An Open-Source Tool for Automated Human-Level Circling Behavior Detection.

Quantifying behavior and relating it to underlying biological states is of paramount importance in many life science fields. Although barriers to recording postural data have been reduced by progress in deep-learning-based computer vision tools for keypoint tracking, extracting specific behaviors from this data remains challenging. Manual behavior coding, the present gold standard, is labor-intensive and subject to intra- and inter-observer variability. Automatic methods are stymied by the difficulty of explicitly defining complex behaviors, even ones which appear obvious to the human eye. Here, we demonstrate an effective technique for detecting one such behavior, a form of locomotion characterized by stereotyped spinning, termed 'circling'. Though circling has an extensive history as a behavioral marker, at present there exists no standard automated detection method. Accordingly, we developed a technique to identify instances of the behavior by applying simple postprocessing to markerless keypoint data from videos of freely-exploring (Cib2-/-;Cib3-/-) mutant mice, a strain we previously found to exhibit circling. Our technique agrees with human consensus at the same level as do individual observers, and it achieves >90% accuracy in discriminating videos of wild type mice from videos of mutants. As using this technique requires no experience writing or modifying code, it also provides a convenient, noninvasive, quantitative tool for analyzing circling mouse models. Additionally, as our approach was agnostic to the underlying behavior, these results support the feasibility of algorithmically detecting specific, research-relevant behaviors using readily-interpretable parameters tuned on the basis of human consensus.

Physiology of central pathways.

The relative simplicity of the neural circuits that mediate vestibular reflexes is well suited for linking systems and cellular levels of analyses. Notably, a distinctive feature of the vestibular system is that neurons at the first central stage of sensory processing in the vestibular nuclei are premotor neurons; the same neurons that receive vestibular-nerve input also send direct projections to motor pathways. For example, the simplicity of the three-neuron pathway that mediates the vestibulo-ocular reflex leads to the generation of compensatory eye movements within ~5ms of a head movement. Similarly, relatively direct pathways between the labyrinth and spinal cord control vestibulospinal reflexes. A second distinctive feature of the vestibular system is that the first stage of central processing is strongly multimodal. This is because the vestibular nuclei receive inputs from a wide range of cortical, cerebellar, and other brainstem structures in addition to direct inputs from the vestibular nerve. Recent studies in alert animals have established how extravestibular signals shape these "simple" reflexes to meet the needs of current behavioral goal. Moreover, multimodal interactions at higher levels, such as the vestibular cerebellum, thalamus, and cortex, play a vital role in ensuring accurate self-motion and spatial orientation perception.

Asymmetric recovery in cerebellar-deficient mice following unilateral labyrinthectomy.

The term "vestibular compensation" refers to the resolution of motor deficits resulting from a peripheral vestibular lesion. We investigated the role of the cerebellum in the compensation process by characterizing the vestibuloocular reflex (VOR) evoked by head rotations at frequencies and velocities similar to those in natural behaviors in wild-type (WT) versus cerebellar-deficient Lurcher (Lc/+) mice. We found that during exploratory activity, normal mice produce head rotations largely consisting of frequencies < or =4 Hz and velocities and accelerations as large as 400 degrees/s and 5,000 degrees/s2, respectively. Accordingly, the VOR was characterized using sinusoidal rotations (0.2-4 Hz) as well as transient impulses (approximately 400 degrees/s; approximately 2,000 degrees/s2). Before lesions, WT and Lc/+ mice produced similar VOR responses to sinusoidal rotation. Lc/+ mice, however, had significantly reduced gains for transient stimuli. After unilateral labyrinthectomy, VOR recovery followed a similar course for WT and Lc/+ groups during the first week: gain was reduced by 80% for ipsilesionally directed head rotations on day 1 and improved for both strains to values of approximately 0.4 by day 5. Moreover, responses evoked by contralesionally directed rotations returned to prelesion in both strains within this period. However, unlike WT, which showed improving responses to ipsilesionally directed rotations, recovery plateaued after first week for Lc/+ mice. Our results show that despite nearly normal recovery in the acute phase, long-term compensation is compromised in Lc/+. We conclude that cerebellar pathways are critical for long-term restoration of VOR during head rotation toward the lesioned side, while noncerebellar pathways are sufficient to restore proper gaze stabilization during contralesionally directed movements.

Activity of vestibular nuclei neurons during vestibular and optokinetic stimulation in the alert mouse.

