfaABC1D promoter embedding AAV11 is superior to AAV8 and AAV5 in astrocytic tropism in vivo, combined with bidirectional multi-vector axoastrocytic labeling, AAV11 can be used to study neuron-astrocyte connection. Finally, we showed that AAV11 allows for analyzing circuit connectivity difference in the brains of the Alzheimer’s disease and control mice. These properties make AAV11 a promising tool for mapping and manipulating neural circuits and for gene therapy of some neurological and neurodegenerative disorders.

A selective projection from the subthalamic nucleus to parvalbumin-expressing interneurons of the striatum.

The striatum and subthalamic nucleus (STN) are considered to be the primary input nuclei of the basal ganglia. Projection neurons of both striatum and STN can extensively interact with other basal ganglia nuclei, and there is growing anatomical evidence of direct axonal connections from the STN to striatum. There remains, however, a pressing need to elucidate the organization and impact of these subthalamostriatal projections in the context of the diverse cell types constituting the striatum. To address this, we carried out monosynaptic retrograde tracing from genetically-defined populations of dorsal striatal neurons in adult male and female mice, quantifying the connectivity from STN neurons to spiny projection neurons, GABAergic interneurons, and cholinergic interneurons. In parallel, we used a combination of ex vivo electrophysiology and optogenetics to characterize the responses of a complementary range of dorsal striatal neuron types to activation of STN axons. Our tracing studies showed that the connectivity from STN neurons to striatal parvalbumin-expressing interneurons is significantly higher (∼ four- to eight-fold) than that from STN to any of the four other striatal cell types examined. In agreement, our recording experiments showed that parvalbumin-expressing interneurons, but not the other cell types tested, commonly exhibited robust monosynaptic excitatory responses to subthalamostriatal inputs. Taken together, our data collectively demonstrate that the subthalamostriatal projection is highly selective for target cell type. We conclude that glutamatergic STN neurons are positioned to directly and powerfully influence striatal activity dynamics by virtue of their enriched innervation of GABAergic parvalbumin-expressing interneurons.Significance StatementPlacing the subthalamostriatal projection within schemes of basal ganglia circuit organization is challenging because of the diversity of cell types within striatum. Here, we shed new light on the structural and electrophysiological substrates by which STN neurons can exert direct and biased influences on the striatal microcircuit. We discovered that STN innervation of parvalbumin-expressing interneurons is relatively enriched and impactful as compared to innervation of other types of striatal neuron. Accordingly, the STN joins a growing list of subcortical structures that, although not considered 'canonical' sources of inputs to striatum, selectively target striatal interneurons. Our results are important in supporting the concept that the glutamatergic subthalamostriatal projection is positioned to fulfil diverse and likely unique roles within basal ganglia circuits.

Whole-brain neural connectivity to cholinergic neurons in the nucleus basalis of Meynert.

The cholinergic neurons in the nucleus basalis of Meynert (NBM) are a key structure in cognition, the dysfunction of which is associated with various neurological disorders, especially dementias. However, the whole-brain neural connectivity to cholinergic neurons in the NBM remains to be further and comprehensively researched. Using virus-based, specific, retrograde, and anterograde tracing, we illustrated the monosynaptic inputs and axon projections of NBM cholinergic neurons in choline acetyltransferase (ChAT)-Cre transgenic mice. Our results showed that NBM cholinergic neurons received mainly inputs from the caudate putamen and the posterior limb of the anterior commissure in the subcortex. Moreover, the majority of cholinergic terminals from the NBM were observed in the cortex mantle, including the motor cortex, sensory cortex, and visual cortex. Interestingly, although NBM cholinergic neurons received input projections from the caudate putamen, interstitial nucleus of the posterior limb of the anterior commissure, and central amygdaloid nucleus, NBM cholinergic neurons sparsely sent axon projection to innervate these areas. Furthermore, primary motor cortex, secondary motor cortex, and primary somatosensory cortex received abundant inputs from the NBM but sent few outputs to the NBM. Taken together, our results reveal the detailed and specific connectivity of cholinergic neurons of the NBM and provide a neuroanatomic foundation for further studies to explore the important physiological functions of NBM cholinergic neurons.

