Awake Nonhuman Primates Research Articles

A 3D-Printed helmet for precise and repeatable neuromodulation targeting in awake non-human primates

The application of non-invasive brain stimulation (NIBS) in non-human primates (NHPs) is critical for advancing understanding of brain networks and developing treatments for neurological diseases. Improving the precision of targeting can significantly enhance the efficacy of these interventions. Here, we introduce a 3D-printed helmet designed to achieve repeatable and precise neuromodulation targeting in awake rhesus monkeys, eliminating the need of head fixation. Imaging studies confirmed that the helmet consistently targets the primary motor cortex (M1) with a margin of error less than 1 mm. Evaluations of stimulation efficacy revealed high resolution and stability. Additionally, physiological evaluations under propofol anesthesia showed that the helmet effectively facilitated the generation of recruitment curves for motor area, confirming successful neuromodulation. Collectively, our findings present a straightforward and effective method for achieving consistent and precise NIBS targeting in awake NHPs, potentially advancing both basic neuroscience research and the development of clinical neuromodulation therapies.

A Physiological Marker for Deep Brain Ultrasonic Neuromodulation

ObjectivesTranscranial ultrasound neuromodulation (TUSN) is a noninvasive and spatially specific therapy that promises to deliver treatments tailored to the specific needs of individuals. To fulfill this promise, each treatment must be modified to adequately correct for variation across individual skulls and neural anatomy. This study examines the use of ultrasound-induced voltage potentials (measured with electroencephalography [EEG]) to guide TUSN therapies. Materials and MethodsWe measured EEG responses in two awake nonhuman primates during sonication of 12 targets surrounding two deep brain nuclei, the left and right lateral geniculate nucleus. ResultsWe report reliable ultrasound evoked potentials measured with EEG after the deep brain ultrasonic modulation in nonhuman primates. Robust responses are observed after just ten repetitions of the ultrasonic stimuli. Moreover, these potentials are only evoked for specific deep brain targets. Furthermore, a behavioral study in one subject shows a direct correspondence between the target with maximal EEG response and ultrasound-based modulation of visual choice behavior. Thus, this study provides evidence for the feasibility of EEG-based guidance for ultrasound neuromodulation therapies.

Hierarchical differences in the encoding of amplitude modulation in the subcortical auditory system of awake nonhuman primates.

Sinusoidal amplitude modulation (SAM) is a key feature of complex sounds. Although psychophysical studies have characterized SAM perception, and neurophysiological studies in anesthetized animals report a transformation from the cochlear nucleus' (CN; brainstem) temporal code to the inferior colliculus' (IC; midbrain's) rate code, none have used awake animals or nonhuman primates to compare CN and IC's coding strategies to modulation-frequency perception. To address this, we recorded single-unit responses and compared derived neurometric measures in the CN and IC to psychometric measures of modulation frequency (MF) discrimination in macaques. IC and CN neurons often exhibited tuned responses to SAM in rate and spike-timing measures of modulation coding. Neurometric thresholds spanned a large range (2-200 Hz ΔMF). The lowest 40% of IC thresholds were less than or equal to psychometric thresholds, regardless of which code was used, whereas CN thresholds were greater than psychometric thresholds. Discrimination at 10-20 Hz could be explained by indiscriminately pooling 30 units in either structure, whereas discrimination at higher MFs was best explained by more selective pooling. This suggests that pooled CN activity was sufficient for AM discrimination. Psychometric and neurometric thresholds decreased as stimulus duration increased, but IC and CN thresholds were higher and more variable than behavior at short durations. This slower subcortical temporal integration compared with behavior was consistent with a drift diffusion model that reproduced individual differences in performance and can constrain future neurophysiological studies of temporal integration. These measures provide an account of AM perception at the neurophysiological, computational, and behavioral levels.NEW & NOTEWORTHY In everyday environments, the brain is tasked with extracting information from sound envelopes, which involves both sensory encoding and perceptual decision-making. Different neural codes for envelope representation have been characterized in midbrain and cortex, but studies of brainstem nuclei such as the cochlear nucleus (CN) have usually been conducted under anesthesia in nonprimate species. Here, we found that subcortical activity in awake monkeys and a biologically plausible perceptual decision-making model accounted for sound envelope discrimination behavior.

