Decomposition of retinal ganglion cell electrical images for cell type and functional inference.

Identifying neuronal cell types and their biophysical properties based on their extracellular electrical features is a major challenge for experimental neuroscience and the development of high-resolution brain-machine interfaces. One example is identification of retinal ganglion cell (RGC) types and their visual response properties, which is fundamental for developing future electronic implants that can restore vision. The electrical image (EI) of a RGC, or the mean spatio-temporal voltage footprint of its recorded spikes on a high-density electrode array, contains substantial information about its anatomical, morphological, and functional properties. However, the analysis of these properties is complex because of the high-dimensional nature of the EI. We present a novel optimization-based algorithm to decompose electrical image into a low-dimensional, biophysically-based representation: the temporally-shifted superposition of three learned basis waveforms corresponding to spike waveforms produced in the somatic, dendritic and axonal cellular compartments. Large-scale multi-electrode recordings from the macaque retina were used to test the effectiveness of the decomposition. The decomposition accurately localized the somatic and dendritic compartments of the cell. The imputed dendritic fields of RGCs correctly predicted the location and shape of their visual receptive fields. The inferred waveform amplitudes and shapes accurately identified the four major primate RGC types (ON and OFF midget and parasol cells), a substantial advance. Together, these findings may contribute to more accurate inference of RGC types and their original light responses in the degenerated retina, with possible implications for other electrical imaging applications.

A single-cell multi-omic atlas spanning the adult rhesus macaque brain.

Cataloging the diverse cellular architecture of the primate brain is crucial for understanding cognition, behavior, and disease in humans. Here, we generated a brain-wide single-cell multimodal molecular atlas of the rhesus macaque brain. Together, we profiled 2.58 M transcriptomes and 1.59 M epigenomes from single nuclei sampled from 30 regions across the adult brain. Cell composition differed extensively across the brain, revealing cellular signatures of region-specific functions. We also identified 1.19 M candidate regulatory elements, many previously unidentified, allowing us to explore the landscape of cis-regulatory grammar and neurological disease risk in a cell type-specific manner. Altogether, this multi-omic atlas provides an open resource for investigating the evolution of the human brain and identifying novel targets for disease interventions.

Direct Comparison of Epifluorescence and Immunostaining for Assessing Viral Mediated Gene Expression in the Primate Brain.

Viral vector technologies are commonly used in neuroscience research to understand and manipulate neural circuits, but successful applications of these technologies in non-human primate models have been inconsistent. An essential component to improve these technologies is an impartial and accurate assessment of the effectiveness of different viral constructs in the primate brain. We tested a diverse array of viral vectors delivered to the brain and extraocular muscles of macaques and compared three methods for histological assessment of viral-mediated fluorescent transgene expression: epifluorescence (Epi), immunofluorescence (IF), and immunohistochemistry (IHC). Importantly, IF and IHC identified a greater number of transduced neurons compared to Epi. Furthermore, IF and IHC reliably provided enhanced visualization of transgene in most cellular compartments (i.e., dendritic, axonal, and terminal fields), whereas the degree of labeling provided by Epi was inconsistent and predominantly restricted to somas and apical dendrites. Because Epi signals are unamplified (in contrast to IF and IHC), Epi may provide a more veridical assessment for the amount of accumulated transgene and, thus, the potential to chemogenetically or optogenetically manipulate neuronal activity. The comparatively weak Epi signals suggest that the current generations of viral constructs, regardless of delivered transgene, are not optimized for primates. This reinforces an emerging viewpoint that viral vectors tailored for the primate brain are necessary for basic research and human gene therapy.

Is the central mesencephalic reticular formation a purely horizontal gaze center?

