Anterograde Tract Tracing Research Articles

The effect of the lateral hypothalamic leptin circuit on cardiorespiratory control

In addition to its well-known roles in regulating metabolic homeostasis, leptin signaling in the brain has also been critically involved in cardiovascular and respiration functions. Although the central mechanisms mediating leptin’s effect on metabolic control have been extensively studied and elucidated, the neural substrates by which leptin exert its effect on cardiovascular and respiratory functions remain incompletely understood. Leptin receptors (LepRs) are expressed distinct hypothalamic nuclei, including the lateral hypothalamic area (LHA), a brain region historically known as the “feeding center”. We recently performed anterograde tract-tracing of LHA LepR-positive neurons and observed a broad but regionally enriched pattern of projections throughout the brain, including, but not limited to the brain regions involved in sympathetic control of cardiovascular function and breathing, such as the periaqueductal gray and ventrolateral medullar. Based on these preliminary neuroanatomical observations, we hypothesized that the LHA leptin circuit is critically involved in cardiorespiratory control. To test this hypothesis, we generated mice expressing hM3Dq-mCherry or mCherry (control) specifically in LHA LepR+ neurons by performing stereotaxic microinjection of Cre-dependent AAV-DIO-hM3Dq-mCherry or AAV-DIO-mCherry into the LHA of LepR-Cre+ mice. These mice were then subjected to radiotelemetry recording of blood pressure and whole-body plethysmography to evaluate the respiratory function. We found that chemogenetic activation of LHA LepR+ neurons by intraperitoneal injection of deschloroclozapine (DCZ, 0.5 mg/Kg) significantly elevated blood pressure (p<0.01) and heart rate (p<0.01) independently of locomotor activity. Whole-body plethysmography further revealed that activation of LHA LepR+ neurons also drastically increased the respiratory rate (p<0.0001) and minute ventilation (p<0.001) without a significant change in the tidal volume. Increased respiratory rate was associated with shortening both inspiratory and expiratory durations. These results identify the LHA as a novel hypothalamic node through which the circulating metabolic hormone leptin might influence cardiorespiratory control. This work is supported by the grants from the National Institute of Health (HL127673 and HL153274 to HC). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Lithium promotes long-term neurological recovery after spinal cord injury in mice by enhancing neuronal survival, gray and white matter remodeling, and long-distance axonal regeneration.

Spinal cord injury (SCI) induces neurological deficits associated with long-term functional impairments. Since the current treatments remain ineffective, novel therapeutic options are needed. Besides its effect on bipolar mood disorder, lithium was reported to have neuroprotective activity in different neurodegenerative conditions, including SCI. In SCI, the effects of lithium on long-term neurological recovery and neuroplasticity have not been assessed. We herein investigated the effects of intraperitoneally administered lithium chloride (LiCl) on motor coordination recovery, electromyography (EMG) responses, histopathological injury and remodeling, and axonal plasticity in mice exposed to spinal cord transection. At a dose of 0.2, but not 2.0 mmol/kg, LiCl enhanced motor coordination and locomotor activity starting at 28 days post-injury (dpi), as assessed by a set of behavioral tests. Following electrical stimulation proximal to the hemitransection, LiCl at 0.2 mmol/kg decreased the latency and increased the amplitude of EMG responses in the denervated hindlimb at 56 dpi. Functional recovery was associated with reduced gray and white matter atrophy rostral and caudal to the hemitransection, increased neuronal survival and reduced astrogliosis in the dorsal and ventral horns caudal to the hemitransection, and increased regeneration of long-distance axons proximal and distal to the lesion site in mice receiving 0.2 mmol/kg, but not 2 mmol/kg LiCl, as assessed by histochemical and immunohistochemical studies combined with anterograde tract tracing. Our results indicate that LiCl induces long-term neurological recovery and neuroplasticity following SCI.

Sulforaphane promotes white matter plasticity and improves long-term neurological outcomes after ischemic stroke via the Nrf2 pathway.

