Preclinical target validation for non-addictive therapeutics development for pain.

INTRODUCTION: The Helping to End Addiction Long-termSM Initiative supports a wide range of programs to develop new or improved prevention and opioid addiction treatment strategies. An essential component of this effort is to accelerate development of non-opioid pain therapeutics. In all fields of medicine, therapeutics development is an arduous process and late-stage translational efforts such as clinical trials to validate targets are particularly complex and costly. While there are plentiful novel targets for pain treatment, successful clinical validation is rare. It is therefore crucial to develop processes whereby therapeutic targets can be reasonably 'de-risked' prior to substantial late-stage validation efforts. Such rigorous validation of novel therapeutic targets in the preclinical space will give potential private sector partners the confidence to pursue clinical validation of promising therapeutic concepts and compounds. AREAS COVERED: In 2020, the National Institutes of Health (NIH) held the Target Validation for Non-Addictive Therapeutics Development for Pain workshop to gather insights from key opinion leaders in academia, industry, and venture-financing. EXPERT OPINION: The result was a roadmap for pain target validation focusing on three modalities: 1) human evidence; 2) assay development in vitro; 3) assay development in vivo.

A focus on the neural exposome.

Many neurological disorders have complex etiologies that include noninheritable factors, collectively called the neural exposome. The National Institute of Neurological Disorders and Stroke is developing a new office with goals to advance our understanding of the multiple causes of neurological illness and to enable the development of more effective interventions.

Cascaded effects of spatial adaptation in the early visual system.

Virtually all stages of the visual system exhibit adaptation: neurons adjust their responses based on the recent stimulus history. While some of these adjustments occur at specific stages, others may be inherited from earlier stages. How do adaptation effects cascade along the visual system? We measured spatially selective adaptation at two successive stages in the mouse visual system: visual thalamus (LGN) and primary visual cortex (V1). This form of adaptation affected both stages but in drastically different ways: in LGN it only changed response gain, while in V1 it also shifted spatial tuning away from the adaptor. These effects, however, are reconciled by a simple model whereby V1 neurons summate LGN inputs with a fixed, unadaptable weighting profile. These results indicate that adaptation effects cascade through the visual system, that this cascading can shape selectivity, and that the rules of integration from one stage to the next are not themselves adaptable.

Fast hemodynamic responses in the visual cortex of the awake mouse.

Hemodynamic responses in mice and other species are typically measured under anesthesia. However, anesthesia could influence their relationship to neural activity. To investigate this relationship, we used optical imaging in mouse primary visual cortex (V1). Hemodynamic responses yielded clear maps of retinotopy in both anesthetized and awake mice. However, during wakefulness, responses were four times larger and twice as fast. These differences held whether we induced anesthesia with urethane or isoflurane and whether awake mice were stationary or running on a treadmill. With electrode recordings, we established that the effects of wakefulness reflect changes in neurovascular coupling, not in neural activity. By activating V1 directly via optogenetics, we replicated the effects of wakefulness in terms of timing but not of amplitude. We conclude that neurovascular coupling depends critically on anesthesia and wakefulness: during wakefulness, neural activity is followed by much stronger and quicker hemodynamic responses.

Visual response properties of V1 neurons projecting to V2 in macaque.

Visual area V2 of the primate cortex receives the largest projection from area V1. V2 is thought to use its striate inputs as the basis for computations that are important for visual form processing, such as signaling angles, object borders, illusory contours, and relative binocular disparity. However, it remains unclear how selectivity for these stimulus properties emerges in V2, in part because the functional properties of the inputs are unknown. We used antidromic electrical stimulation to identify V1 neurons that project directly to V2 (10% of all V1 neurons recorded) and characterized their electrical and visual responses. V2-projecting neurons were concentrated in the superficial and middle layers of striate cortex, consistent with the known anatomy of this cortico-cortical circuit. Most were fast conducting and temporally precise in their electrical responses, and had broad spike waveforms consistent with pyramidal regular-spiking excitatory neurons. Overall, projection neurons were functionally diverse. Most, however, were tuned for orientation and binocular disparity and were strongly suppressed by large stimuli. Projection neurons included those selective and invariant to spatial phase, with roughly equal proportions. Projection neurons found in superficial layers had longer conduction times, broader spike waveforms, and were more responsive to chromatic stimuli; those found in middle layers were more strongly selective for motion direction and binocular disparity. Collectively, these response properties may be well suited for generating complex feature selectivity in and beyond V2.

Multiple adaptable mechanisms early in the primate visual pathway.