Effects of chronic naltrexone treatment on relapse-related behavior and neural responses to fentanyl in awake nonhuman primates.

Naltrexone, an opioid antagonist that blocks the reinforcing properties of opioid agonists, is often prescribed to preclude relapse to opioid use disorder (OUD) following detoxification. However, few laboratory studies have directly investigated the ability of naltrexone to alter relapse-inducing effects of opioid agonists, including their priming strength in reinstatement studies and their impact in brain regions known to be involved in drug-induced reinforcement in MRI studies. Here we directly address this issue by investigating the effects of continuous exposure to naltrexone on 1) fentanyl-induced reinstatement of drug-seeking behavior, 2) fentanyl-induced patterns of blood oxygenation level dependent (BOLD) activation in the nucleus accumbens (NAcc), and 3) fentanyl-induced changes in NAcc functional connectivity (FC) in awake non-human primates that are engaged in ongoing opioid self-administration studies. We found that naltrexone antagonizes the priming strength of fentanyl as shown by a rightward shift in its reinstatement dose-effect curve and that naltrexone surmountably antagonizes the BOLD response induced by fentanyl. However, while naltrexone also countered fentanyl's effects on NAcc FC, the effects were not surmounted by a higher dose of fentanyl. Together, these data suggest that, in contrast to naltrexone's modulation of fentanyl's effects on behavior and BOLD responses, their interactive effects on FC between multiple brain regions do not reflect their receptor-mediated activity. Additionally, we demonstrated opposing effects in the absence and presence of naltrexone on NAcc FC at baseline (i.e., in the absence of any fentanyl prime) suggesting that naltrexone alters FC at baseline, even though naltrexone appears behaviorally silent in the absence of an agonist prime. Together these data provide additional insight into ways in which naltrexone interacts with opioid agonists, both behaviorally and in the brain. Further understanding the effects of opioid agonists on patterns of FC could help elucidate our understanding of the neural processes that contribute to the initiation of and relapse to opioid-seeking behavior in OUD.

Electroencephalography Measurements in Awake Marmosets Listening to Conspecific Vocalizations.

Vocal communication plays a crucial role in the social interactions of primates, particularly in survival and social organization. Humans have developed a unique and advanced vocal communication strategy in the form of language. To study the evolution of human language, it is necessary to investigate the neural mechanisms underlying vocal processing in humans, as well as to understand how brain mechanisms have evolved by comparing them with those in nonhuman primates. Herein, we developed a method to noninvasively measure the electroencephalography (EEG) of awake nonhuman primates. This recording method allows for long-term studies without harming the animals, and, importantly, allows us to directly compare nonhuman primate EEG data with human data, providing insights into the evolution of human language. In the current study, we used the scalp EEG recording method to investigate brain activity in response to species-specific vocalizations in marmosets. This study provides novel insights by using scalp EEG to capture widespread neural representations in marmosets during vocal perception, filling gaps in existing knowledge.

Dynamic off-resonance correction improves functional image analysis in fMRI of awake behaving non-human primates.

Use of functional MRI in awake non-human primate (NHPs) has recently increased. Scanning animals while awake makes data collection possible in the absence of anesthetic modulation and with an extended range of possible experimental designs. Robust awake NHP imaging however is challenging due to the strong artifacts caused by time-varying off-resonance changes introduced by the animal's body motion. In this study, we sought to thoroughly investigate the effect of a newly proposed dynamic off-resonance correction method on brain activation estimates using extended awake NHP data. We correct for dynamic B0 changes in reconstruction of highly accelerated simultaneous multi-slice EPI acquisitions by estimating and correcting for dynamic field perturbations. Functional MRI data were collected in four male rhesus monkeys performing a decision-making task in the scanner, and analyses of improvements in sensitivity and reliability were performed compared to conventional image reconstruction. Applying the correction resulted in reduced bias and improved temporal stability in the reconstructed time-series data. We found increased sensitivity to functional activation at the individual and group levels, as well as improved reliability of statistical parameter estimates. Our results show significant improvements in image fidelity using our proposed correction strategy, as well as greatly enhanced and more reliable activation estimates in GLM analyses.

