Allosteric modulation of α1ß3γ2 GABAA receptors by farnesol through the neurosteroid sites.

In mammalian cells, all-trans farnesol, a 15-carbon isoprenol, is a product of the mevalonate pathway. It is the natural substrate of alcohol dehydrogenase and a substrate for CYP2E1, two enzymes implicated in ethanol metabolism. Studies have shown that farnesol is present in the human brain and inhibits voltage-gated Ca2+ channels at much lower concentrations than ethanol. Here we show that farnesol modulates the activity of γ-aminobutyric acid type A receptors (GABAARs), some of which also mediate the sedative activity of ethanol. Electrophysiology experiments performed in HEK cells expressing human α1ß3γ2 or α6ß3γ2 GABAARs revealed that farnesol increased chloride currents through positive allosteric modulation of these receptors and showed dependence on both the alcoholic functional group of farnesol and the length of the alkyl chain for activity. In silico studies using long-timescale unbiased all-atom molecular dynamics (MD) simulations of the human α1ß3γ2 GABAA receptors revealed that farnesol modulates the channel by directly binding to the transmembrane neurosteroid-binding site, after partitioning into the surrounding membrane and reaching the receptor by lateral diffusion. Channel activation by farnesol was further characterized by several structural and dynamic variables, such as global twisting of the receptor's extracellular domain, tilting of the transmembrane M2 helices, radius, cross-sectional area, hydration status, and electrostatic potential of the channel pore. Our results expand the pharmacological activities of farnesol to yet another class of ion channels implicated in neurotransmission, thus providing a novel path for understanding and treatment of diseases involving GABAA receptor dysfunction.

CCR -NB-LRR proteins MdRNL2 and MdRNL6 interact physically to confer broad-spectrum fungal resistance in apple (Malus × domestica).

Apple leaf spot, a disease caused by Alternaria alternata f. sp. mali and other fungal species, leads to severe defoliation and results in tremendous losses to the apple (Malus × domestica) industry in China. We previously identified three RPW8, nucleotide-binding, and leucine-rich repeat domain CCR -NB-LRR proteins (RNLs), named MdRNL1, MdRNL2, and MdRNL3, that contribute to Alternaria leaf spot (ALT1) resistance in apple. However, the role of NB-LRR proteins in resistance to fungal diseases in apple remains poorly understood. We therefore used MdRNL1/2/3 as baits to screen ALT1-inoculated leaves for interacting proteins and identified only MdRNL6 (another RNL) as an interactor of MdRNL2. Protein interaction assays demonstrated that MdRNL2 and MdRNL6 interact through their NB-ARC domains. Transient expression assays in apple indicated that complexes containing both MdRNL2 and MdRNL6 are necessary for resistance to Alternaria leaf spot. Intriguingly, the same complexes were also required to confer resistance to Glomerella leaf spot and Marssonina leaf spot in transient expression assays. Furthermore, stable transgenic apple plants with suppressed expression of MdRNL6 showed hypersensitivity to Alternaria leaf spot, Glomerella leaf spot, and Marssonina leaf spot; these effects were similar to the effects of suppressing MdRNL2 expression in transgenic apple plantlets. The identification of these novel broad-spectrum fungal resistance genes will facilitate breeding for fungal disease resistance in apple.

Isolation and Identification of Antioxidative Peptide from Goose Liver Hydrolysate to Ameliorate Alcohol-Mediated Oxidative Stress Damage in HHL-5 Hepatocytes.

The aim of this study was to isolate and identify antioxidative peptide from goose liver hydrolysate (GLHP) for ameliorating oxidative stress damage by alcohol in HHL-5 hepatocytes. In this research, the target antioxidative peptides in GLHP were separated, purified, and identified via a tangential flow ultrafiltration system combined with size exclusion chromatography (SEC), ion exchange chromatography (IEC), reversed-phase liquid chromatography (RP-LC), and LC-MS/MS. The results suggested that the amino acid sequence of the target antioxidative peptide for ameliorating alcohol-mediated oxidative stress damage in HHL-5 hepatocytes was Leu-Pro-Leu-Pro-Phe-Pro (LPLPFP), which had a molecular weight of 683.41 Da, and was derived from NADH-ubiquinone oxidoreductase chain 1 in goose liver. In addition, LPLPFP was confirmed to have a satisfactory stability and maintained high hepatic protective activity in a simulated gastrointestinal digestion. Moreover, the mechanism of LPLPFP prevented against oxidative stress damage in HHL-5 hepatocytes was attributed to inhibiting the production of reactive oxide species (ROS) by upregulating genes expression in the Ahr-NQO1 signal pathway. In conclusion, these results indicated that dietary GLHP supplementation could ameliorate alcohol-mediated oxidative stress damage and provide an affordable dietary intervention strategy to prevent alcohol-mediated hepatocyte damage.