We describe experiments that isolate and characterize multiple adaptable mechanisms that influence responses of orientation-selective neurons in primary visual cortex (V1) of anesthetized macaque (Macaca fascicularis). The results suggest that three adaptable stages of machinery shape neural responses in V1: a broadly tuned early stage and a spatio-temporally tuned later stage, both of which provide excitatory input, and a normalization pool that is also broadly tuned. The early stage and the normalization pool are revealed by adapting gratings that themselves fail to evoke a response from the neuron: either low temporal frequency gratings at the null orientation or gratings of any orientation drifting at high temporal frequencies. When effective, adapting stimuli that altered the sensitivity of these two mechanisms caused reductions of contrast gain and often brought about a paradoxical increase in response gain due to a relatively greater desensitization of the normalization pool. The tuned mechanism is desensitized only by stimuli well matched to a neuron's receptive field. We could thus infer desensitization of the tuned mechanism by comparing effects obtained with adapting gratings of preferred and null orientation modulated at low temporal frequencies.

The detection of visual contrast in the behaving mouse.

The mouse is becoming a key species for research on the neural circuits of the early visual system. To relate such circuits to perception, one must measure visually guided behavior and ask how it depends on fundamental stimulus attributes such as visual contrast. Using operant conditioning, we trained mice to detect visual contrast in a two-alternative forced-choice task. After 3-4 weeks of training, mice performed hundreds of trials in each session. Numerous sessions yielded high-quality psychometric curves from which we inferred measures of contrast sensitivity. In multiple sessions, however, choices were influenced not only by contrast, but also by estimates of reward value and by irrelevant factors such as recent failures and rewards. This behavior was captured by a generalized linear model involving not only the visual responses to the current stimulus but also a bias term and history terms depending on the outcome of the previous trial. We compared the behavioral performance of the mice to predictions of a simple decoder applied to neural responses measured in primary visual cortex of awake mice during passive viewing. The decoder performed better than the animal, suggesting that mice might not use optimally the information contained in the activity of visual cortex.

Nonlinear signal summation in magnocellular neurons of the macaque lateral geniculate nucleus.

Magnocellular (M-), but not parvocellular (P-), neurons of the macaque lateral geniculate nucleus (LGN) differ distinctively in their responses to counterphase-modulated and drifting gratings. Relative to stimulation with drifting gratings, counterphase modulation reduces the responses of M- cells in a band around 25 Hz, producing a "notch" in the temporal modulation transfer function (tMTF). The notch is prominent in nearly every M- cell with little variation in the temporal frequency at which it is deepest. The machinery responsible for the notch lies mostly outside the classical linear center. Directly driving the notching mechanism with annular gratings evokes no linear response but elicits a second harmonic (F2) modulation of the discharge accompanied by a drop in the mean discharge (F0). Analysis of the S- potential, which reveals inputs from ganglion cells, shows that 1) tMTFs of the afferent retinal ganglion cells are not notched and 2) during stimulation with annular gratings, the second harmonic component is present, but the drop in the F0 is largely absent from the responses of parasol ganglion cells. These results suggest that the notch is caused by the combined action of the linear response and the second harmonic response, both inherited from retina, and a suppression that originates after the retina. Our results reveal a distinctive signal transformation in the LGN and they show that nearly every M- cell exhibits a spatial nonlinearity like that observed in Y cells of the cat.

Habituation reveals fundamental chromatic mechanisms in striate cortex of macaque.

Prolonged viewing of a chromatically modulated stimulus usually leads to changes in its appearance, and that of similar stimuli. These aftereffects of habituation have been thought to reflect the activity of two populations of neurons in visual cortex that have particular importance in color vision, one sensitive to red-green modulation, the other to blue-yellow, but they have not been identified. We show here, in recordings from macaque primary visual cortex (V1), that prolonged exposure to chromatic modulation reveals two fundamental mechanisms with distinctive chromatic signatures that match those of the mechanisms identified by perceptual observations. In nearly all neurons, these mechanisms contribute to both excitation and to regulatory gain controls, and as a result their habituation can have paradoxical effects on response. The mechanisms must be located near the input layers of V1, before their distinct chromatic signatures diffuse. Our observations suggest that the fundamental mechanisms do not give rise to two distinct L-M and S chromatic pathways. Rather, the mechanisms are better understood as stages in the elaboration of chromatic tuning, expressed in varying proportions in all cells in V1 (and beyond), and made accessible to physiological and perceptual investigation only through habituation.

A new code for contrast in the primate visual pathway.

We characterize a hitherto undocumented type of neuron present in the regions bordering the principal layers of the macaque lateral geniculate nucleus. Neurons of this type were distinguished by a high and unusually regular maintained discharge that was suppressed by spatiotemporal modulation of luminance or chromaticity within the receptive field. The response to any effective stimulus was a reduction in discharge, reminiscent of the "suppressed-by-contrast" cells of the cat retina. To a counterphase-modulated grating, the response was a phase-insensitive suppression modulated at twice the stimulus frequency, implying a receptive field comprised of multiple mechanisms that generate rectifying responses. This distinctive nonlinearity makes the neurons well suited to computing a measure of contrast energy; such a signal might be important in regulating sensitivity early in visual cortex.