Intracranial EEG Recordings of High-Frequency Activity From a Wireless Implantable BMI Device in Awake Nonhuman Primates.

Wireless implantable brain machine interfaces (BMIs) are a promising tool to restore communication and motor functions for individuals with severe motor disability. Prior to clinical application, recording performance must be sufficiently confirmed by animal experiments. In this paper, we aimed to evaluate the performance of a novel BMI wireless device for recording brain activity in two nonhuman primates. We customized a wireless device for implantable BMIs for clinical application. We used a battery instead of a wireless power charging system. Thirty-two electrodes were subdurally implanted over the left temporoparietal cortex. We evaluated the recording performance of the wireless device by auditory steady-state responses (ASSRs) and ketamine-induced responses. The devices successfully recorded broadband oscillatory activities up to the high-frequency band from the temporal cortex in two awake macaque monkeys. Spectral analysis of raw signals demonstrated that the devices detected characteristic results of a 40-Hz ASSR and prominent high-frequency band activity induced by ketamine injection. We confirmed the functionality of the wireless device in recording and transmitting electrocorticography (ECoG) signals with both millisecond precision and recording stability. These results provide confidence that this wireless device can be a translational tool for other fundamental neuroscientific studies in free-moving models.

Responses of Cortical Neurons to Intracortical Microstimulation in Awake Primates.

Intracortical microstimulation (ICMS) is commonly used in many experimental and clinical paradigms; however, its effects on the activation of neurons are still not completely understood. To document the responses of cortical neurons in awake nonhuman primates to stimulation, we recorded single-unit activity while delivering single-pulse stimulation via Utah arrays implanted in primary motor cortex (M1) of three macaque monkeys. Stimuli between 5 and 50 μA delivered to single channels reliably evoked spikes in neurons recorded throughout the array with delays of up to 12 ms. ICMS pulses also induced a period of inhibition lasting up to 150 ms that typically followed the initial excitatory response. Higher current amplitudes led to a greater probability of evoking a spike and extended the duration of inhibition. The likelihood of evoking a spike in a neuron was dependent on the spontaneous firing rate as well as the delay between its most recent spike time and stimulus onset. Tonic repetitive stimulation between 2 and 20 Hz often modulated both the probability of evoking spikes and the duration of inhibition; high-frequency stimulation was more likely to change both responses. On a trial-by-trial basis, whether a stimulus evoked a spike did not affect the subsequent inhibitory response; however, their changes over time were often positively or negatively correlated. Our results document the complex dynamics of cortical neural responses to electrical stimulation that need to be considered when using ICMS for scientific and clinical applications.

Estimating and mitigating the effects of systemic low frequency oscillations (sLFO) on resting state networks in awake non-human primates using time lag dependent methodology.

Resting-state fMRI (rs-fMRI) is often used to infer regional brain interactions from the degree of temporal correlation between spontaneous low-frequency fluctuations, thought to reflect local changes in the BOLD signal due to neuronal activity. One complication in the analysis and interpretation of rs-fMRI data is the existence of non-neuronal low frequency physiological noise (systemic low frequency oscillations; sLFOs) which occurs within the same low frequency band as the signal used to compute functional connectivity. Here, we demonstrate the use of a time lag mapping technique to estimate and mitigate the effects of the sLFO signal on resting state functional connectivity of awake squirrel monkeys. Twelve squirrel monkeys (6 male/6 female) were acclimated to awake scanning procedures; whole-brain fMRI images were acquired with a 9.4 Tesla scanner. Rs-fMRI data was preprocessed using an in-house pipeline and sLFOs were detected using a seed regressor generated by averaging BOLD signal across all voxels in the brain, which was then refined recursively within a time window of -16-12 s. The refined regressor was then used to estimate the voxel-wise sLFOs; these regressors were subsequently included in the general linear model to remove these moving hemodynamic components from the rs-fMRI data using general linear model filtering. Group level independent component analysis (ICA) with dual regression was used to detect resting-state networks and compare networks before and after sLFO denoising. Results show sLFOs constitute ~64% of the low frequency fMRI signal in squirrel monkey gray matter; they arrive earlier in regions in proximity to the middle cerebral arteries (e.g., somatosensory cortex) and later in regions close to draining vessels (e.g., cerebellum). Dual regression results showed that the physiological noise was significantly reduced after removing sLFOs and the extent of reduction was determined by the brain region contained in the resting-state network. These results highlight the need to estimate and remove sLFOs from fMRI data before further analysis.