Historically, the central mesencephalic reticular formation has been regarded as a purely horizontal gaze center based on the fact that electrical stimulation of this region produces horizontal saccades, it provides monosynaptic input to medial rectus motoneurons, and cells recorded in this region often display a peak in firing when horizontal saccades are made. We tested the proposition that the central mesencephalic reticular formation is purely a horizontal gaze center by examining whether this region also supplies terminals to superior rectus and levator palpebrae superioris motoneurons, both of which fire when making vertical eye movements. The experiments were carried out using dual tracer techniques at the light and electron microscopic level in macaque monkeys. Injections of biotinylated dextran amine or Phaseolus vulgaris leukoagglutinin into the central mesencephalic reticular formation produced anterogradely labeled terminals that were in synaptic contact with superior rectus and levator palpebrae superioris motoneurons that had been retrogradely labeled. These results indicate that this region is not purely connected with horizontal gaze motoneurons. In addition, we found that the number of contacts on vertical gaze motoneurons increased with more rostral injections involving the mesencephalic reticular formation adjacent to the interstitial nucleus of Cajal. This suggests that there is a caudal to rostral gradient for horizontal to vertical saccades, respectively, represented within the midbrain reticular formation. Finally, we utilized post-embedding immunohistochemistry to show that a portion of the labeled terminals were GABAergic, indicating they likely originate from downgaze premotor neurons.

Cerebellar projections to the macaque midbrain tegmentum: Possible near response connections.

Since most gaze shifts are to targets that lie at a different distance from the viewer than the current target, gaze changes commonly require a change in the angle between the eyes. As part of this response, lens curvature must also be adjusted with respect to target distance by the ciliary muscle. It has been suggested that projections by the cerebellar fastigial and posterior interposed nuclei to the supraoculomotor area (SOA), which lies immediately dorsal to the oculomotor nucleus and contains near response neurons, support this behavior. However, the SOA also contains motoneurons that supply multiply innervated muscle fibers (MIFs) and the dendrites of levator palpebrae superioris motoneurons. To better determine the targets of the fastigial nucleus in the SOA, we placed an anterograde tracer into this cerebellar nucleus in Macaca fascicularis monkeys and a retrograde tracer into their contralateral medial rectus, superior rectus, and levator palpebrae muscles. We only observed close associations between anterogradely labeled boutons and the dendrites of medial rectus MIF and levator palpebrae motoneurons. However, relatively few of these associations were present, suggesting these are not the main cerebellar targets. In contrast, labeled boutons in SOA, and in the adjacent central mesencephalic reticular formation (cMRF), densely innervated a subpopulation of neurons. Based on their location, these cells may represent premotor near response neurons that supply medial rectus and preganglionic Edinger-Westphal motoneurons. We also identified lens accommodation-related cerebellar afferent neurons via retrograde trans-synaptic transport of the N2c rabies virus from the ciliary muscle. They were found bilaterally in the fastigial and posterior interposed nuclei, in a distribution which mirrored that of neurons retrogradely labeled from the SOA and cMRF. Our results suggest these cerebellar neurons coordinate elements of the near response during symmetric vergence and disjunctive saccades by targeting cMRF and SOA premotor neurons.

Superior colliculus projections to target populations in the supraoculomotor area of the macaque monkey.

A projection by the superior colliculus to the supraoculomotor area (SOA) located dorsal to the oculomotor complex was first described in 1978. This projection's targets have yet to be identified, although the initial study suggested that vertical gaze motoneuron dendrites might receive this input. Defining the tectal targets is complicated by the fact the SOA contains a number of different cell populations. In the present study, we used anterograde tracers to characterize collicular axonal arbors and retrograde tracers to label prospective SOA target populations in macaque monkeys. Close associations were not found with either superior or medial rectus motoneurons whose axons supply singly innervated muscle fibers. S-group motoneurons, which supply superior rectus multiply innervated muscle fibers, appeared to receive a very minor input, but C-group motoneurons, which supply medial rectus multiply innervated muscle fibers, received no input. A number of labeled boutons were observed in close association with SOA neurons projecting to the spinal cord, or the reticular formation in the pons and medulla. These descending output neurons are presumed to be peptidergic cells within the centrally projecting Edinger-Westphal population. It is possible the collicular input provides a signaling function for neurons in this population that serve roles in either stress responses, or in eating and drinking behavior. Finally, a number of close associations were observed between tectal terminals and levator palpebrae superioris motoneurons, suggesting the possibility that the superior colliculus provides a modest direct input for raising the eyelids during upward saccades.