Early and late mechanisms of surround suppression in striate cortex of macaque.

The response of a neuron in striate cortex to an optimally configured visual stimulus is generally reduced when the stimulus is enlarged to encroach on a suppressive region that surrounds its classical receptive field (CRF). To characterize the mechanism that gives rise to this suppression, we measured its spatiotemporal tuning, its susceptibility to contrast adaptation, and its capacity for interocular transfer. Responses to an optimally configured grating confined to the CRF were strongly suppressed by annular surrounding gratings drifting at a wide range of temporal and spatial frequencies (including spatially uniform fields) that extended from well below to well above the range that drives most cortical neurons. Suppression from gratings capable of driving cortical CRFs was profoundly reduced by contrast adaptation and showed substantial interocular transfer. Suppression from stimuli that lay outside the spatiotemporal passband of most cortical CRFs was relatively stronger when the stimulus on the CRF was of low contrast, was generally insusceptible to contrast adaptation, and showed little interocular transfer. Our findings point to the existence of two mechanisms of surround suppression: one that is prominent when high-contrast stimuli drive the CRF, is orientation selective, has relatively sharp spatiotemporal tuning, is binocularly driven, and can be substantially desensitized by adaptation; the other is relatively more prominent when low-contrast stimuli drive the CRF, has very broad spatiotemporal tuning, is monocularly driven, and is insusceptible to adaptation. Its character suggests an origin in the input layers of primary visual cortex, or earlier.

Profound contrast adaptation early in the visual pathway.

Prior exposure to a moving grating of high contrast led to a substantial and persistent reduction in the contrast sensitivity of neurons in the lateral geniculate nucleus (LGN) of macaque. This slow contrast adaptation was potent in all magnocellular (M) cells but essentially absent in parvocellular (P) cells and neurons that received input from S cones. Simultaneous recordings of M cells and the potentials of ganglion cells driving them showed that adaptation originated in ganglion cells. As expected from the spatiotemporal tuning of M cells, adaptation was broadly tuned for spatial frequency and lacked orientation selectivity. Adaptation could be induced by high temporal frequencies to which cortical neurons do not respond, but not by low temporal frequencies that can strongly adapt cortical neurons. Our observations confirm that contrast adaptation occurs at multiple levels in the visual system, and they provide a new way to reveal the function and perceptual significance of the M pathway.

Noise-enhanced balance control in older adults.

Somatosensory information is critical to balance control and fall prevention in older adults. Recently, it has been shown that low-level input noise (electrical or mechanical) can enhance the sensitivity of the human somatosensory system. In this study, we tested the effect of low-level electrical noise, applied at the knee, on balance control in 13 healthy elderly volunteers. Subjects performed multiple single-legged stance trials with imperceptible electrical noise applied at the knee during half of the trials. Balance performance was characterized using a force platform to measure the displacement of the center of pressure (COP) under the subject's stance foot. Seven sway parameters were extracted from the COP time series. Improved balance was defined as a reduction in postural sway as indicated by decreases in the COP measures. Six of the seven sway parameters decreased with electrical noise. Three of these parameters decreased significantly ( < 0.05), and a fourth parameter was borderline significant. Averaged across subjects, the application of electrical noise resulted in a 3.8% reduction in mediolateral COP standard deviation ( = 0.04), a 5.4% decrease in the maximum anteroposterior COP excursion ( = 0.03), a 3.1% reduction in the COP path length ( = 0.04), and a 7.8% decrease in swept area ( = 0.05). The results suggest that imperceptible electrical noise, when applied to the knee, can enhance the balance performance of healthy older adults. These findings suggest that electrical noise-based devices may be effective in improving balance control in elderly people.

Enhancing tactile sensation in older adults with electrical noise stimulation.

Older adults often suffer from diminished somatosensation stemming from age-related neuropathy. Recently, localized low-level electrical noise stimulation was shown to enhance tactile sensitivity in healthy young subjects. Here, we hypothesized that fine-touch sensitivity in older adults can be similarly improved. Semmes-Weinstein monofilaments were used to evaluate fine-touch sensitivity on the first metatarsal phalangeal joint with four electrical stimulus conditions and a null (no-noise) condition in nine healthy elderly subjects. Electrical noise stimulation resulted in a statistically significant increase in the number of detections below the null-condition detection threshold, for five of the nine subjects, as well as across the entire population. This work suggests that electrical noise-based techniques may enable people to overcome functional difficulties due to age-related sensory loss.