Post-stroke cognitive impairment (PSCI) is a common condition following ischemic stroke. Neuronal loss and white matter injury are among the most common neuropathological characteristics in patients with PSCI. The present study tested our hypothesis that activation of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) reduces neuronal loss, white matter injury, and neurobehavioral deficits in a mouse model of PSCI and investigated the underlying protective mechanisms. PSCI was modeled in wildtype (WT) and Nrf2 knockout (KO), male and female mice, by distal middle cerebral artery occlusion (dMCAO), with intraperitoneal injections of the Nrf2 activator sulforaphane (Sfn) or vehicle. Long-term (35 days) sensorimotor and cognitive performances, white matter integrity, oligodendrogenesis by BrdU incorporation, and neurite sprouting using anterograde tract-tracing were evaluated up to 35 days after dMCAO. Neuronal apoptosis was evaluated three days after dMCAO. In vitro, primary neuronal cultures were applied to validate the in vivo findings. Compared to vehicle-injected controls, Sfn treatment improved long-term sensorimotor and cognitive deficits after dMCAO in WT male and female mice. Sfn-treated WT mice also had less myelin loss/axonal injury and showed evidence of Nrf2 activation. Sfn treatment failed to provide the same level of protection in Nrf2 KO mice. Mechanistically, the ability of Sfn to reduce neuronal death after ischemia in vitro and in vivo, augment axonal sprouting and enhance oligodendrogenesis after dMCAO was dependent on Nrf2 activation. Our results support that Nrf2 is critical for Sfn-afforded neuroprotection after ischemic stroke. Thus, targeting Nrf2 may be a promising strategy for the treatment of PSCI.

Leveling up: a long-range olivary projection to the medial geniculate without collaterals to the central nucleus of the inferior colliculus in rats.

The medial nucleus of the trapezoid body (MNTB) is one of the monaural cell groups situated within the superior olivary complex (SOC), a constellation of brainstem nuclei with numerous roles in hearing. Principal MNTB neurons are glycinergic and express the calcium-binding protein, calbindin (CB). The MNTB receives its main glutamatergic, excitatory input from the contralateral cochlear nucleus via the calyx of Held and converts this into glycinergic inhibition directed toward nuclei in the SOC and the ventral and intermediate nuclei of the lateral lemniscus (VNLL and INLL). Through this inhibition, the MNTB plays essential roles in localization of sound sources and encoding spectral and temporal features of sound. In rats, very few MNTB neurons project to the inferior colliculus. However, our recent study of SOC projections to the auditory thalamus revealed a substantial number of retrogradely labeled MNTB neurons. This observation led us to examine whether the rat MNTB provides a long-range projection to the medial geniculate body (MGB). We examined this possible projection using retrograde and anterograde tract tracing and immunohistochemistry for CB and the glycine receptor. Our results demonstrate a significant projection to the MGB from the ipsilateral MNTB that does not involve a collateral projection to the inferior colliculus.

Prenatal androgen treatment impairs the suprachiasmatic nucleus arginine-vasopressin to kisspeptin neuron circuit in female mice.

Polycystic ovary syndrome (PCOS) is associated with elevated androgen and luteinizing hormone (LH) secretion and with oligo/anovulation. Evidence indicates that elevated androgens impair sex steroid hormone feedback regulation of pulsatile LH secretion. Hyperandrogenemia in PCOS may also disrupt the preovulatory LH surge. The mechanisms through which this might occur, however, are not fully understood. Kisspeptin (KISS1) neurons of the rostral periventricular area of the third ventricle (RP3V) convey hormonal cues to gonadotropin-releasing hormone (GnRH) neurons. In rodents, the preovulatory surge is triggered by these hormonal cues and coincident timing signals from the central circadian clock in the suprachiasmatic nucleus (SCN). Timing signals are relayed to GnRH neurons, in part, via projections from SCN arginine-vasopressin (AVP) neurons to RP3VKISS1 neurons. Because rodent SCN cells express androgen receptors (AR), we hypothesized that these circuits are impaired by elevated androgens in a mouse model of PCOS. In prenatally androgen-treated (PNA) female mice, SCN Ar expression was significantly increased compared to that found in prenatally vehicle-treated mice. A similar trend was seen in the number of Avp-positive SCN cells expressing Ar. In the RP3V, the number of kisspeptin neurons was preserved. Anterograde tract-tracing, however, revealed reduced SCNAVP neuron projections to the RP3V and a significantly lower proportion of RP3VKISS1 neurons with close appositions from SCNAVP fibers. Functional assessments showed, on the other hand, that RP3VKISS1 neuron responses to AVP were maintained in PNA mice. These findings indicate that PNA changes some of the neural circuits that regulate the preovulatory surge. These impairments might contribute to ovulatory dysfunction in PNA mice modeling PCOS.