Modulation of phase preference of single units in awake non-human primate using tACS

Modulation of phase preference of single units in awake non-human primate using tACS

Suppressive effects of ketamine on auditory steady-state responses in intact, awake macaques: A non-human primate model of schizophrenia

Auditory steady-state responses (ASSRs) are recurrent neural activities entrained to regular cyclic auditory stimulation. ASSRs are altered in individuals with schizophrenia, and may be related to hypofunction of the N-methyl-D-aspartate (NMDA) glutamate receptor. Noncompetitive NMDA receptor antagonists, including ketamine, have been used in ASSR studies of rodent models of schizophrenia. Although animal studies using non-human primates are required to complement rodent studies, the effects of ketamine on ASSRs are unknown in intact awake non-human primates. In this study, after administration of vehicle or ketamine, click trains at 20–83.3 Hz were presented to elicit ASSRs during recording of electroencephalograms in intact, awake macaque monkeys. The results indicated that ASSRs quantified by event-related spectral perturbation and inter-trial coherence were maximal at 83.3 Hz after vehicle administration, and that ketamine reduced ASSRs at 58.8 and 83.3 Hz, but not at 20 and 40 Hz. The present results demonstrated a reduction of ASSRs by the NMDA receptor antagonist at optimal frequencies with maximal responses in intact, awake macaques, comparable to ASSR reduction in patients with schizophrenia. These findings suggest that ASSR can be used as a neurophysiological biomarker of the disturbance of gamma-oscillatory neural circuits in this ketamine model of schizophrenia using intact, awake macaques. Thus, this model with ASSRs would be useful in the investigation of human brain pathophysiology as well as in preclinical translational research.

Primate monocular vision is intrinsically unstable: a side-effect of binocular homeostasis

Short-term deprivation of one eye of 1-2 hours in human adults results in reciprocal changes in the interocular balance that last 30-90 minutes, assumed to be the consequence of a change in the contrast gain within the binocular circuitry. What is not known is how these changes emerge and with what dynamic. One possibility is that they are initiated once the patch is removed. The alternative is that they result from a dynamic build-up from the moment the patch is applied. In this study, we measure the monocular dynamics of the non-deprived eye activation during the deprivation of the other eye in humans, using psychophysics, and in awake non-human primates, using functional ultrasound imaging (fUS) of the primary visual cortex (V1). In humans, we show that the contrast threshold increases overtime during the deprivation of the other eye. This observation indicates that the patching effect slowly builds up over the deprivation period at a binocular site. In the awake macaque, we show that during monocular deprivation the cerebral blood volume (CBV) increases in the non-deprived eye ocular-dominance bands, but not the deprived eye bands with similar dynamics as observed in human psychophysics. We show that human behavioral results can be predicted by a simple interocular normalization model that uses non-human primate CBV dynamics as a descriptor of the normalization pool activity. In conclusion, our results highlight a hitherto unknown feature of primate vision: monocular vision per se is intrinsically unstable.

Fentanyl-induced changes in brain activity in awake nonhuman primates at 9.4 Tesla.