A systematic strategy for estimating hERG block potency and its implications in a new cardiac safety paradigm.

INTRODUCTION: hERG block potency is widely used to calculate a drug's safety margin against its torsadogenic potential. Previous studies are confounded by use of different patch clamp electrophysiology protocols and a lack of statistical quantification of experimental variability. Since the new cardiac safety paradigm being discussed by the International Council for Harmonisation promotes a tighter integration of nonclinical and clinical data for torsadogenic risk assessment, a more systematic approach to estimate the hERG block potency and safety margin is needed. METHODS: A cross-industry study was performed to collect hERG data on 28 drugs with known torsadogenic risk using a standardized experimental protocol. A Bayesian hierarchical modeling (BHM) approach was used to assess the hERG block potency of these drugs by quantifying both the inter-site and intra-site variability. A modeling and simulation study was also done to evaluate protocol-dependent changes in hERG potency estimates. RESULTS: A systematic approach to estimate hERG block potency is established. The impact of choosing a safety margin threshold on torsadogenic risk evaluation is explored based on the posterior distributions of hERG potency estimated by this method. The modeling and simulation results suggest any potency estimate is specific to the protocol used. DISCUSSION: This methodology can estimate hERG block potency specific to a given voltage protocol. The relationship between safety margin thresholds and torsadogenic risk predictivity suggests the threshold should be tailored to each specific context of use, and safety margin evaluation may need to be integrated with other information to form a more comprehensive risk assessment.

The Mechanism of Ovule Abortion in Self-Pollinated 'Hanfu' Apple Fruits and Related Gene Screening.

Apples exhibit S-RNase-mediated self-incompatibility and typically require cross-pollination in nature. 'Hanfu' is a cultivar that produces abundant fruit after self-pollination, although it also shows a high rate of seed abortion afterwards, which greatly reduces fruit quality. In this study, we investigated the ovule development process and the mechanism of ovule abortion in apples after self-pollination. Using a DIC microscope and biomicroscope, we found that the abortion of apple ovules occurs before embryo formation and results from the failure of sperm-egg fusion. Further, we used laser-assisted microdissection (LAM) cutting and sperm and egg cell sequencing at different periods after pollination to obtain the genes related to ovule abortion. The top 40 differentially expressed genes (DEGs) were further verified, and the results were consistent with switching the mechanism at the 5' end of the RNA transcript (SMART-seq). Through this study, we can preliminarily clarify the mechanism of ovule abortion in self-pollinated apple fruits and provide a gene reserve for further study and improvement of 'Hanfu' apple fruit quality.

Performance characterization and biocompatibility assessment of silicone polyurethanes for polymer heart valve applications.

Silicone polyurethanes have gained widespread application in the biomedical field due to their excellent biocompatibility. This study comprehensively investigates four silicone polyurethane materials suitable for polymer heart valves, each exhibiting distinct chemical compositions and structural characteristics, leading to significant differences, particularly in mechanical performance and biocompatibility. Surface analysis reveals an elevated surface silicon element content in all materials compared to the bulk, indicating a migration of silicon elements towards the surface, providing a structural basis for enhancing biological stability and biocompatibility. However, higher silicon content leads to a decrease in mechanical performance, potentially resulting in mechanical failure and rupture in artificial heart valves. Concerning biocompatibility, an increase in silicone content diminishes the material's adsorption capability for cells and proteins, consequently improving its biocompatibility and biological stability. In summary, while high silicone content leads to a reduction in mechanical performance, the formation of a "silicon protective layer" on the material surface mitigates cell and protein adsorption, thereby enhancing biocompatibility and biological stability. Through comprehensive testing of the four silicone polyurethane materials, this study aims to provide insightful perspectives and methods for selecting materials suitable for polymer heart valves. Additionally, the thorough performance exploration of these materials serves as a crucial reference for the performance assessment and biocompatibility research of polymeric artificial heart valve materials.