Artemin sensitizes nociceptors that innervate the osteoarthritic joint to produce pain

There have been significant developments in understanding artemin/GFRα3 signaling in recent years, and there is now accumulating evidence that artemin has important roles to play in pain signaling, including that derived from joint and bone, and that associated with OA. A total of 163 Sprague Dawley rats were used in this study. We used an animal model of mono-iodoacetate (MIA)-induced OA, in combination with electrophysiology, behavioral testing, Western blot analysis, and retrograde tracing and immunohistochemistry, to identify roles for artemin/GFRα3 signaling in the pathogenesis of OA pain. We have found that: 1) GFRα3 is expressed in a substantial proportion of knee joint afferent neurons; 2) exogenous artemin sensitizes knee joint afferent neurons in naïve rats; 3) artemin is expressed in articular tissues of the joint, but not surrounding bone, early in MIA-induced OA; 4) artemin expression increases in bone later in MIA-induced OA when pathology involves subchondral bone; and 5) sequestration of artemin reverses MIA-induced sensitization of both knee joint and bone afferent neurons late in disease when there is inflammation of knee joint tissues and damage to the subchondral bone. Our findings show that artemin/GFRα3 signaling has a role to play in the pathogenesis of OA pain, through effects on both knee joint and bone afferent neurons, and suggest that targeted manipulation of artemin/GFRα3 signaling may provide therapeutic benefit for the management of OA pain.

301-OR: Estrogen Signaling in the Ventromedial Hypothalamus Modulates Adipose Tissue Metabolic Adaptation

Brain estrogen receptor α (ERα) signaling plays a vital role in the regulation of energy homeostasis and adipose tissue metabolism. It has been demonstrated that ERα is abundantly expressed in the ventrolateral region of the ventromedial hypothalamus (vlVMH), a sex-dimorphic structure that directly modulates brown adipose tissue (BAT) thermogenesis. We previously showed that an ERαvlVMH-originated neural circuit responds to changes in ambient temperature and nutritional states, suggesting a potential role of this neural circuit in metabolic adaptation. Here, we found that chronic activation of ERαvlVMH neurons in male mice reduces body weight, decreases adiposity, and prevents diet-induced obesity (DIO), which is associated with increases in BAT thermogenesis and core temperature. Conversely, chronic inhibition of ERαvlVMH neurons increases body weight, promotes adiposity, and decreases baseline BAT temperature. Notably, ERαvlVMH neural inhibition also impairs cold-induced food consumption and BAT thermogenesis, resulting in lethal phenotypes during chronic cold exposure. Post hoc histology analysis further revealed that ERαvlVMH neural inhibition induces adipose tissue whitening. Finally, we explored the anatomical distribution of ERαvlVMH downstream neural circuitries. Anterograde tracing revealed ERαvlVMH-originated projections to the medial preoptic area (MPA), paraventricular nucleus (PVN), substantia nigra (SNR) and dorsal raphe nucleus (DRN). Interestingly, transsynaptic retrograde tracing further demonstrated that these brain regions send projections to inguinal white adipose tissue (iWAT), suggesting potential ERαvlVMH-originated circuits involved in adipose tissue adaptation. Together, these findings support a model that estrogens act through ERαvlVMH neurons to modulate fat-specific outputs and subsequently regulate adipose tissue adaptation to cold challenges. Disclosure V.C.Torres irizarry: None. P.Xu: None. H.Ye: None. P.Luo: None. L.Carrillo-sáenz: None. X.Yang: None. M.D.Munoz: None. N.Antony: None. Y.Jiang: None. Y.He: None. Funding National Institutes of Health (R01DK123098, T32AA026577); U.S. Department of Defense (W81XWH-19-PRMRP-DA); National Institute of Diabetes and Digestive and Kidney Diseases (DK020595); American Heart Association (915789)

Two Ascending Thermosensory Pathways from the Lateral Parabrachial Nucleus That Mediate Behavioral and Autonomous Thermoregulation.