Functional magnetic resonance imaging (fMRI) has been used to study the influence of opioids on neural circuitry implicated in opioid use disorder, such as the cortico-striatal-thalamo-cortical (CSTC) circuit. Given the increase in fentanyl-related deaths, this study was conducted to characterize the effects of fentanyl on patterns of brain activation in awake nonhuman primates. Four squirrel monkeys were acclimated to awake scanning procedures conducted at 9.4 Tesla. Subsequently, test sessions were conducted in which a dose of fentanyl that reliably maintains intravenous (IV) self-administration behavior in monkeys, 1μg/kg, was administered and the effects on patterns of brain activity were assessed using: (1) a pharmacological regressor to elucidate fentanyl-induced patterns of neural activity, and (2) seed-based approaches targeting bilateral anterior cingulate, thalamus, or nucleus accumbens (NAc) to determine alterations in CSTC functional connectivity. Results showed a functional inhibition of BOLD signal in brain regions that mediate behavioral effects of opioid agonists, such as cingulate cortex, striatum and midbrain. Functional connectivity between each of the seed regions and areas involved in motoric, sensory and cognition-related behavior generally decreased. In contrast, NAc functional connectivity with other striatal regions increased. These results indicate that fentanyl produces changes within CSTC circuitry that may reflect key features of opioid use disorder (e.g. persistent drug-taking/seeking) and thereby contribute to long-term disruptions in behavior and addiction. They also indicate that fMRI in alert nonhuman primates can detect drug-induced changes in neural circuits and, in turn, may be useful for investigating the effectiveness of medications to reverse drug-induced dysregulation.

Effects of Cortical Cooling on Sound Processing in Auditory Cortex and Thalamus of Awake Marmosets.

The auditory thalamus is the central nexus of bottom-up connections from the inferior colliculus and top-down connections from auditory cortical areas. While considerable efforts have been made to investigate feedforward processing of sounds in the auditory thalamus (medial geniculate body, MGB) of non-human primates, little is known about the role of corticofugal feedback in the MGB of awake non-human primates. Therefore, we developed a small, repositionable cooling probe to manipulate corticofugal feedback and studied neural responses in both auditory cortex and thalamus to sounds under conditions of normal and reduced cortical temperature. Cooling-induced increases in the width of extracellularly recorded spikes in auditory cortex were observed over the distance of several hundred micrometers away from the cooling probe. Cortical neurons displayed reduction in both spontaneous and stimulus driven firing rates with decreased cortical temperatures. In thalamus, cortical cooling led to increased spontaneous firing and either increased or decreased stimulus driven activity. Furthermore, response tuning to modulation frequencies of temporally modulated sounds and spatial tuning to sound source location could be altered (increased or decreased) by cortical cooling. Specifically, best modulation frequencies of individual MGB neurons could shift either toward higher or lower frequencies based on the vector strength or the firing rate. The tuning of MGB neurons for spatial location could both sharpen or widen. Elevation preference could shift toward higher or lower elevations and azimuth tuning could move toward ipsilateral or contralateral locations. Such bidirectional changes were observed in many parameters which suggests that the auditory thalamus acts as a filter that could be adjusted according to behaviorally driven signals from auditory cortex. Future work will have to delineate the circuit elements responsible for the observed effects.

Population receptive fields in nonhuman primates from whole-brain fMRI and large-scale neurophysiology in visual cortex.

Population receptive field (pRF) modeling is a popular fMRI method to map the retinotopic organization of the human brain. While fMRI-based pRF maps are qualitatively similar to invasively recorded single-cell receptive fields in animals, it remains unclear what neuronal signal they represent. We addressed this question in awake nonhuman primates comparing whole-brain fMRI and large-scale neurophysiological recordings in areas V1 and V4 of the visual cortex. We examined the fits of several pRF models based on the fMRI blood-oxygen-level-dependent (BOLD) signal, multi-unit spiking activity (MUA), and local field potential (LFP) power in different frequency bands. We found that pRFs derived from BOLD-fMRI were most similar to MUA-pRFs in V1 and V4, while pRFs based on LFP gamma power also gave a good approximation. fMRI-based pRFs thus reliably reflect neuronal receptive field properties in the primate brain. In addition to our results in V1 and V4, the whole-brain fMRI measurements revealed retinotopic tuning in many other cortical and subcortical areas with a consistent increase in pRF size with increasing eccentricity, as well as a retinotopically specific deactivation of default mode network nodes similar to previous observations in humans.

Orientation-dependent effects of transcranial alternating current stimulation (tACS) on spike timing in awake non-human primates

Orientation-dependent effects of transcranial alternating current stimulation (tACS) on spike timing in awake non-human primates

Distinct neuron populations for simple and compound calls in the primary auditory cortex of awake marmosets.