Nutrition strategies to control post-weaning diarrhea of piglets: From the perspective of feeds.

Post-weaning diarrhea (PWD) is a globally significant threat to the swine industry. Historically, antibiotics as well as high doses of zinc oxide and copper sulfate have been commonly used to control PWD. However, the development of bacterial resistance and environmental pollution have created an interest in alternative strategies. In recent years, the research surrounding these alternative strategies and the mechanisms of piglet diarrhea has been continually updated. Mechanically, diarrhea in piglets is a result of an imbalance in intestinal fluid and electrolyte absorption and secretion. In general, enterotoxigenic Escherichia coli (ETEC) and diarrheal viruses are known to cause an imbalance in the absorption and secretion of intestinal fluids and electrolytes in piglets, resulting in diarrhea when Cl- secretion-driven fluid secretion surpasses absorptive capacity. From a perspective of feedstuffs, factors that contribute to imbalances in fluid absorption and secretion in the intestines of weaned piglets include high levels of crude protein (CP), stimulation by certain antigenic proteins, high acid-binding capacity (ABC), and contamination with deoxynivalenol (DON) in the diet. In response, efforts to reduce CP levels in diets, select feedstuffs with lower ABC values, and process feedstuffs using physical, chemical, and biological approaches are important strategies for alleviating PWD in piglets. Additionally, the diet supplementation with additives such as vitamins and natural products can also play a role in reducing the diarrhea incidence in weaned piglets. Here, we examine the mechanisms of absorption and secretion of intestinal fluids and electrolytes in piglets, summarize nutritional strategies to control PWD in piglets from the perspective of feeds, and provide new insights towards future research directions.

Optimal treatment of tannic acid for the anti-calcification of bovine jugular veins and the underlying mechanism.

OBJECTIVES: To evaluate the effects of combined treatment with tannic acid and ferric ions on the biomechanical and anti-calcification properties of glutaraldehyde-fixed bovine jugular veins after xenografting. METHODS: Two-point bending test and uniaxial tensile test were used to evaluate the flexural and biomechanical properties; Subcutaneous implantation in rat and right ventricular outflow tract reconstruction of sheep were used to evaluate the anti-calcification effects; The performance of the graft in sheep models was evaluated every month after the surgery with echocardiography examination. Markers of macrophages, T lymphocytes, smooth muscle cell osteogenic differentiation and matrix metalloproteinases in sheep explants were detected by immunohistochemistry. RESULTS: The flexibility of the bovine jugular veins cotreated with ferric ions-tannic acid was improved while maintaining biomechanical properties and excellent anti-calcification effects. Echocardiography results showed that the grafts functioned well in the animals without stenosis or reflux of the valve. Immunohistochemical studies showed that the osteogenic differentiation marker (Runx2) was detected in calcified regions and colocalised with the SMC marker (α-SMA). Compared to the glutaraldehyde-treated samples, T-cell marker (CD3), matrix metalloproteinase-2 and 9 expressions were reduced in the ferric ions-tannic acid treated group. CONCLUSION: Ferric ions-tannic acid treatment can give the conduits better flexibility with excellent biomechanical properties and anti-calcification effects, making it a promising bovine jugular veins processing method.

Thalamic burst firing propensity: a comparison of the dorsal lateral geniculate and pulvinar nuclei in the tree shrew.

Relay neurons in dorsal thalamic nuclei can fire high-frequency bursts of action potentials that ride the crest of voltage-dependent transient (T-type) calcium currents [low-threshold spike (LTS)]. To explore potential nucleus-specific burst features, we compared the membrane properties of dorsal lateral geniculate nucleus (dLGN) and pulvinar nucleus relay neurons using in vitro whole-cell recording in juvenile and adult tree shrew (Tupaia) tissue slices. We injected current ramps of variable slope into neurons that were sufficiently hyperpolarized to de-inactivate T-type calcium channels. In a small percentage of juvenile pulvinar and dLGN neurons, an LTS could not be evoked. In the remaining juvenile neurons and in all adult dLGN neurons, a single LTS could be evoked by current ramps. However, in the adult pulvinar, current ramps evoked multiple LTSs in >70% of recorded neurons. Using immunohistochemistry, Western blot techniques, unbiased stereology, and confocal and electron microscopy, we found that pulvinar neurons expressed more T-type calcium channels (Ca(v) 3.2) and more small conductance potassium channels (SK2) than dLGN neurons and that the pulvinar nucleus contained a higher glia-to-neuron ratio than the dLGN. Hodgkin-Huxley-type compartmental models revealed that the distinct firing modes could be replicated by manipulating T-type calcium and SK2 channel density, distribution, and kinetics. The intrinsic properties of pulvinar neurons that promote burst firing in the adult may be relevant to the treatment of conditions that involve the adult onset of aberrant thalamocortical interactions.