Thermoregulatory behavior in homeothermic animals is an innate behavior to defend body core temperature from environmental thermal challenges in coordination with autonomous thermoregulatory responses. In contrast to the progress in understanding the central mechanisms of autonomous thermoregulation, those of behavioral thermoregulation remain poorly understood. We have previously shown that the lateral parabrachial nucleus (LPB) mediates cutaneous thermosensory afferent signaling for thermoregulation. To understand the thermosensory neural network for behavioral thermoregulation, in the present study, we investigated the roles of ascending thermosensory pathways from the LPB in avoidance behavior from innocuous heat and cold in male rats. Neuronal tracing revealed two segregated groups of LPB neurons projecting to the median preoptic nucleus (MnPO), a thermoregulatory center (LPB→MnPO neurons), and those projecting to the central amygdaloid nucleus (CeA), a limbic emotion center (LPB→CeA neurons). While LPB→MnPO neurons include separate subgroups activated by heat or cold exposure of rats, LPB→CeA neurons were only activated by cold exposure. By selectively inhibiting LPB→MnPO or LPB→CeA neurons using tetanus toxin light chain or chemogenetic or optogenetic techniques, we found that LPB→MnPO transmission mediates heat avoidance, whereas LPB→CeA transmission contributes to cold avoidance. In vivo electrophysiological experiments showed that skin cooling-evoked thermogenesis in brown adipose tissue requires not only LPB→MnPO neurons but also LPB→CeA neurons, providing a novel insight into the central mechanism of autonomous thermoregulation. Our findings reveal an important framework of central thermosensory afferent pathways to coordinate behavioral and autonomous thermoregulation and to generate the emotions of thermal comfort and discomfort that drive thermoregulatory behavior.SIGNIFICANCE STATEMENT Coordination of behavioral and autonomous thermoregulation is important for maintaining thermal homeostasis in homeothermic animals. However, the central mechanism of thermoregulatory behaviors remains poorly understood. We have previously shown that the lateral parabrachial nucleus (LPB) mediates ascending thermosensory signaling that drives thermoregulatory behavior. In this study, we found that one pathway from the LPB to the median preoptic nucleus mediates heat avoidance, whereas the other pathway from the LPB to the central amygdaloid nucleus is required for cold avoidance. Surprisingly, both pathways are required for skin cooling-evoked thermogenesis in brown adipose tissue, an autonomous thermoregulatory response. This study provides a central thermosensory network that coordinates behavioral and autonomous thermoregulation and generates thermal comfort and discomfort that drive thermoregulatory behavior.

Opioid suppression of an excitatory pontomedullary respiratory circuit by convergent mechanisms.

Opioids depress breathing by inhibition of interconnected respiratory nuclei in the pons and medulla. Mu opioid receptor (MOR) agonists directly hyperpolarize a population of neurons in the dorsolateral pons, particularly the Kölliker-Fuse (KF) nucleus, that are key mediators of opioid-induced respiratory depression. However, the projection target and synaptic connections of MOR-expressing KF neurons are unknown. Here, we used retrograde labeling and brain slice electrophysiology to determine that MOR-expressing KF neurons project to respiratory nuclei in the ventrolateral medulla, including the preBötzinger complex (preBötC) and rostral ventral respiratory group (rVRG). These medullary-projecting, MOR-expressing dorsolateral pontine neurons express FoxP2 and are distinct from calcitonin gene-related peptide-expressing lateral parabrachial neurons. Furthermore, dorsolateral pontine neurons release glutamate onto excitatory preBötC and rVRG neurons via monosynaptic projections, which is inhibited by presynaptic opioid receptors. Surprisingly, the majority of excitatory preBötC and rVRG neurons receiving MOR-sensitive glutamatergic synaptic input from the dorsolateral pons are themselves hyperpolarized by opioids, suggesting a selective opioid-sensitive circuit from the KF to the ventrolateral medulla. Opioids inhibit this excitatory pontomedullary respiratory circuit by three distinct mechanisms-somatodendritic MORs on dorsolateral pontine and ventrolateral medullary neurons and presynaptic MORs on dorsolateral pontine neuron terminals in the ventrolateral medulla-all of which could contribute to opioid-induced respiratory depression.