As a result of the availability of genetic mutant strains and development of noninvasive eye movements recording techniques, the mouse stands as a very interesting model for bridging the gap among behavioral responses, neuronal response dynamics studied in vivo, and cellular mechanisms investigated in vitro. Here we characterized the responses of individual neurons in the mouse vestibular nuclei during vestibular (horizontal whole body rotations) and full field visual stimulation. The majority of neurons ( approximately 2/3) were sensitive to vestibular stimulation but not to eye movements. During the vestibular-ocular reflex (VOR), these neurons discharged in a manner comparable to the "vestibular only" (VO) neurons that have been previously described in primates. The remaining neurons [eye-movement-sensitive (ES) neurons] encoded both head-velocity and eye-position information during the VOR. When vestibular and visual stimulation were applied so that there was sensory conflict, the behavioral gain of the VOR was reduced. In turn, the modulation of sensitivity of VO neurons remained unaffected, whereas that of ES neurons was reduced. ES neurons were also modulated in response to full field visual stimulation that evoked the optokinetic reflex (OKR). Mouse VO neurons, however, unlike their primate counterpart, were not modulated during OKR. Taken together, our results show that the integration of visual and vestibular information in the mouse vestibular nucleus is limited to a subpopulation of neurons which likely supports gaze stabilization for both VOR and OKR.

Postural and locomotor control in normal and vestibularly deficient mice.

We investigated how vestibular information is used to maintain posture and control movement by studying vestibularly deficient mice (IsK-/- mutant). In these mutants, microscopy showed degeneration of the cristae of the semicircular canals and of the maculae of the utriculi and sacculi, while behavioural and vestibulo-ocular reflex testing showed that vestibular function was completely absent. However, the histology of Scarpa's ganglia and the vestibular nerves was normal in mutant mice, indicating the presence of intact central pathways. Using X-ray and high-speed cineradiography, we compared resting postures and locomotion patterns between these vestibularly deficient mice and vestibularly normal mice (wild-type and IsK+/-). The absence of vestibular function did not affect resting posture but had profound effects on locomotion. At rest, the S-shaped, sagittal posture of the vertebral column was the same for wild-type and mutant mice. Both held the head with the atlanto-occipital joint fully flexed, the cervico-thoracic junction fully flexed, and the cervical column upright. Wild-type mice extended the head and vertebral column and could walk in a straight line. In marked contrast, locomotion in vestibularly deficient mice was characterized by circling episodes, during which the vertebral column maintained an S-shaped posture. Thus, vestibular information is not required to control resting posture but is mandatory for normal locomotion. We propose that vestibular inputs are required to signal the completion of a planned trajectory because mutant mice continued rotating after changing heading direction. Our findings support the hypothesis that vertebrates limit the number of degrees of freedom to be controlled by adopting just a few of the possible skeletal configurations.

Effects of electrode penetrations into the abducens nucleus of the monkey: eye movement recordings and histopathological evaluation of the nuclei and lateral rectus muscles.

Two adult rhesus monkeys that had undergone 2 years of electrode penetrations into their abducens and vestibular nuclei, for chronic eye movement studies, were examined histologically. An analysis of their VIth nucleus neurons and lateral rectus muscles revealed the following. Twenty-two percent of the large neurons (approximately 30 microm in diameter), on average, were missing and extensive neuropil disruption and gliosis was evident in the experimental side abducens nuclei as compared with the control side in each animal. While the lateral rectus muscles showed small, but inconsistent, changes in total fiber number, the muscle fiber diameters were altered, leading to a more homogenous muscle and making the typical orbital and global subdivisions of the muscle less distinct. Eye movement records from before and after the electrophysiological studies were comparable. We discuss how the complex architecture of the extraocular muscles as well as the possibility of polyneuronal innervation of single muscle fibers could explain our results.

Signal processing by vestibular nuclei neurons is dependent on the current behavioral goal.