Synaptic organization of connections between the temporal cortex and pulvinar nucleus of the tree shrew.

We examined the synaptic organization of reciprocal connections between the temporal cortex and the dorsal (Pd) and central (Pc) subdivisions of the tree shrew pulvinar nucleus, regions innervated by the medial and lateral superior colliculus, respectively. Both Pd and Pc subdivisions project topographically to 2 separate regions of the temporal cortex; small injections of anterograde tracers placed in either Pd or Pc labeled 2 foci of terminals in the temporal cortex. Pulvinocortical pathways innervated layers I-IV, with beaded axons oriented perpendicular to the cortical surface, where they synapsed with spines that did not contain gamma amino butyric acid (GABA), likely located on the apical dendrites of pyramidal cells. Projections from the temporal cortex to the Pd and Pc originate from layer VI cells, and form small terminals that contact small caliber non-GABAergic dendrites. These results suggest that cortical terminals are located distal to tectopulvinar terminals on the dendritic arbors of Pd and Pc projection cells, which subsequently contact pyramidal cells in the temporal cortex. This circuitry could provide a mechanism for the pulvinar nucleus to activate subcortical visuomotor circuits and modulate the activity of other visual cortical areas. The potential relation to primate tecto-pulvino-cortical pathways is discussed.

Two pathogenesis-related proteins interact with leucine-rich repeat proteins to promote Alternaria leaf spot resistance in apple.

Alternaria leaf spot in apple (Malus x domestica), caused by the fungal pathogen Alternaria alternata f. sp. mali (also called A. mali), is a devastating disease resulting in substantial economic losses. We previously established that the resistance (R) protein MdRNL2, containing a coiled-coil, nucleotide-binding, and leucine-rich repeat (CCR-NB-LRR) domain, interacts with another CCR-NB-LRR protein, MdRNL6, to form a MdRNL2-MdRNL6 complex that confers resistance to A. mali. Here, to investigate the function of the MdRNL2-MdRNL6 complex, we identified two novel pathogenesis-related (PR) proteins, MdPR10-1 and MdPR10-2, that interact with MdRNL2. Yeast two-hybrid (Y2H) assays and bimolecular fluorescence complementation (BiFC) assays confirmed that MdPR10-1 and MdPR10-2 interact with MdRNL2 and MdRNL6 at the leucine-rich repeat domain. Transient expression assays demonstrated that accumulation of MdPR10-1 and MdPR10-2 enhanced the resistance of apple to four strains of A. mali that we tested: ALT1, GBYB2, BXSB5, and BXSB7. In vitro antifungal activity assays demonstrated that both the proteins contribute to Alternaria leaf spot resistance by inhibiting fungal growth. Our data provide evidence for a novel regulatory mechanism in which MdRNL2 and MdRNL6 interact with MdPR10-1 and MdPR10-2 to inhibit fungal growth, thereby contributing to Alternaria leaf spot resistance in apple. The identification of these two novel PR proteins will facilitate breeding for fungal disease resistance in apple.

Diabetic albuminuria is due to a small fraction of nephrons distinguished by albumin-stained tubules and glomerular adhesions.

OVE26 diabetic mice develop severe albuminuria. Immunohistochemical analysis revealed a pattern of intense albumin staining in a small subset of OVE26 tubules. Immunostaining was strikingly heterogeneous; some tubules stained intensely for albumin, but most tubules had weak or no staining. Serial sectioning showed that staining patterns were distinctive for each nephron. Electron microscopy revealed that albumin accumulated in villi and at the base of the brush border. Tubule cell injury, as shown by loss of villi, tubule dilation, and cellular protrusions into the tubule lumen, was unambiguously associated with albumin staining. Examination of albumin staining of proteinuric human kidneys also showed a heterogeneous pattern of staining. Analysis of OVE26 serial sections indicated that all glomeruli connected to albumin-positive tubules were identified by albumin-stained lesions in the tuft that adhered to Bowman's capsule, implicating this as a critical feature of heavy albumin leakage. These results indicate that albumin accumulation provides a marker of damaged nephrons, and confirm that albumin leakage produces significant tubular damage. This study shows that that formation of sclerotic glomerular adhesions is a critical step leading to severe albuminuria.