Hippocampal CA3 inhibitory neurons receive extensive noncanonical synaptic inputs from CA1 and subicular complex.

Hippocampal CA3 is traditionally conceptualized as a brain region within a unidirectional feedforward trisynaptic pathway that links major hippocampal subregions. Recent genomic and viral tracing studies indicate that the anatomical connectivity of CA3 and the trisynaptic pathway is more complex than initially expected and suggests that there may be cell type-specific input gradients throughout the three-dimensional hippocampal structure. In several recent studies using multiple viral tracing approaches, we describe subdivisions of the subiculum complex and ventral hippocampal CA1 that show significant back projections to CA1 and CA3 excitatory neurons. These novel connections form "noncanonical" circuits that run in the opposite direction relative to the well-characterized feedforward pathway. Diverse subtypes of GABAergic inhibitory neurons participate within the trisynaptic pathway. In the present study, we have applied monosynaptic retrograde viral tracing to examine noncanonical synaptic inputs from CA1 and subicular complex to the inhibitory neuron in hippocampal CA3. We quantitatively mapped synaptic inputs to CA3 inhibitory neurons to understand how they are connected within and beyond the hippocampus formation. Major brain regions that provide typical inputs to CA3 inhibitory neurons include the medial septum, the dentate gyrus, the entorhinal cortex, and CA3. Noncanonical inputs from ventral CA1 and subicular complex to CA3 inhibitory neurons follow a proximodistal topographic gradient with regard to CA3 subregions. We find novel noncanonical circuit connections between inhibitory CA3 neurons and ventral CA1, subiculum complex, and other brain regions. These results provide a new anatomical connectivity basis to further study the function of CA3 inhibitory neurons.

Directed stepwise tracing of polysynaptic neuronal circuits with replication-deficient pseudorabies virus

Brain functions are accomplished by polysynaptic circuits formed by neurons wired together through multiple orders of synaptic connections. Polysynaptic connectivity has been difficult to examine due to a lack of methods of continuously tracing the pathways in a controlled manner. Here, we demonstrate directed, stepwise retrograde polysynaptic tracing by inducible reconstitution of replication-deficient trans-neuronal pseudorabies virus (PRVΔIE) in the brain. Furthermore, PRVΔIE replication can be temporally restricted to minimize its neurotoxicity. With this tool, we delineate a wiring diagram between the hippocampus and striatum-two major brain systems for learning, memory, and navigation-that consists of projections from specific hippocampal domains to specific striatal areas via distinct intermediate brain regions. Therefore, this inducible PRVΔIE system provides a tool for dissecting polysynaptic circuits underlying complex brain functions.

WNK1 promotes water homeostasis by acting as a central osmolality sensor for arginine vasopressin release.

Maintaining internal osmolality constancy is essential for life. Release of arginine vasopressin (AVP) in response to hyperosmolality is critical. Current hypotheses for osmolality sensors in circumventricular organs (CVOs) of the brain focus on mechanosensitive membrane proteins. The present study demonstrated that intracellular protein kinase WNK1 was involved. Focusing on vascular-organ-of-lamina-terminalis (OVLT) nuclei, we showed that WNK1 kinase was activated by water restriction. Neuron-specific conditional KO (cKO) of Wnk1 caused polyuria with decreased urine osmolality that persisted in water restriction and blunted water restriction-induced AVP release. Wnk1 cKO also blunted mannitol-induced AVP release but had no effect on osmotic thirst response. The role of WNK1 in the osmosensory neurons in CVOs was supported by neuronal pathway tracing. Hyperosmolality-induced increases in action potential firing in OVLT neurons was blunted by Wnk1 deletion or pharmacological WNK inhibitors. Knockdown of Kv3.1 channel in OVLT by shRNA reproduced the phenotypes. Thus, WNK1 in osmosensory neurons in CVOs detects extracellular hypertonicity and mediates the increase in AVP release by activating Kv3.1 and increasing action potential firing from osmosensory neurons.

