Corticocortical connections of the rostral forelimb area in rats: a quantitative tract-tracing study.

The rostral forelimb area (RFA) in the rat is a premotor cortical region based on its dense efferent projections to primary motor cortex. This study describes corticocortical connections of RFA and the relative strength of connections with other cortical areas. The goal was to provide a better understanding of the cortical network in which RFA participates, and thus, determine its function in sensorimotor behavior. The RFA of adult male Long-Evans rats (n = 6) was identified using intracortical microstimulation techniques and injected with the tract-tracer, biotinylated dextran amine (BDA). In post-mortem tissue, locations of BDA-labeled terminal boutons and neuronal somata were plotted and superimposed on cortical field boundaries. Quantitative estimates of terminal boutons in each region of interest were based on unbiased stereological methods. The results demonstrate that RFA has dense connections with primary motor cortex and frontal cortex medial and lateral to RFA. Moderate connections were found with insular cortex, primary somatosensory cortex (S1), the M1/S1 overlap zone, and lateral somatosensory areas. Cortical connections of RFA in rat are strikingly similar to cortical connections of the ventral premotor cortex in non-human primates, suggesting that these areas share similar functions and allow greater translation of rodent premotor cortex studies to primates.

Photoacoustic imaging of squirrel monkey cortical responses induced by peripheral mechanical stimulation.

Non-human primates (NHPs) are crucial models for studies of neuronal activity. Emerging photoacoustic imaging modalities offer excellent tools for studying NHP brains with high sensitivity and high spatial resolution. In this research, a photoacoustic microscopy (PAM) device was used to provide a label-free quantitative characterization of cerebral hemodynamic changes due to peripheral mechanical stimulation. A 5 × 5 mm area within the somatosensory cortex region of an adult squirrel monkey was imaged. A deep, fully connected neural network was characterized and applied to the PAM images of the cortex to enhance the vessel structures after mechanical stimulation on the forelimb digits. The quality of the PAM images was improved significantly with a neural network while preserving the hemodynamic responses. The functional responses to the mechanical stimulation were characterized based on the improved PAM images. This study demonstrates capability of PAM combined with machine learning for functional imaging of the NHP brain.

Collagen IV of basement membranes: III. Chloride pressure is a primordial innovation that drives and maintains the assembly of scaffolds.

Collagen IV scaffold is a primordial innovation enabling the assembly of a fundamental architectural unit of epithelial tissues-a basement membrane attached to polarized cells. A family of six α-chains (α1 to α6) coassemble into three distinct protomers that form supramolecular scaffolds, noted as collagen IVα121, collagen IVα345, and collagen IVα121-α556. Chloride ions play a pivotal role in scaffold assembly, based on studies of NC1 hexamers from mammalian tissues. First, Cl- activates a molecular switch within trimeric NC1 domains that initiates protomer oligomerization, forming an NC1 hexamer between adjoining protomers. Second, Cl- stabilizes the hexamer structure. Whether this Cl--dependent mechanism is of fundamental importance in animal evolution is unknown. Here, we developed a simple in vitro method of SDS-PAGE to determine the role of solution Cl- in hexamer stability. Hexamers were characterized from 34 animal species across 15 major phyla, including the basal Cnidarian and Ctenophora phyla. We found that solution Cl- stabilized the quaternary hexamer structure across all phyla except Ctenophora, Ecdysozoa, and Rotifera. Further analysis of hexamers from peroxidasin knockout mice, a model for decreasing hexamer crosslinks, showed that solution Cl- also stabilized the hexamer surface conformation. The presence of sufficient chloride concentration in solution or "chloride pressure" dynamically maintains the native form of the hexamer. Collectively, our findings revealed that chloride pressure on the outside of cells is a primordial innovation that drives and maintains the quaternary and conformational structure of NC1 hexamers of collagen IV scaffolds.

Broad Therapeutic Time Window for Driving Motor Recovery After TBI Using Activity-Dependent Stimulation.

BACKGROUND: After an acquired injury to the motor cortex, the ability to generate skilled movements is impaired, leading to long-term motor impairment and disability. While rehabilitative therapy can improve outcomes in some individuals, there are no treatments currently available that are able to fully restore lost function. OBJECTIVE: We previously used activity-dependent stimulation (ADS), initiated immediately after an injury, to drive motor recovery. The objective of this study was to determine if delayed application of ADS would still lead to recovery and if the recovery would persist after treatment was stopped. METHODS: Rats received a controlled cortical impact over primary motor cortex, microelectrode arrays were implanted in ipsilesional premotor and somatosensory areas, and a custom brain-machine interface was attached to perform the ADS. Stimulation was initiated either 1, 2, or 3 weeks after injury and delivered constantly over a 4-week period. An additional group was monitored for 8 weeks after terminating ADS to assess persistence of effect. Results were compared to rats receiving no stimulation. RESULTS: ADS was delayed up to 3 weeks from injury onset and still resulted in significant motor recovery, with maximal recovery occurring in the 1-week delay group. The improvements in motor performance persisted for at least 8 weeks following the end of treatment. CONCLUSIONS: ADS is an effective method to treat motor impairments following acquired brain injury in rats. This study demonstrates the clinical relevance of this technique as it could be initiated in the post-acute period and could be explanted/ceased once recovery has occurred.