An Open Resource for Non-human Primate Optogenetics.

Optogenetics has revolutionized neuroscience in small laboratory animals, but its effect on animal models more closely related to humans, such as non-human primates (NHPs), has been mixed. To make evidence-based decisions in primate optogenetics, the scientific community would benefit from a centralized database listing all attempts, successful and unsuccessful, of using optogenetics in the primate brain. We contacted members of the community to ask for their contributions to an open science initiative. As of this writing, 45 laboratories around the world contributed more than 1,000 injection experiments, including precise details regarding their methods and outcomes. Of those entries, more than half had not been published. The resource is free for everyone to consult and contribute to on the Open Science Framework website. Here we review some of the insights from this initial release of the database and discuss methodological considerations to improve the success of optogenetic experiments in NHPs.

Adeno-Associated Virus Capsid-Promoter Interactions in the Brain Translate from Rat to the Nonhuman Primate.

Recently, we established an adeno-associated virus (AAV9) capsid-promoter interaction that directly determined cell-specific gene expression across two synthetic promoters, Cbh and CBA, in the rat striatum. These studies not only expand this capsid-promoter interaction to include another promoter in the rat striatum but also establish AAV capsid-promoter interactions in the nonhuman primate brain. When AAV serotype 9 (AAV9) vectors were injected into the rat striatum, the minimal synthetic promoter JetI drove green fluorescent protein (GFP) gene expression predominantly in oligodendrocytes. However, similar to our previous findings, the insertion of six alanines into VP1/VP2 of the AAV9 capsid (AAV9AU) significantly shifted JetI-driven GFP gene expression to neurons. In addition, previous retrograde tracing studies in the nonhuman primate brain also revealed the existence of a capsid-promoter interaction. When rAAV2-Retro vectors were infused into the frontal eye field (FEF) of rhesus macaques, local gene expression was prominent using either the hybrid chicken beta actin (CAG) or human synapsin (hSyn) promoters. However, only the CAG promoter, not the hSyn promoter, led to gene expression in the ipsilateral claustrum and contralateral FEF. Conversely, infusion of rAAV2-retro-hSyn vectors, but not rAAV2-retro-CAG, into the macaque superior colliculus led to differential and selective retrograde gene expression in cerebellotectal afferent cells. Clearly, this differential promoter/capsid expression profile could not be attributed to promoter inactivation from retrograde transport of the rAAV2-Retro vector. In summary, we document the potential for AAV capsid/promoter interactions to impact cell-specific gene expression across species, experimental manipulations, and engineered capsids, independent of capsid permissivity.

Using rAAV2-retro in rhesus macaques: Promise and caveats for circuit manipulation.

BACKGROUND: Recent genetic technologies such as opto- and chemogenetics allow for the manipulation of brain circuits with unprecedented precision. Most studies employing these techniques have been undertaken in rodents, but a more human-homologous model for studying the brain is the nonhuman primate (NHP). Optimizing viral delivery of transgenes encoding actuator proteins could revolutionize the way we study neuronal circuits in NHPs. NEW METHOD: rAAV2-retro, a popular new capsid variant, produces robust retrograde labeling in rodents. Whether rAAV2-retro's highly efficient retrograde transport would translate to NHPs was unknown. Here, we characterized the anatomical distribution of labeling following injections of rAAV2-retro encoding opsins or DREADDs in the cortico-basal ganglia and oculomotor circuits of rhesus macaques. RESULTS: rAAV2-retro injections in striatum, frontal eye field, and superior colliculus produced local labeling at injection sites and robust retrograde labeling in many afferent regions. In every case, however, a few brain regions with well-established projections to the injected structure lacked retrogradely labeled cells. We also observed robust terminal field labeling in downstream structures. COMPARISON WITH EXISTING METHOD(S): Patterns of labeling were similar to those obtained with traditional tract-tracers, except for some afferent labeling that was noticeably absent. CONCLUSIONS: rAAV2-retro promises to be useful for circuit manipulation via retrograde transduction in NHPs, but caveats were revealed by our findings. Some afferently connected regions lacked retrogradely labeled cells, showed robust axon terminal labeling, or both. This highlights the importance of anatomically characterizing rAAV2-retro's expression in target circuits in NHPs before moving to manipulation studies.