Activation of the Mesencephalic Trigeminal Nucleus Contributes to Masseter Hyperactivity Induced by Chronic Restraint Stress.

Psychological stress is commonly accepted to be closely associated with masticatory muscle disorder, which is the main symptom of temporomandibular disorder (TMD). Previous studies have confirmed that exposure to stress may cause masticatory muscle hyperactivity. However, the central mechanism underlying this process remains unclear. The mesencephalic trigeminal nucleus (Vme), which resides in the brainstem, is the primary afferent center for masticatory proprioception and plays a key role in oral–motor movements by projecting to the trigeminal motor nucleus (Vmo). Therefore, the present study was designed to examine the role of Vme neurons in masseter overactivity induced by chronic stress. We found that subjecting mice to restraint stress (6 h/day) for 14 days caused significant anxiety-like behavior, obvious masseter overactivity, and markedly enhanced electrophysiological excitability of Vme neurons. By using anterograde tract tracing combined with immunofluorescence staining methods, we observed vesicular glutamate transporter 1 (VGLUT1)-positive glutamatergic projections from the Vme to the Vmo. Moreover, chronic restraint stress (CRS) elevated the expression of VGLUT1 and choline acetyltransferase (ChAT) in Vmo. Furthermore, administration of VGLUT1-targeted short hairpin RNA (shRNA) into the bilateral Vme significantly suppressed the enhanced overexcitability of Vme neurons, downregulated the overexpression of VGLUT1 and ChAT in the Vmo, and attenuated the elevated overactivity of the masseter caused by CRS. Taken together, we showed that CRS can excite neurons in the Vme, enhancing glutamatergic excitatory projections from the Vme to the Vmo and resulting in masseter muscle overactivity. These findings provide us with a novel central mechanism underlying the correlation between psychological factors and TMD.

GAD2 Expression Defines a Class of Excitatory Lateral Habenula Neurons in Mice that Project to the Raphe and Pontine Tegmentum.

The lateral habenula (LHb) sends complex projections to several areas of the mesopontine tegmentum, the raphe, and the hypothalamus. However, few markers have been available to distinguish subsets of LHb neurons that may serve these pathways. In order to address this complexity, we examined the mouse and rat LHb for neurons that express the GABA biosynthesis enzymes glutamate decarboxylase 1 (GAD1) and GAD2, and the vesicular GABA transporter (VGAT). The mouse LHb contains a population of neurons that express GAD2, while the rat LHb contains discrete populations of neurons that express GAD1 and VGAT. However, we could not detect single neurons in either species that co-express a GABA synthetic enzyme and VGAT, suggesting that these LHb neurons do not use GABA for conventional synaptic transmission. Instead, all of the neuronal types expressing a GABAergic marker in both species showed co-expression of the glutamate transporter VGluT2. Anterograde tract-tracing of the projections of GAD2-expressing LHb neurons in Gad2Cre mice, combined with retrograde tracing from selected downstream nuclei, show that LHb-GAD2 neurons project selectively to the midline structures in the mesopontine tegmentum, including the median raphe (MnR) and nucleus incertus (NI), and only sparsely innervate the hypothalamus, rostromedial tegmental nucleus (RMTg), and ventral tegmental area (VTA). Postsynaptic recording of LHb-GAD2 neuronal input to tegmental neurons confirms that glutamate, not GABA, is the fast neurotransmitter in this circuit. Thus, GAD2 expression can serve as a marker for functional studies of excitatory neurons serving specific LHb output pathways in mice.