Marmosets are highly social non-human primates that live in families. They exhibit rich vocalization, but the neural basis underlying this complex vocal communication is largely unknown. Here we report the existence of specific neuron populations in marmoset A1 that respond selectively to distinct simple or compound calls made by conspecific marmosets. These neurons were spatially dispersed within A1 but distinct from those responsive to pure tones. Call-selective responses were markedly diminished when individual domains of the call were deleted or the domain sequence was altered, indicating the importance of the global rather than local spectral-temporal properties of the sound. Compound call-selective responses also disappeared when the sequence of the two simple-call components was reversed or their interval was extended beyond 1 s. Light anesthesia largely abolished call-selective responses. Our findings demonstrate extensive inhibitory and facilitatory interactions among call-evoked responses, and provide the basis for further study of circuit mechanisms underlying vocal communication in awake non-human primates.

Long-term stability of avalanche scaling and integrative network organization in prefrontal and premotor cortex.

Ongoing neuronal activity in the brain establishes functional networks that reflect normal and pathological brain function. Most estimates of these functional networks suffer from low spatiotemporal resolution and indirect measures of neuronal population activity, limiting the accuracy and reliability in their reconstruction over time. Here, we studied the stability of neuronal avalanche dynamics and corresponding reconstructed functional networks in the adult brain. Using chronically implanted high-density microelectrode arrays, the local field potential (LFP) of resting-state activity was recorded in prefrontal and premotor cortex of awake nonhuman primates. Avalanche dynamics revealed stable scaling exhibiting an inverted parabolic profile and collapse exponent of 2 in line with a critical branching process over many days and weeks. Functional networks were based on a Bayesian-derived estimator and demonstrated stable integrative properties characterized by nontrivial high neighborhood overlap between strongly connected nodes and robustness to weak-link pruning. Entropy-based mixing analysis revealed significant changes in strong link weights over weeks. The long-term stability in avalanche scaling and integrative network organization in the face of individual link weight changes should support the development of noninvasive biomarkers to characterize normal and abnormal brain states in the adult brain.

Selective Participation of Single Cortical Neurons in Neuronal Avalanches.

Neuronal avalanches are scale-invariant neuronal population activity patterns in the cortex that emerge in vivo in the awake state and in vitro during balanced excitation and inhibition. Theory and experiments suggest that avalanches indicate a state of cortex that improves numerous aspects of information processing by allowing for the transient and selective formation of local as well as system-wide spanning neuronal groups. If avalanches are indeed involved with information processing, one might expect that single neurons would participate in avalanche patterns selectively. Alternatively, all neurons could participate proportionally to their own activity in each avalanche as would be expected for a population rate code. Distinguishing these hypotheses, however, has been difficult as robust avalanche analysis requires technically challenging measures of their intricate organization in space and time at the population level, while also recording sub- or suprathreshold activity from individual neurons with high temporal resolution. Here, we identify repeated avalanches in the ongoing local field potential (LFP) measured with high-density microelectrode arrays in the cortex of awake nonhuman primates and in acute cortex slices from young and adult rats. We studied extracellular unit firing in vivo and intracellular responses of pyramidal neurons in vitro. We found that single neurons participate selectively in specific LFP-based avalanche patterns. Furthermore, we show in vitro that manipulating the balance of excitation and inhibition abolishes this selectivity. Our results support the view that avalanches represent the selective, scale-invariant formation of neuronal groups in line with the idea of Hebbian cell assemblies underlying cortical information processing.

Intracellular neuronal recording in awake nonhuman primates.

Intracellular neuronal recordings from the brain of awake nonhuman primates have remained difficult to obtain because of several formidable technical challenges, such as poor recording stability and difficulties in maintaining long-term recording conditions. We have developed a technique to record neuronal activity by using a coaxial guide tube and sharp electrode assembly, which allows researchers to repeatedly and reliably perform intracellular recordings in the cortex of awake marmosets. Recordings from individual neurons last from several minutes to more than an hour. A key advantage of this approach is that it does not require dura removal, permitting recordings over weeks and months in a single animal. This protocol describes the step-by-step procedures for construction of a custom-made marmoset chair, head-cap implantation, preparation of the sharp electrode and guide tube, neuronal recording and data analysis. As the technique is practical and easy to adapt, we anticipate that it can also be applied to other mammalian models, including larger-size nonhuman primates.