Studying Nicotinic Acetylcholine Receptors Using the IonFlux™ Microfluidic-Based Automated Patch-Clamp System with Continuous Perfusion and Fast Solution Exchange.

Automated patch-clamp (APC) systems have become indispensable tools of drug-discovery programs by allowing high-throughput electrophysiology-based screening of ion channel compounds. The recent development and introduction of microfluidics-based APC systems have made it possible to study the interactions of ligand-gated ion channels with pharmacological reagents, such as agonists, antagonists, or positive allosteric modulators (PAMs), with reliable pharmacological results comparable to those of the gold-standard manual patch-clamp technique while maintaining high-throughput capacity. Many ligand-gated ion channels exhibit rapid desensitization upon repetitive introduction of ligands; this loss of channel activity in the absence of pharmacological interaction poses a challenge for developing accurate, precise, and robust assays with high success rate, low run-down, and reliable pharmacological results. Here we present procedures to study nicotinic acetylcholine receptors (nAChRs) with the IonFlux™, an automated patch-clamp system with continuous flow and precise fluidic exchange; these procedures can also be generalized to the study of other ligand-gated ion channels. We present protocols to study agonist, antagonist, and PAM activities on nAChRs, particularly the rapidly desensitizing nAChR α7 receptors. The data demonstrate that the IonFlux™ system is a fast, robust, and reliable platform for the study of nAChRs and other ligand-gated ion channels, generating data that closely mimic those from manual patch-clamp conditions. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Measuring agonist concentration-dependent response Basic Protocol 2: Measuring antagonist concentration-dependent response Basic Protocol 3: Measuring positive allosteric modulator (PAM) concentration-dependent response Support Protocol 1: Basic IonFlux system operation Support Protocol 2: Plate care and filling Support Protocol 3: Plate preparation for water rinsing Support Protocol 4: Water rinsing of plates Support Protocol 5: Plate priming Support Protocol 6: General assay Support Protocol 7: Editing the compound addition sequence (compound list) Support Protocol 8: Creating compound list for agonist concentration-dependent response Support Protocol 9: Creating compound list for antagonist or PAM concentration-dependent response Support Protocol 10: Defining the different compounds used or compound list Support Protocol 11: Maintenance Support Protocol 12: Data analysis Support Protocol 13: Cell culture.

Chronic morphine exposure impairs short-term synaptic depression of geniculo-cortical visual pathway in vivo.

Chronic morphine exposure can induce addiction and affect synaptic plasticity, but the underlying neuronal mechanisms remain unknown. Two forms of short-term synaptic depression (paired-pulse depression (PPD) and frequency depression) were investigated in vivo in the geniculo-cortical visual pathway of morphine-treated and saline-treated (as control) adult rats. Acute exposure to morphine had no effect on paired-pulse synaptic depression and 10-40 Hz induced frequency synaptic depression. However, chronic morphine exposure reduced markedly the paired-pulse depression and frequency depression at 40 Hz. The effect of chronic morphine exposure on short-term synaptic plasticity in the geniculo-cortical visual pathway was sensitization given that morphine re-exposure further significantly reduced the short-term synaptic depression. Interestingly, the further reduction in short-term synaptic depression due to re-exposure of morphine was recovered to normal (control) levels at 3 to 6h after morphine re-exposure. These findings suggest that chronic morphine treatment could significantly degrade the short-term synaptic plasticity of geniculo-cortical visual pathway.

Short-term synaptic plasticity in the rat geniculo-cortical pathway during development in vivo.