Connections of the sheep basolateral amygdala: A diffusion tensor imaging study

BackgroundA large amount of anatomic data published over the past decade has provided novel insight into the connections of the basolateral amygdala (BLA) in the rat, cat, and monkey. The mammalian (rat, cat, and monkey) BLA has strong connections with the cortex (especially piriform, and frontal cortices), the hippocampal region (especially perirhinal cortex, entorhinal cortex, and subiculum), the thalamus (in particular, the posterior internuclear nucleus and medial geniculate nucleus) and, to some extent, the hypothalamus. An important question remains as to how well the data obtained in rodents and primates can be extrapolated to ruminants. New methodTo address this issue the connections of the sheep BLA has been determined by Magnetic Resonance Imaging (MRI) and Diffusion Tensor Imaging (DTI, Tractography). ResultsTractography showed ipsilateral connections between the BLA and several areas. Comparison with existing method(s)Reviews based mainly on description of the results obtained using anterograde and retrograde neuronal tracers. In the present research, we prefer to use a non-invasive technique (DTI). ConclusionsThis report shows the existence of specific amygdaloid connections in the sheep.

Cholinergic projections to the preBötzinger complex.

Rhythmic inspiratory activity is generated in the preBötzinger complex (preBötC), a neuronal network located bilaterally in the ventrolateral medulla. Cholinergic neurotransmission affects respiratory rhythmogenic neurons and inhibitory glycinergic neurons in the preBötC. Acetylcholine has been extensively investigated given that cholinergic fibers and receptors are present and functional in the preBötC, are important in sleep/wake cycling, and modulate inspiratory frequency through its action on preBötC neurons. Despite its role in modulating inspiratory rhythm, the source of acetylcholine input to the preBötC is not known. In the present study, we used retrograde and anterograde viral tracing approaches in transgenic mice expressing Cre-recombinase driven by the choline acetyltransferase promoter to identify the source of cholinergic inputs to the preBötC. Surprisingly, we observed very few, if any, cholinergic projections originating from the laterodorsal and pedunculopontine tegmental nuclei (LDT/PPT), two main cholinergic, state-dependent systems long hypothesized as the main source of cholinergic inputs to the preBötC. On the contrary, we identified glutamatergic and GABAergic/glycinergic neurons in the PPT/LDT that send projections to the preBötC. Although these neurons contribute minimally to the direct cholinergic modulation of preBötC neurons, they could be involved in state-dependent regulation of breathing. Our data also suggest that the source of cholinergic inputs to the preBötC appears to originate from cholinergic neurons in neighboring regions of the medulla, the intermediate reticular formation, the lateral paragigantocellularis, and the nucleus of the solitary tract.

Perinatal high-fat diet exposure alters oxytocin and corticotropin releasing factor inputs onto vagal neurocircuits controlling gastric motility.