The vestibular sensory apparatus and associated vestibular nuclei are generally thought to encode angular head velocity during our daily activities. However, in addition to direct inputs from vestibular afferents, the vestibular nuclei receive substantial projections from cortical, cerebellar, and other brainstem structures. Given this diversity of inputs, the question arises: How are the responses of vestibular nuclei neurons to head velocity modified by these additional inputs during naturally occurring behaviors? Here we have focused on the signal processing done by two specific classes of neurons in the vestibular nuclei: (1) position-vestibular-pause (PVP) neurons that mediate the vestibulo-ocular reflex (VOR), and (2) vestibular-only (VO) neurons that are thought to mediate, at least in part, the vestibulo-collic reflex (VCR). We first characterized neuronal responses to passive rotation in the head-restrained condition, and then released the head to record the discharges of the same neurons during self-generated head movements. VOR interneurons (i.e., PVP neurons) faithfully transmitted head velocity signals when the animal stabilized its gaze, regardless of whether the head motion was actively or passively generated; their responses were attenuated only when the monkey's behavioral goal was to redirect its axis of gaze relative to space. In contrast, VCR interneurons (i.e., VO neurons) faithfully transmitted head velocity signals during passive head motion, but their responses were greatly (and similarly) attenuated during all behaviors (i.e., gaze shifts, gaze pursuit, gaze stabilization) during which the monkey's behavioral goal was to move its head relative to the body. To characterize the mechanism(s) that underlie this differential processing, we tested neurons during passive rotation of the head relative to the body, as well as during a task in which a monkey actively "drove" both its head and body together in space. We conclude that neither passive activation of neck proprioceptors nor knowledge of self-generated head-in-space motion directly mediate the observed reductions in head-velocity-related modulation. Instead, we propose that the VOR and VCR pathways use efference copies of oculomotor and neck movement commands, respectively, for the differential processing of vestibular information.

Selective processing of vestibular reafference during self-generated head motion.

The vestibular sensory apparatus and associated vestibular nuclei are generally thought to encode head-in-space motion. Angular head-in-space velocity is detected by vestibular hair cells that are located within the semicircular canals of the inner ear. In turn, the afferent fibers of the vestibular nerve project to neurons in the vestibular nuclei, which, in head-restrained animals, similarly encode head-in-space velocity during passive whole-body rotation. However, during the active head-on-body movements made to generate orienting gaze shifts, neurons in the vestibular nuclei do not reliably encode head-in-space motion. The mechanism that underlies this differential processing of vestibular information is not known. To address this issue, we studied vestibular nuclei neural responses during passive head rotations and during a variety of tasks in which alert rhesus monkeys voluntarily moved their heads relative to space. Neurons similarly encoded head-in-space velocity during passive rotations of the head relative to the body and during passive rotations of the head and body together in space. During all movements that were generated by activation of the neck musculature (voluntary head-on-body movements), neurons were poorly modulated. In contrast, during a task in which each monkey actively "drove" its head and body together in space by rotating a steering wheel with its arm, neurons reliably encoded head-in-space motion. Our results suggest that, during active head-on-body motion, an efferent copy of the neck motor command, rather than the monkey's knowledge of its self-generated head-in-space motion or neck proprioceptive information, gates the differential processing of vestibular information at the level of the vestibular nuclei.

A comparison of head-unrestrained and head-restrained pursuit: influence of eye position and target velocity on latency.

Horizontal step-ramp target trajectories were used to study the initiation of head-unrestrained and head-restrained pursuit in the monkey. In a first series of experiments, initial target position (0 degrees, 5 degrees, or 30 degrees, contraversive to the direction of pursuit), fixation duration, target velocity (20 degrees, 40 degrees, 60 degrees and 80 degrees/s), and target direction were randomized in order to minimize predictive responses. Animals pursued the target either with their eyes alone (head-restrained: HR condition) or with a combination of eye and head movements (head-unrestrained: HU condition). Head motion onset consistently lagged pursuit onset (i.e., eye motion) by 50 ms or more in the HU condition, and was influenced by target velocity as well as by initial target position. Pursuit onset latencies did not vary systematically as a function of target velocity in either the HR or HU conditions. However, pursuit initiation latencies tended to be longer in the HU condition as compared to the HR condition when target motion started from the most contraversive position. A second series of experiments revealed that this difference in HR and HU pursuit onset latencies could be explained by the effects of initial eye-in-head position; more contraversive initial eye positions yielded shorter pursuit latencies in both conditions, and the monkeys generally moved their head towards the target in the HU condition, resulting in smaller eye-in-head eccentricities. Furthermore, we found that initial gaze and head positions had little or no effect on pursuit latencies. We conclude that the latency for pursuit initiation is similar irrespective of whether an animal is head-restrained or head-unrestrained, when initial eye position is held constant.

Comparing extraocular motoneuron discharges during head-restrained saccades and head-unrestrained gaze shifts.