Hypothalamus mediates cardioinhibitory pathway from the paraventricular nucleus of the thalamus

The paraventricular nucleus of the thalamus (PVT) is known to function as a site of integration of brainstem, limbic and cortical afferent information involved in affective behaviors, and visceral and homeostatic regulation. This study investigated the role of the hypothalamus in mediating the arterial pressure (AP) and heart rate (HR) changes elicited by activation of PVT neurons. In the first series of experiments, cardiovascular responsive sites were identified in PVT using L‐glutamate (Glu; 10 nl, 0.25 M) microinjections in chloralose‐anesthetized Wistar rats. Glu injections in the anterior PVT elicited decreases in mean AP (MAP, −20 ± 3 mmHg) and HR (−15 ± 5 bpm). In the second series, iontophoretic injections of the anterograde tract tracer PHA‐L (2.5%) were made at these depressor sites in the anterior PVT in Wistar rats. Within the hypothalamus, PHA‐L labelled fibers and presumptive terminals were predominantly found bilaterally in and around the anterior, dorsolateral and the ventromedial parvocellular components of the paraventricular nucleus of the hypothalamus (PVH). A few scattered PHA‐L labelled fibers were also observed within the posterior magnocellular component of PVH, the supraoptic nucleus, the lateral and dorsomedial hypothalamic areas, and the area of the tuber cinereum. In the final series, bilateral microinjection (100 nl) of either the synaptic blocker CoCl2, the non‐specific Glu antagonist kynurenic acid or the selective NMDA receptor antagonists 2‐amino‐5‐phosphonovalerate were made into the PVH in chloralose‐anesthetized Wistar rats. Injections of these compounds did not significantly alter the resting level of AP or HR. However, all injected compounds attenuated the MAP responses (grouped data; −15 ± 5 mmHg; p<0.01), and completely blocked the HR responses (grouped data, −3 ± 5 bpm; p<0.01) to PVT neuronal activation (control responses; MAP, −25 ± 3 mmHg; HR, −19 ± 7). These data suggest that PVT functions to alter cardiac sympathoinhibitory responses associated with arousal, stress, and affective behaviors through a glutamatergic pathway relaying in PVH.Support or Funding InformationSupported in part by the Heart and Stroke Foundation of Ontario

Network topology of the marmoset connectome.

The brain is a complex network of interconnected and interacting neuronal populations. Global efforts to understand the emergence of behavior and the effect of perturbations depend on accurate reconstruction of white matter pathways, both in humans and in model organisms. An emerging animal model for next-generation applied neuroscience is the common marmoset (Callithrix jacchus). A recent open respository of retrograde and anterograde tract tracing presents an opportunity to systematically study the network architecture of the marmoset brain (Marmoset Brain Architecture Project; http://www.marmosetbrain.org). Here we comprehensively chart the topological organization of the mesoscale marmoset cortico-cortical connectome. The network possesses multiple nonrandom attributes that promote a balance between segregation and integration, including near-minimal path length, multiscale community structure, a connective core, a unique motif composition, and multiple cavities. Altogether, these structural attributes suggest a link between network architecture and function. Our findings are consistent with previous reports across a range of species, scales, and reconstruction technologies, suggesting a small set of organizational principles universal across phylogeny. Collectively, these results provide a foundation for future anatomical, functional, and behavioral studies in this model organism.