Perinatal high-fat diet (pHFD) exposure alters the development of vagal neurocircuits that control gastrointestinal (GI) motility and reduce stress resiliency in offspring. Descending oxytocin (OXT; prototypical anti-stress peptide) and corticotropin releasing factor (CRF; prototypical stress peptide) inputs from the paraventricular nucleus (PVN) of the hypothalamus to the dorsal motor nucleus of the vagus (DMV) modulate the GI stress response. How these descending inputs, and their associated changes to GI motility and stress responses, are altered following pHFD exposure are, however, unknown. The present study used retrograde neuronal tracing experiments, cerebrospinal fluid extraction, in vivo recordings of gastric tone, motility and gastric emptying rates, and in vitro electrophysiological recordings from brainstem slice preparations to investigate the hypothesis that pHFD alters descending PVN-DMV inputs and dysregulates vagal brain-gut responses to stress. Compared to controls, rats exposed to pHFD had slower gastric emptying rates and did not respond to acute stress with the expected delay in gastric emptying. Neuronal tracing experiments demonstrated that pHFD reduced the number of PVNOXT neurons that project to the DMV, but increased PVNCRF neurons. Both in vitro electrophysiology recordings of DMV neurons and in vivo recordings of gastric motility and tone demonstrated that, following pHFD, PVNCRF -DMV projections were tonically active, and that pharmacological antagonism of brainstem CRF1 receptors restored the appropriate gastric response to brainstem OXT application. These results suggest that pHFD exposure disrupts descending PVN-DMV inputs, leading to a dysregulated vagal brain-gut response to stress. KEY POINTS: Maternal high-fat diet exposure is associated with gastric dysregulation and stress sensitivity in offspring. The present study demonstrates that perinatal high-fat diet exposure downregulates hypothalamic-vagal oxytocin (OXT) inputs but upregulates hypothalamic-vagal corticotropin releasing factor (CRF) inputs. Both in vitro and in vivo studies demonstrated that, following perinatal high-fat diet, CRF receptors were tonically active at NTS-DMV synapses, and that pharmacological antagonism of these receptors restored the appropriate gastric response to OXT. The current study suggests that perinatal high-fat diet exposure disrupts descending PVN-DMV inputs, leading to a dysregulated vagal brain-gut response to stress.

Retro-Tango enables versatile retrograde circuit tracing in Drosophila.

Transsynaptic tracing methods are crucial tools in studying neural circuits. Although a couple of anterograde tracing methods and a targeted retrograde tool have been developed in Drosophila melanogaster, there is still need for an unbiased, user-friendly, and flexible retrograde tracing system. Here, we describe retro-Tango, a method for transsynaptic, retrograde circuit tracing and manipulation in Drosophila. In this genetically encoded system, a ligand-receptor interaction at the synapse triggers an intracellular signaling cascade that results in reporter gene expression in presynaptic neurons. Importantly, panneuronal expression of the elements of the cascade renders this method versatile, enabling its use not only to test hypotheses but also to generate them. We validate retro-Tango in various circuits and benchmark it by comparing our findings with the electron microscopy reconstruction of the Drosophila hemibrain. Our experiments establish retro-Tango as a key method for circuit tracing in neuroscience research.

Drop-off in axonal regeneration along the length of a cross-face nerve graft: An experimental study in rats.

The average nerve graft length utilized in cross-face nerve grafting for reconstruction of facial nerve palsy is 20-22 cm. While the graft length is thought to be one of the greatest determinants of muscle strength, the mechanism through which this happens remains unknown. We studied changes in axonal regeneration along the length of a 2 cm cross-face nerve graft in a rat model. The hypothesis was that axon count would decrease along the length of the graft. A 2 cm nerve graft (sciatic nerve) was used as a cross-face nerve graft in 16 adult female, 210-250 g, Sprague Dawley rats. Thirteen weeks later, 5 mm nerve biopsies were taken at four sites: the facial nerve trunk (control), proximal graft, midpoint of graft (1 cm distal to coaptation) and distal graft (2 cm distal to coaptation). Retrograde nerve labeling with FluoroGold was performed at the biopsied nerve site and the facial motor nucleus was taken 1 week later. Microscopic imaging and manual counting of axons and labeled motor nuclei was performed. Retrograde-labeled motor neuron counts were decreased at the midway point of the graft compared to the facial trunk (1517 ± 335 axons, Δ% = 92.5, p = .01) and even further decreased at the distal end of the graft (269 ± 293 axons, Δ% = 175.5, p = .006). Analysis of the nerve biopsies demonstrated no significant differences in myelinated axon count between the nerve trunk and over the length of the nerve graft (range 6207-7179 axons, Δ% = 14.5, p = .07). In a rat model, the number of regenerating motor neurons drops off along the length of the graft and axon count is preserved due to axon sprouting. How this pattern correlates to ultimate muscle strength remains unknown, but this study provides insight into why shorter grafts may afford better outcomes.