The critical period for visual system development in rats normally peaks at postnatal three weeks and ends at postnatal five weeks. However, the change of short-term synaptic plasticity during this period has rarely been investigated. In the present study, we compared the short-term plasticity of visual cortical responses to lateral geniculate nucleus stimulation in rats at different development stages (P20, P30 and adult) in vivo. The results show that paired-pulse depression (PPD) and frequency-dependent depression of evoked field potentials (FP) are present in P20 rats and increase in magnitude with development. The time course of this maturation of synaptic depression parallels that of the visual critical period. The weak synaptic depression observed in juvenile rats may be important in enhancing excitatory neurotransmission at a time when synapses are immature; this could endow immature synapses with wide integrative capabilities. In contrast, suppressive temporal interactions could provide an important substrate for neuronal processing of visual information in the mature cortex.

Slice XVIvo™: a novel electrophysiology system with the capability for 16 independent brain slice recordings.

Here we validate the design and use of a novel, customized electrophysiology system (Slice XVIvo™) that is capable of recording from 16 independent brain slices. The system consists of 16 independent recording chambers in which individual electrodes can be manually manipulated and fixed in order to stimulate and record extracellular responses from 16 brain slices simultaneously. Responses from each brain slice are elicited with individual stimulus isolator units and recorded through separate channels, thus allowing for independent control and analysis of the evoked extracellular activity from each slice. The system was designed to fit on a standard anti-vibration table, thus the Slice XVIvo™ system occupies considerably less space than other currently available multi-slice recording systems. We have demonstrated the utility of the system to obtain stable, extracellular responses from the CA1 region of the hippocampus, as well as induce long-term potentiation. Additionally, we show the utility of the Slice XVIvo™ system to significantly improved throughput for testing compounds in an oxygen and glucose deprivation assay. Overall, we have designed, created and validated a considerably cost- and space-efficient electrophysiology system that greatly improves throughput while minimizing the number of animals used in experiments.

Validation of a high-throughput, automated electrophysiology platform for the screening of nicotinic agonists and antagonists.

High-throughput compound screening using electrophysiology-based assays represents an important tool for biomedical research and drug discovery programs. The recent development and availability of devices capable of performing high-throughput electrophysiology-based screening have brought the need to validate these tools by producing data that are consistent with results obtained with conventional electrophysiological methods. In this study, we compared the response properties of hα3ß4 and hα4ß2 nicotinic receptors to their endogenous ligand acetylcholine (ACh) using three separate electrophysiology platforms: Dynaflow (low-throughput, manual system), PatchXpress 7000A (medium-throughput automated platform), and IonWorks Barracuda (high-throughput automated platform). We found that despite the differences in methodological approaches between these technologies, the EC(50) values from the ACh dose-response curves were consistent between all three platforms. In addition, we have validated the IonWorks Barracuda for both competitive and uncompetitive inhibition assays by using the competitive nicotinic antagonist dihydro-beta-erythroidin (DHßE) and uncompetitive nicotinic antagonist mecamylamine. Furthermore, we have demonstrated the utility of a custom-written algorithm for generating dose-response curves from multiple extrapolated current metrics that allows for discriminating between competitive and uncompetitive inhibition while maintaining high-throughput capacity. This study provides validation of the consistency of results using low-, medium-, and high-throughput electrophysiology platforms and supports their use for screening nicotinic compounds.

Cortical projections to the superior colliculus in tree shrews (Tupaia belangeri).

The visuomotor functions of the superior colliculus depend not only on direct inputs from the retina, but also on inputs from neocortex. As mammals vary in the areal organization of neocortex, and in the organization of the number of visual and visuomotor areas, patterns of corticotectal projections vary. Primates in particular have a large number of visual areas projecting to the superior colliculus. As tree shrews are close relatives of primates, and they are also highly visual, we studied the distribution of cortical neurons projecting to the superior colliculus by injecting anatomical tracers into the colliculus. Since projections from visuotopically organized visual areas are expected to match the visuotopy of the superior colliculus, injections at different retinotopic locations in the superior colliculus provide information about the locations and organization of topographic areas in extrastriate cortex. Small injections in the superior colliculus labeled neurons in locations within areas 17 (V1) and 18 (V2) that are consistent with the known topography of these areas and the superior colliculus. In addition, the separate locations of clusters of labeled cells in temporal visual cortex provide evidence for five or more topographically organized areas. Injections that included deeper layers of the superior colliculus also labeled neurons in medial frontal cortex, likely in premotor cortex. Only occasional labeled neurons were observed in somatosensory or auditory cortex. Regardless of tracer injection location, we found that, unlike primates, a substantial projection to the superior colliculus from posterior parietal cortex is not a characteristic of tree shrews.