Transduction of Craniofacial Motoneurons Following Intramuscular Injections of Canine Adenovirus Type-2 (CAV-2) in Rhesus Macaques.

Reliable viral vector-mediated transgene expression in primate motoneurons would improve our ability to anatomically and physiologically interrogate motor systems. We therefore investigated the efficacy of replication defective, early region 1-deleted canine adenovirus type-2 (CAV-2) vectors for mediating transgene expression of fluorescent proteins into brainstem motoneurons following craniofacial intramuscular injections in four rhesus monkeys (Macaca mulatta). Vector injections were placed into surgically identified and isolated craniofacial muscles. After a 1- to 2-month survival time, animals were sacrificed and transgene expression was assessed with immunohistochemistry in the corresponding motoneuronal populations. We found that injections of CAV-2 into individual craniofacial muscles at doses in the range of ∼1010 to 1011 physical particles/muscle resulted in robust motoneuronal transduction and expression of immunohistochemically identified fluorescent proteins across multiple animals. By using different titers in separate muscles, with the resulting transduction patterns tracked via fluorophore expression and labeled motoneuron location, we established qualitative dose-response relationships in two animals. In one animal that received an atypically high titer (5.7 × 1011 total CAV-2 physical particles) distributed across numerous injection sites, no transduction was detected, likely due to a retaliatory immune response. We conclude that CAV-2 vectors show promise for genetic modification of primate motoneurons following craniofacial intramuscular injections. Our findings warrant focused attention toward the use of CAV-2 vectors to deliver opsins, DREADDs, and other molecular probes to improve genetics-based methods for primate research. Further work is required to optimize CAV-2 transduction parameters. CAV-2 vectors encoding proteins could provide a new, reliable route for modifying activity in targeted neuronal populations of the primate central nervous system.

Mouse Extraocular Muscles and the Musculotopic Organization of Their Innervation.

The organization of extraocular muscles (EOMs) and their motor nuclei was investigated in the mouse due to the increased importance of this model for oculomotor research. Mice showed a standard EOM organization pattern, although their eyes are set at the side of the head. They do have more prominent oblique muscles, whose insertion points differ from those of frontal-eyed species. Retrograde tracers revealed that the motoneuron layout aligns with the general vertebrate plan with respect to nuclei and laterality. The mouse departed in some significant respects from previously studied species. First, more overlap between the distributions of muscle-specific motoneuronal pools was present in the oculomotor nucleus (III). Furthermore, motoneuron dendrites for each pool filled the entire III and extended beyond the edge of the abducens nucleus (VI). This suggests mouse extraocular motoneuron afferents must target specific pools based on features other than dendritic distribution and nuclear borders. Second, abducens internuclear neurons are located outside the VI. We concluded this because no unlabeled abducens internuclear neurons were observed following lateral rectus muscle injections and because retrograde tracer injections into the III labeled cells immediately ventral and ventrolateral to the VI, not within it. This may provide an anatomical substrate for differential input to motoneurons and internuclear neurons that allows rodents to move their eyes more independently. Finally, while soma size measurements suggested motoneuron subpopulations supplying multiply and singly innervated muscle fibers are present, markers for neurofilaments and perineuronal nets indicated overlap in the size distributions of the two populations. Anat Rec, 302:1865-1885, 2019. © 2019 American Association for Anatomy.

Probabilistic inferential decision-making under time pressure in rhesus macaques (Macaca mulatta).