Burst neurons (BNs) in the paramedian pontine reticular formation provide the primary input to the extraocular motoneurons (MNs) during head-restrained saccades and combined eye-head gaze shifts. Prior studies have shown that BNs carry eye movement-related signals during saccades and carry head as well as eye movement-related signals during gaze shifts. Therefore MNs receive signals related to head motion during gaze shifts, yet they solely drive eye motion. Here we addressed whether the relationship between MN firing rates and eye movements is influenced by the additional premotor signals present during gaze shifts. Neurons in the abducens nucleus of monkeys were first studied during saccades made with the head stationary. We then recorded from the same neurons during voluntary combined eye-head gaze shifts. We conclude that the activity of MNs, in contrast to that of BNs, is related to eye motion by the same dynamic relationship during head-restrained saccades and head-unrestrained gaze shifts. In addition, we show that a standard metric-based analysis [i.e., counting the number of spikes (NOS) in a burst] yields misleading results when applied to the same data set. We argue that this latter approach fails because it does not properly consider the system's dynamics or the strong interactions between eye and head motion.

Quantitative analysis of abducens neuron discharge dynamics during saccadic and slow eye movements.

The mechanics of the eyeball and its surrounding tissues, which together form the oculomotor plant, have been shown to be the same for smooth pursuit and saccadic eye movements. Hence it was postulated that similar signals would be carried by motoneurons during slow and rapid eye movements. In the present study, we directly addressed this proposal by determining which eye movement-based models best describe the discharge dynamics of primate abducens neurons during a variety of eye movement behaviors. We first characterized abducens neuron spike trains, as has been classically done, during fixation and sinusoidal smooth pursuit. We then systematically analyzed the discharge dynamics of abducens neurons during and following saccades, during step-ramp pursuit and during high velocity slow-phase vestibular nystagmus. We found that the commonly utilized first-order description of abducens neuron firing rates (FR = b + kE + r, where FR is firing rate, E and are eye position and velocity, respectively, and b, k, and r are constants) provided an adequate model of neuronal activity during saccades, smooth pursuit, and slow phase vestibular nystagmus. However, the use of a second-order model, which included an exponentially decaying term or "slide" (FR = b + kE + r + uE - c), notably improved our ability to describe neuronal activity when the eye was moving and also enabled us to model abducens neuron discharges during the postsaccadic interval. We also found that, for a given model, a single set of parameters could not be used to describe neuronal firing rates during both slow and rapid eye movements. Specifically, the eye velocity and position coefficients (r and k in the above models, respectively) consistently decreased as a function of the mean (and peak) eye velocity that was generated. In contrast, the bias (b, firing rate when looking straight ahead) invariably increased with eye velocity. Although these trends are likely to reflect, in part, nonlinearities that are intrinsic to the extraocular muscles, we propose that these results can also be explained by considering the time-varying resistance to movement that is generated by the antagonist muscle. We conclude that to create realistic and meaningful models of the neural control of horizontal eye movements, it is essential to consider the activation of the antagonist, as well as agonist motoneuron pools.

A neural correlate for vestibulo-ocular reflex suppression during voluntary eye-head gaze shifts.

The vestibulo-ocular reflex (VOR) is classically associated with stabilizing the visual world on the retina by producing an eye movement of equal and opposite amplitude to the motion of the head. Here we have directly measured the efficacy of VOR pathways during voluntary combined eye-head gaze shifts by recording from individual vestibular neurons in monkeys whose heads were unrestrained. We found that the head-velocity signal carried by VOR pathways is reduced during gaze shifts in an amplitude-dependent manner, consistent with results from behavioral studies in humans and monkeys. Our data support the hypothesis that the VOR is not a hard-wired reflex, but rather a pathway that is modulated in a manner that depends on the current gaze strategy.

Regulation of hsp expression during rodent spermatogenesis.