Layer specificity of inputs from supplementary motor area and dorsal premotor cortex to primary motor cortex in macaque monkeys

The primate frontal lobe processes diverse motor information in parallel through multiple motor-related areas. For example, the supplementary motor area (SMA) is mainly involved in internally-triggered movements, whereas the premotor cortex (PM) is highly responsible for externally-guided movements. The primary motor cortex (M1) deals with both aspects of movements to execute a single motor behavior. To elucidate how the cortical motor system is structured to process a variety of information, the laminar distribution patterns of signals were examined between SMA and M1, or PM and M1 in macaque monkeys by using dual anterograde tract-tracing. Dense terminal labeling was observed in layers 1 and upper 2/3 of M1 after one tracer injection into SMA, another tracer injection into the dorsal division of PM resulted in prominent labeling in the deeper portion of layer 2/3. Weaker labeling was also visible in layer 5 in both cases. On the other hand, inputs from M1 terminated in both the superficial and the deep layers of SMA and PM. The present data indicate that distinct types of motor information are arranged in M1 in a layer-specific fashion to be orchestrated through a microcircuit within M1.

Uncovering a Role for the Dorsal Hippocampal Commissure in Recognition Memory.

The dorsal hippocampal commissure (DHC) is a white matter tract that provides interhemispheric connections between temporal lobe brain regions. Despite the importance of these regions for learning and memory, there is scant evidence of a role for the DHC in successful memory performance. We used diffusion-weighted magnetic resonance imaging (DW-MRI) and white matter tractography to reconstruct the DHC in both humans (in vivo) and nonhuman primates (ex vivo). Across species, our findings demonstrate a close consistency between the known anatomy and tract reconstructions of the DHC. Anterograde tract-tracer techniques also highlighted the parahippocampal origins of DHC fibers in nonhuman primates. Finally, we derived diffusion tensor MRI metrics from the DHC in a large sample of human subjects to investigate whether interindividual variation in DHC microstructure is predictive of memory performance. The mean diffusivity of the DHC correlated with performance in a standardized recognition memory task, an effect that was not reproduced in a comparison commissure tract—the anterior commissure. These findings highlight a potential role for the DHC in recognition memory, and our tract reconstruction approach has the potential to generate further novel insights into the role of this previously understudied white matter tract in both health and disease.

Ultrastructure of giant thalamic terminals in the auditory cortex.

The auditory system comprises some very large axonal terminals like the endbulb and calyx of Held and "giant" corticothalamic synapses. Previously, we described a hitherto unknown population of giant thalamocortical boutons arising from the medial division of the medial geniculate body (MGm) in the Mongolian gerbil, which terminate over a wide cortical range but in a columnar manner particularly in the extragranular layers of the auditory cortex. As a first step towards an understanding of their potential functional role, we here describe their ultrastructure combining anterograde tract-tracing with biocytin and electron microscopy. Quantitative ultrastructural analyses revealed that biocytin-labelled MGm boutons reach much larger sizes than other, non-labelled boutons. Also, mitochondria occupy more space within labelled boutons whereas synapses are of similar size. Labelled boutons are very heterogeneous in size but homogeneous with respect to their ultrastructural characteristics, with asymmetric synapses containing clear, round vesicles and targeting dendritic spines. Functionally, the ultrastructure of the MGm terminals indicates that they form excitatory contacts, which may transmit their information in a rapid, powerful and high-fidelity manner onto strategically advantageous compartments of their cortical target cells.

Abstract 086: Neuroanatomical Basis of PVN MC4R-Expressing Neurons for Sympathetic Cardiovascular Control