Decisions often involve the consideration of multiple cues, each of which may inform selection on the basis of learned probabilities. Our ability to use probabilistic inference for decisions is bounded by uncertainty and constraints such as time pressure. Previous work showed that when humans choose between visual objects in a multiple-cue, probabilistic task, they cope with time pressure by discounting the least informative cues, an example of satisficing or "good enough" decision-making. We tested two rhesus macaques (Macaca mulatta) on a similar task to assess their capacity for probabilistic inference and satisficing in comparison with humans. In each trial, a monkey viewed two compound stimuli consisting of four cue dimensions. Each dimension (e.g., color) had two possible states (e.g., red or blue) with different probabilistic weights. Selecting the stimulus with highest total weight yielded higher odds of receiving reward. Both monkeys learned the assigned weights at high accuracy. Under time pressure, both monkeys were less accurate as a result of decreased use of cue information. One monkey adopted the same satisficing strategy used by humans, ignoring the least informative cue dimension. Both monkeys, however, exhibited a strategy not reported for humans, a "group-the-best" strategy in which the top two cues were used similarly despite their different assigned weights. The results validate macaques as an animal model of probabilistic decision-making, establishing their capacity to discriminate between objects using at least four visual dimensions simultaneously. The time pressure data suggest caution, however, in using macaques as models of human satisficing. (PsycINFO Database Record (c) 2019 APA, all rights reserved).

A central mesencephalic reticular formation projection to medial rectus motoneurons supplying singly and multiply innervated extraocular muscle fibers.

We recently demonstrated a bilateral projection to the supraoculomotor area from the central mesencephalic reticular formation (cMRF), a region implicated in horizontal gaze changes. C-group motoneurons, which supply multiply innervated fibers in the medial rectus muscle, are located within the primate supraoculomotor area, but their inputs and function are poorly understood. Here, we tested whether C-group motoneurons in Macaca fascicularis monkeys receive a direct cMRF input by injecting this portion of the reticular formation with anterograde tracers in combination with injection of retrograde tracer into the medial rectus muscle. The results indicate that the cMRF provides a dense, bilateral projection to the region of the medial rectus C-group motoneurons. Numerous close associations between labeled terminals and each multiply innervated fiber motoneuron were present. Within the oculomotor nucleus, a much sparser ipsilateral projection onto some of the A- and B- group medial rectus motoneurons that supply singly innervated fibers was observed. Ultrastructural analysis demonstrated a direct synaptic linkage between anterogradely labeled reticular terminals and retrogradely labeled medial rectus motoneurons in all three groups. These findings reinforce the notion that the cMRF is a critical hub for oculomotility by proving that it contains premotor neurons supplying horizontal extraocular muscle motoneurons. The differences between the cMRF input patterns for C-group versus A- and B-group motoneurons suggest the C-group motoneurons serve a different oculomotor role than the others. The similar patterns of cMRF input to C-group motoneurons and preganglionic Edinger-Westphal motoneurons suggest that medial rectus C-group motoneurons may play a role in accommodation-related vergence.

Examination of feline extraocular motoneuron pools as a function of muscle fiber innervation type and muscle layer.

This study explores two points related to the pattern of innervation of the extraocular muscles. First, species differences exist in the location of the motoneurons supplying multiply innervated fibers (MIFs) and singly innervated fibers (SIFs) in eye muscles. MIF motoneurons are located outside the extraocular nuclei in primates, but are intermixed with SIF motoneurons within rat extraocular nuclei. To test whether this difference is related to visual capacity and frontal placement of eyes, we injected retrograde tracers into the medial rectus muscle of the cat, a highly visual nonprimate with frontally placed eyes. Distal injections labeled smaller MIF motoneurons located ventrolaterally and rostrally within the oculomotor nucleus (III). More central injections also labeled a separate population of larger cells located dorsally in III. Thus, the cat shares with the nocturnal rat the feature of having MIF motoneurons located within the bounds of III. On the other hand, just as with monkeys, cats show segregation of the MIF and SIF medial rectus motoneuron pools, albeit in a different pattern. Second, extraocular muscles are divided into two layers; the inner, global layer inserts into the sclera, and the outer, orbital layer inserts into the connective tissue pulley. To test whether these layers are supplied by anatomically discrete motoneuron pools, we injected tracer into the orbital layer of the cat lateral rectus muscle. No evidence of either morphological or distributional differences was found, suggesting that the functional differences in these layers may be due mainly to their orbital anatomy, not their innervation. J. Comp. Neurol. 525:919-935, 2017. © 2016 Wiley Periodicals, Inc.