Spermatogenesis is the process by which immature male germ cells, through a complex series of events involving mitosis, meiosis, and cellular differentiation, eventually become mature spermatozoa capable of fertilizing an ovum. This process involves the developmental progression of male germ cells through a number of spermatogenetic cell types, each of which is characterized by unique features of morphology, cellular associations, and specialized functions. The unique features of each germ cell type are dictated, to a large degree, by the patterns of protein expression characteristic of each cell type. This review will examine two different aspects of the regulated expression of heat shock proteins in spermatogenic cells. First, we will review studies showing that the expression of several different members of both the hsp70 as well as hsp90 families of heat shock proteins is regulated during the differentiation of these cells. Second, we will review studies which have examined the induction of hsp expression in spermatogenic cells following exposure to elevated temperatures. Next, we will review the role of the transcription factors, heat shock factor 1 (HSF1) and HSF2 in the regulation of expression of hsps in the testis. One interesting and unique function of the male reproductive system in many species is the maintenance of the testes at a temperature below that of the other tissues of the animal. The importance of precise thermoregulation of the testis is evidenced by the fact that even slight elevations of scrotal temperature are associated with infertility. The results of recent studies have suggested a potential involvement of the cellular stress response in the mechanism responsible for these inhibitory effects of elevated testis temperature on spermatogenesis. Possible mechanisms are discussed.

Characterization of hypothermia-induced cellular stress response in mouse tissues.

Cells respond to adverse environmental conditions by expressing heat shock proteins, which serve to protect cells from harmful effects of the stress conditions. In this study we demonstrated that mice subjected to whole body hypothermia induced the cellular stress response, resulting in the increased expression of hsp72 mRNA in brain, heart, kidney, liver, and lung. We performed a detailed analysis of the major parameters of the stress response and found that cold induction of hsp expression is mediated by heat shock factor 1 (HSF1), which is also responsible for heat induction of the cellular stress response. However, there are differences in the mechanisms of HSF1 activation by hypothermia versus hyperthermia, as hypothermia does not cause the hyperphosphorylation of HSF1 that is characteristic of heat-activated HSF1.

Analysis of primate IBN spike trains using system identification techniques. I. Relationship To eye movement dynamics during head-fixed saccades.

The dynamic behavior of primate (Macaca fascicularis) inhibitory burst neurons (IBNs) during head-fixed saccades was analyzed by using system identification techniques. Neurons were categorized as IBNs on the basis of their anatomic location as well as by their activity during horizontal head-fixed saccadic and smooth pursuit eye movements and vestibular nystagmus. Each IBN's latency or "dynamic lead time" (td) was determined by shifting the unit discharge in time until an optimal fit to the firing rate frequency B(t) profile was obtained by using the simple model based on eye movement dynamics,B(t) = r + b1(t); where is eye velocity. For the population of IBNs, the dynamic estimate of lead time provided a significantly lower value than a method that used the onset of the first spike. We then compared the relative abilities of different eye movement-based models to predict B(t) by using objective optimization algorithms. The most important terms for predicting B(t) were eye velocity gain (b1) and bias terms (r) mentioned above. The contributions of higher-order velocity, acceleration, and/or eye position terms to model fits were found to be negligible. The addition of a pole term [the time derivative of B(t)] in conjunction with an acceleration term significantly improved model fits to IBN spike trains, particularly when the firing rates at the beginning of each saccade [initial conditions (ICs)] were estimated as parameters. Such a model fit the data well (a fit comparable to a linear regression analysis with a R2 value of 0.5, or equivalently, a correlation coefficient of 0.74). A simplified version of this model [B(t) = rk + b1(t)], which did not contain a pole term, but in which the bias term (rk) was estimated separately for each saccade, provided nearly equivalent fits of the data. However, models in which ICs or rks were estimated separately for each saccade contained too many parameters to be considered as useful models of IBN discharges. We discovered that estimated ICs and rks were correlated with saccade amplitude for the majority of short-lead IBNs (SLIBNs; 56%) and many long-lead IBNs (LLIBNs; 42%). This observation led us to construct a more simple model that included a term that was inversely related to the amplitude of the saccade, in addition to eye velocity and constant bias terms. Such a model better described neuron discharges than more complex models based on a third-order nonlinear function of eye velocity. Given the small number of parameters required by this model (only 3) and its ability to fit the data, we suggest that it provides the most valuable description of IBN discharges. This model emphasizes that the IBN discharges are dependent on saccade amplitude and implies further that a mechanism must exist, at the motoneuron (MN) level, to offset the effect of the bias and amplitude-dependent terms. In addition, we did not find a significant difference in the variance accounted for by any of the downstream models tested for SLIBNs versus LLIBNs. Therefore we conclude that the eye movement signals encoded dynamically by SLIBNs and LLIBNs are similar in nature. Put another way, SLIBNs are not closer, dynamically, to MNs than LLIBNs.