It is well established that the central melanocortin system is critical for autonomic functions and energy homeostasis mainly via signaling at melanocortin-4 receptor (MC4R). Importantly, while obesity is commonly associated with elevated sympathetic tone and blood pressure, severely obese humans and rodents due to genetic MC4R deficiency exhibit normal to low sympathetic tone and blood pressure, suggesting a significant role of MC4R pathway in mediating obesity-associated sympathoexcitation and hypertension. MC4R is widely expressed in the brain including hypothalamic paraventricular nucleus (PVN) which regulates feeding and sympathetic traffic. However, the neuroanatomical basis of PVN MC4R neurons for sympathetic regulation is unclear. The goal of current study is to map the PVN MC4R neural circuits affecting sympathetic tone in mice. To this end, we injected Cre-dependent AAV driving eYFP-fused channel rhodopsin-2 expression into the PVN of MC4R-t2a-Cre knock-in mice, which allow targeted anterograde tract-tracing of PVN MC4R neurons throughout the brain. In addition to known brain region for feeding behavior (i.e. parabrachial nucleus), we found broad innervations of PVN MC4R neurons to various brain regions important for autonomic-cardiovascular control, including, but not limited to, nucleus of solitary tractus, dorsomotor nucleus of vagus, and ventrolateral medulla. Considerable innervation was also evident in spinal cord, which is further confirmed by Fluoro-gold (FG)-mediated retrograde tracing in the spinal cord (thoracic T6-10) of MC4R-GFP transgenic mouse. Double immunofluorescence labeling of GFP and FG revealed that ~50% (58 out of 116) MC4R neurons in the posterior parvocellular subdivision of PVN project to thoracic spinal cord. Furthermore, microinjection of synthetic MC4R agonist (MTII) into the PVN evokes ~42% increase (from baseline) in renal sympathetic nerve activity in anesthetized mice. These results provide important insights into understanding the divergent neural circuits by which PVN MC4R signaling differentially regulates metabolic and cardiovascular functions. Functional dissection of these diverging neural pathways using optogenetic/chemogenetic approaches is ongoing.

New Corticopontine Connections in the Primate Brain: Contralateral Projections From the Arm/Hand Area of the Precentral Motor Region.

The ipsilateral corticopontine projection (iCPP) represents a massive descending axon system terminating in the pontine nuclei (PN). In the primate, this projection is well known for its dominant influence on contralateral upper limb movements through the classical cerebrocerebellar circuity system. Although a much weaker contralateral corticopontine projection (cCPP) from motor cortex to the paramedian region has been reported in the non-human primate brain, we provide the first comprehensive description of the cCPP from the lateral motor cortex using high resolution anterograde tract tracing in Macaca mulatta. We found a relatively light cCPP from the hand/arm area of the primary motor cortex (M1), comparatively moderate cCPP from ventrolateral premotor cortex (LPMCv) and a more robust and widespread cCPP from the dorsolateral premotor cortex (LPMCd) that involved all nine contralateral PN. The M1 projection primarily targeted the dorsal pontine region, the LPMCv projection targeted the medial pontine region and LPMCd targeted both regions. These results show the first stage of the primate frontomotor cerebrocerebellar projection is bilateral, and may affect both ipsilateral and contralateral limbs. Clinically, the cCPP originating in the non-injured hemisphere may influence the recovery process of the more affected upper extremity following subtotal unilateral damage to the lateral cortical region. The cCPP may also contribute to the mild impairment of the upper limb contralateral to a unilateral cerebellar injury.

Long-Range, Border-Crossing, Horizontal Axon Radiations Are a Common Feature of Rat Neocortical Regions That Differ in Cytoarchitecture.

Employing wide-field optical imaging techniques supported by electrophysiological recordings, previous studies have demonstrated that stimulation of a spatially restricted area (point) in the sensory periphery results in a large evoked neuronal activity spread in mammalian primary cortices. In rats’ primary cortices, such large evoked spreads extend diffusely in multiple directions, cross cortical cytoarchitectural borders and can trespass into other unimodal sensory areas. These point spreads are supported by a spatially matching, diffuse set of long-range horizontal projections within gray matter that extend in multiple directions and cross borders to interconnect different cortical areas. This horizontal projection system is in addition to well-known area-to-area clustered projections to defined targets through white matter. Could similar two-projection cortical systems also be found in cortical regions that differ in their cytoarchitectural structure? To address this question, an adeno-associated viral vector expressing green fluorescent protein (GFP) was injected as an anterograde tract tracer into granular somatosensory cortex (trunk area), dysgranular cortex (somatosensory dysgranular zone and extrastriate cortex) and agranular motor cortex (MCx). Irrespective of the injection site the same two projection systems were found, and their quantification revealed a close similarity to findings in primary sensory cortices. Following detailed reconstruction, the diffuse horizontal axon radiation was found to possess numerous varicosities and to include short, medium and long axons, the latter extending up to 5.2 mm. These “proof of concept” findings suggest that the similarity of the two projection systems among different cortical areas could potentially constitute a canonical motif of neocortical organization.