Disordered zonal and cellular CYP11B2 enzyme expression in familial hyperaldosteronism type 3.

Three forms of familial primary aldosteronism have been recognized. Familial Hyperaldosteronism type 1 (FH1) or dexamethasone suppressible hyperaldosteronism, FH2, the most common form of as yet unknown cause(s), and FH3. FH3 is due to activating mutations of the potassium channel gene KCNJ5 that increase constitutive and angiotensin II-induced aldosterone synthesis. In this study we examined the cellular distribution of CYP11B2, CYP11B1, CYP17A1 and KCNJ5 in adrenals from two FH3 siblings using immunohistochemistry and immunofluorescence and obtained unexpected results. The adrenals were markedly enlarged with loss of zonation. CYP11B2 was expressed sporadically throughout the adrenal cortex. CYP11B2 was most often expressed by itself, relatively frequently with CYP17A1, and less frequently with CYP11B1. KCNJ5 was co-expressed with CYP11B2 and in some cells with CYP11B1. This aberrant co-expression of enzymes likely explains the abnormally high secretion rate of the hybrid steroid, 18-oxocortisol.

A noninvasive electromagnetic perturbation approach to probe extraocular proprioception.

BACKGROUND: Extraocular proprioception has been shown to participate in spatial perception and binocular alignment. Yet the physiological approaches used to study this sensory signal are limited because proprioceptive signaling takes place at the same time as visuomotor signaling. It is critical to dissociate this sensory signal from other visuomotor events that accompany eye movements. METHODS: We present a novel noninvasive and quantifiable method for probing extraocular proprioception independent of other visuomotor processing by attaching a rare-earth magnet to a real-time model eye and placing an electromagnet <20 mm from the eye. An electromagnet can increase or decrease angular displacements and velocities of the model eye. RESULTS: Electromagnetic activation rapidly affected (<2 ms) the rotation kinematics of the eye, which were correlated linearly with both the current supply and the distance of the electromagnet relative to the eye. CONCLUSIONS: This method circumvented the constraints of conventional physiological manipulation of extraocular proprioception, such as manually or mechanically tugging on the eye ball. It can be applied to produce the discrepancy between the intended and the executed eye movements, so that proprioceptive reafference signals are dissociated from corollary motor discharges and other visuomotor events.

A central mesencephalic reticular formation projection to the supraoculomotor area in macaque monkeys.

The central mesencephalic reticular formation is physiologically implicated in oculomotor function and anatomically interwoven with many parts of the oculomotor system's premotor circuitry. This study in Macaca fascicularis monkeys investigates the pattern of central mesencephalic reticular formation projections to the area in and around the extraocular motor nuclei, with special emphasis on the supraoculomotor area. It also examines the location of the cells responsible for this projection. Injections of biotinylated dextran amine were stereotaxically placed within the central mesencephalic reticular formation to anterogradely label axons and terminals. These revealed bilateral terminal fields in the supraoculomotor area. In addition, dense terminations were found in both the preganglionic Edinger-Westphal nuclei. The dense terminations just dorsal to the oculomotor nucleus overlap with the location of the C-group medial rectus motoneurons projecting to multiply innervated muscle fibers suggesting they may be targeted. Minor terminal fields were observed bilaterally within the borders of the oculomotor and abducens nuclei. Injections including the supraoculomotor area and oculomotor nucleus retrogradely labeled a tight band of neurons crossing the central third of the central mesencephalic reticular formation at all rostrocaudal levels, indicating a subregion of the nucleus provides this projection. Thus, these experiments reveal that a subregion of the central mesencephalic reticular formation may directly project to motoneurons in the oculomotor and abducens nuclei, as well as to preganglionic neurons controlling the tone of intraocular muscles. This pattern of projections suggests an as yet undetermined role in regulating the near triad.