Central Nucleus of the Amygdala Mediates Sympathoinhibitory Responses to Activation of Paraventricular Nucleus of the Thalamus

The paraventricular nucleus of the thalamus (PVT) is known to relay sensory information from the brainstem, and information from limbic and cortical structures involved in visceral and homeostatic regulation, and affective behaviors. In turn, PVT relays this information to a number of forebrain structures, including the amygdala. However, whether PVT is involved in mediating cardiovascular information to other central sites is not clear. This study was done to investigate whether the cardiovascular responses elicited by PVT involved a pathway that included the central nucleus of the amygdala (ACe). In the first series of experiments, a systematic mapping of PVT was done to identify cardiovascular responsive sites using microinjection of glutamate (Glu; 10 nL, 0.25 M) in chloralose anesthetized Wistar rats. Glu injections into PVT elicited decreases in systemic mean arterial pressure (MAP, −20 ± 3 mmHg) and heart rate (HR; −15 ± 5 bpm). These depressor responses were mediated through sympathoinhibition as intravenous injections of the ganglionic blocker hexamethonium bromide prevented the responses. In the second series, iontophoretic injections of the anterograde tract tracer PHA‐L were made at PVT depressor sites in the Wistar rats. Dense PHA‐L fiber and presumptive terminal labeling was found within and around the region of the lateral ACe, while light to moderate labeling was observed within the medial ACe. Additionally, extensive labeling was observed throughout the extended amygdala, including the bed nucleus of the stria terminalis. In the final series, bilateral microinjection (100 nL) of either the synaptic blocker CoCl2, the non‐specific glutamate antagonist kynurenic acid or the specific NMDA receptor antagonists 2‐amino‐5‐phosphonovalerate or MK801 were made into ACe in the chloralose anesthetized Wistar rats. Injections of these compounds did not significantly alter the resting levels of arterial pressure (AP) or HR, but significantly attenuated (p<0.01) the MAP response (mean of all injected compounds; −7 ± 5 mmHg) to activation of PVT. On the other hand, these injections into ACe did not alter the HR response (mean of all injected compounds; −12 ± 3 bpm) to PVT stimulation. These data suggest that PVT may function as a relay of sensory information that affects AP responses associated with motivated and addictive behaviors, arousal and/or stress through a sympathoinhibitory neural circuit involving ACe.Support or Funding InformationSupported in part by Heart and Stroke Foundation of Ontario.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Identification of Neurotensin Receptor Expressing Cells in the Ventral Tegmental Area across the Lifespan.

Neurotensin (Nts) promotes activation of dopamine (DA) neurons in the ventral tegmental area (VTA) via incompletely understood mechanisms. Nts can signal via the G protein-coupled Nts receptors 1 and 2 (NtsR1 and NtsR2), but the lack of methods to detect NtsR1- and NtsR2-expressing cells has limited mechanistic understanding of Nts action. To overcome this challenge, we generated dual recombinase mice that express FlpO-dependent Cre recombinase in NtsR1 or NtsR2 cells. This strategy permitted temporal control over recombination, such that we could identify NtsR1- or NtsR2-expressing cells and determine whether their distributions differed between the developing and adult brain. Using this system, we found that NtsR1 is transiently expressed in nearly all DA neurons and in many non-DA neurons in the VTA during development. However, NtsR1 expression is more restricted within the adult brain, where only two thirds of VTA DA neurons expressed NtsR1. By contrast, NtsR2 expression remains constant throughout lifespan, but it is predominantly expressed within glia. Anterograde tract tracing revealed that NtsR1 is expressed by mesolimbic, not mesocortical DA neurons, suggesting that VTA NtsR1 neurons may represent a functionally unique subset of VTA DA neurons. Collectively, this work reveals a cellular mechanism by which Nts can directly engage NtsR1-expressing DA neurons to modify DA signaling. Going forward, the dual recombinase strategy developed here will be useful to selectively modulate NtsR1- and NtsR2-expressing cells and to parse their contributions to Nts-mediated behaviors.

Anterograde Tract Tracing for Assaying Axonopathy and Transport Deficits in Glaucoma.

Whether to stage degeneration or investigate early pathology in glaucoma, examination of axonal structure and function is essential. There are a wide variety of methods available to investigators using animal models of glaucoma, with varying utilities depending on the questions asked. Here, we describe the use of anterograde neuronal tract tracing using cholera toxin B (CTB) for the determination of axon transport integrity of the retinofugal projection. This method reveals the structure of the retinal axons as well as the functional integrity of anterograde transport systems.

Different effects of running wheel exercise and skilled reaching training on corticofugal tract plasticity in hypertensive rats with cortical infarctions

The approaches that facilitate white matter plasticity may prompt functional recovery after a stroke. The effects of different exercise methods on motor recovery in stroke rats have been investigated. However, it is not clear whether their effects on axonal plasticity different. The aim of this study was to compare the effects of the forced running wheel exercise (RWE) and skilled reaching training (SRT) on axonal plasticity and motor recovery. Cortical infarctions were generated in stroke-prone renovascular hypertensive rats. The rats were randomly divided into the following three groups: the control (Con) group, the RWE group, and the SRT group. A sham group was also included. The mNSS and forelimb grip strength tests were performed on days 3, 7, 14, 21, 28, 35, and 42 after ischemia. The anterograde tract tracer biotinylated dextran amine (BDA) was injected into the rats to trace the axonal plasticity of the contralesional corticofugal tracts. Compared with the Con group, the mNSS scores in the SRT and RWE groups decreased on day 28 (P<0.05) and on days 35 and 42 (P<0.01). The grip strength in the SRT group increased relative to that in the RWE group at 42day post-ischemia (P<0.01). Both the RWE and SRT groups exhibited enhanced plasticity of the contralesional corticofugal tract axons at the level of the red nucleus (P<0.01) and the cervical enlargement (P<0.01). More contralateral corticorubral tract remodeling was observed at the red nucleus level in the SRT group than in the RWE group (P<0.001). Taken together, these results suggest that SRT may enhance axon plasticity in the corticorubral tract more effectively than the forced RWE and is associated with better motor recovery after cerebral ischemia.

Neuronal connections of the central amygdalar nucleus with refeeding-activated brain areas in rats

Following fasting, satiety is accompanied by neuronal activation in brain areas including the central amygdalar nucleus (CEA). Since CEA is known to inhibit food intake, we hypothesized that CEA contributes to the termination of meal during refeeding. To better understand the organization of this satiety-related circuit, the interconnections of the CEA with refeeding-activated neuronal groups were elucidated using retrograde (cholera toxin-β subunit, CTB) and anterograde (phaseolus vulgaris leucoagglutinin, PHA-L) tracers in male rats. C-Fos-immunoreactivity was used as marker of neuronal activation. The refeeding-activated input of the CEA primarily originated from the paraventricular thalamic, parasubthalamic and parabrachial nuclei. Few CTB-c-Fos double-labeled neurons were detected in the prefrontal cortex, lateral hypothalamic area, nucleus of the solitary tract (NTS) and the bed nuclei of the stria terminalis (BNST). Only few refeeding-activated proopiomelanocortin-producing neurons of the arcuate nucleus projected to the CEA. Anterograde tract tracing revealed a high density of PHAL-labeled axons contacted with refeeding-activated neurons in the BNST, lateral hypothalamic area, parasubthalamic, paraventricular thalamic and parabrachial nuclei and NTS; a low density of labeled axons was found in the paraventricular hypothalamic nucleus. Chemogenetic activation of the medial CEA (CEAm) inhibited food intake during the first hour of refeeding, while activation of lateral CEA had no effect. These data demonstrate the existence of reciprocal connections between the CEA and distinct refeeding-activated hypothalamic, thalamic and brainstem nuclei, suggesting the importance of short feedback loops in the regulation of satiety and importance of the CEAm in the regulation of food intake during refeeding.