A central mesencephalic reticular formation projection to the Edinger-Westphal nuclei.

The central mesencephalic reticular formation, a region associated with horizontal gaze control, has recently been shown to project to the supraoculomotor area in primates. The Edinger-Westphal nucleus is found within the supraoculomotor area. It has two functionally and anatomically distinct divisions: (1) the preganglionic division, which contains motoneurons that control both the actions of the ciliary muscle, which focuses the lens, and the sphincter pupillae muscle, which constricts the iris, and (2) the centrally projecting division, which contains peptidergic neurons that play a role in food and fluid intake, and in stress responses. In this study, we used neuroanatomical tracers in conjunction with immunohistochemistry in Macaca fascicularis monkeys to examine whether either of these Edinger-Westphal divisions receives synaptic input from the central mesencephalic reticular formation. Anterogradely labeled reticular axons were observed making numerous boutonal associations with the cholinergic, preganglionic motoneurons of the Edinger-Westphal nucleus. These associations were confirmed to be synaptic contacts through the use of confocal and electron microscopic analysis. The latter indicated that these terminals generally contained pleomorphic vesicles and displayed symmetric, synaptic densities. Examination of urocortin-1-positive cells in the same cases revealed fewer examples of unambiguous synaptic relationships, suggesting the centrally projecting Edinger-Westphal nucleus is not the primary target of the projection from the central mesencephalic reticular formation. We conclude from these data that the central mesencephalic reticular formation must play a here-to-for unexpected role in control of the near triad (vergence, lens accommodation and pupillary constriction), which is used to examine objects in near space.

Attenuation of social interaction-associated ultrasonic vocalizations and spatial working memory performance in rats exposed to chronic unpredictable stress.

Exposure to unpredictable chronic mild stress (CUS) is a commonly used protocol in rats that is reported to evoke antidepressant-reversible behaviors such as loss of preference for a sweetened water solution which is taken as an analog of the anhedonia seen in major depression. However, the induction of anhedonic-like behavior by chronic mild stress, gauged by an animal's preference for sucrose solution, is not fully reproducible and consistent across laboratories. In this study, we compared a widely used behavioral marker of anhedonia - the sucrose preference test, with another phenotypic marker of emotional valence, social interaction-associated ultrasonic vocalizations as well as a marker of an anxiety-like phenotype, novelty-suppressed feeding, and cognitive performance in the eight arm radial maze task in adult male Sprague-Dawley rats. Chronic four-week exposure to unpredictable mild stressors resulted in 1) attenuation of social interaction-associated ultrasonic vocalizations 2) attenuation of spatial memory performance on the radial arm maze 3) attenuation of body weight gain and 4) increased latency to feed in a novelty-suppressed feeding task. However, chronic exposure to CUS did not result in any significant change in sucrose preference at one-week and three-week intervals. Our results argue for the utility of ultrasonic vocalizations in a social interaction context as a comparable alternative or adjunct to the sucrose preference test in determining the efficacy of CUS to generate an anhedonic-like phenotypic state.

The precision of video and photocell tracking systems and the elimination of tracking errors with infrared backlighting.

Automated tracking offers a number of advantages over both manual and photocell tracking methodologies, including increased reliability, validity, and flexibility of application. Despite the advantages that video offers, our experience has been that video systems cannot track a mouse consistently when its coat color is in low contrast with the background. Furthermore, the local lab lighting can influence how well results are quantified. To test the effect of lighting, we built devices that provide a known path length for any given trial duration, at a velocity close to the average speed of a mouse in the open-field and the circular water maze. We found that the validity of results from two commercial video tracking systems (ANY-maze and EthoVision XT) depends greatly on the level of contrast and the quality of the lighting. A photocell detection system was immune to lighting problems but yielded a path length that deviated from the true length. Excellent precision was achieved consistently, however, with video tracking using infrared backlighting in both the open field and water maze. A high correlation (r=0.98) between the two software systems was observed when infrared backlighting was used with live mice.