Stimulation-Evoked Effective Connectivity (SEEC): An in-vivo approach for defining mesoscale corticocortical connectivity.

BACKGROUND: Cortical electrical stimulation is a versatile technique for examining the structure and function of cortical regions and for implementing novel therapies. While electrical stimulation has been used to examine the local spread of neural activity, it may also enable longitudinal examination of mesoscale interregional connectivity. NEW METHOD: Here, we sought to use intracortical microstimulation (ICMS) in conjunction with recordings of multi-unit action potentials to assess the mesoscale effective connectivity within sensorimotor cortex. Neural recordings were made from multielectrode arrays placed into sensory, motor, and premotor regions during surgical experiments in three squirrel monkeys. During each recording, single-pulse ICMS was repeatably delivered to a single region. Mesoscale effective connectivity was calculated from ICMS-evoked changes in multi-unit firing. RESULTS: Multi-unit action potentials were able to be detected on the order of 1 ms after each ICMS pulse. Across sensorimotor regions, short-latency (< 2.5 ms) ICMS-evoked neural activity strongly correlated with known anatomical connections. Additionally, ICMS-evoked responses remained stable across the experimental period, despite small changes in electrode locations and anesthetic state. COMPARISON WITH EXISTING METHODS: Previous imaging studies investigating cross-regional responses to stimulation are limited to utilizing indirect hemodynamic responses and thus lack the temporal specificity of ICMS-evoked responses. CONCLUSIONS: These results show that monitoring ICMS-evoked neural activity, in a technique we refer to as Stimulation-Evoked Effective Connectivity (SEEC), is a viable way to longitudinally assess effective connectivity, enabling studies comparing the time course of connectivity changes with the time course of changes in behavioral function.

Photoacoustic imaging of squirrel monkey cortical and subcortical brain regions during peripheral electrical stimulation.

The investigation of neuronal activity in non-human primate models is of critical importance due to their genetic similarity to human brains. In this study, we tested the feasibility of using photoacoustic imaging for the detection of cortical and subcortical responses due to peripheral electrical stimulation in a squirrel monkey model. Photoacoustic computed tomography and photoacoustic microscopy were applied on squirrel monkeys for real-time deep subcortical imaging and optical-resolution cortical imaging, respectively. The electrically evoked hemodynamic changes in primary somatosensory cortex, premotor cortices, primary motor cortex, and underlying subcortical areas were measured. Hemodynamic responses were observed in both cortical and subcortical brain areas at the cortices during external stimulation, demonstrating the feasibility of photoacoustic technique for functional imaging of non-human primate brain.

A cortical injury model in a non-human primate to assess execution of reach and grasp actions: implications for recovery after traumatic brain injury.

BACKGROUND: Technological advances in developing experimentally controlled models of traumatic brain injury (TBI) are prevalent in rodent models and these models have proven invaluable in characterizing temporal changes in brain and behavior after trauma. To date no long-term studies in non-human primates (NHPs) have been published using an experimentally controlled impact device to follow behavioral performance over time. NEW METHOD: We have employed a controlled cortical impact (CCI) device to create a focal contusion to the hand area in primary motor cortex (M1) of three New World monkeys to characterize changes in reach and grasp function assessed for 3 months after the injury. RESULTS: The CCI destroyed most of M1 hand representation reducing grey matter by 9.6 mm3, 12.9 mm3, and 15.5 mm3 and underlying corona radiata by 7.4 mm3, 6.9 mm3, and 5.6 mm3 respectively. Impaired motor function was confined to the hand contralateral to the injury. Gross hand-use was only mildly affected during the first few days of observation after injury while activity requiring skilled use of the hand was impaired over three months. COMPARISON WITH EXISTING METHOD(S): This study is unique in establishing a CCI model of TBI in an NHP resulting in persistent impairments in motor function evident in volitional use of the hand. CONCLUSIONS: Establishing an NHP model of TBI is essential to extend current rodent models to the complex neural architecture of the primate brain. Moving forward this model can be used to investigate novel therapeutic interventions to improve or restore impaired motor function after trauma.

Representation of individual forelimb muscles in primary motor cortex.

Stimulus-triggered averaging (StTA) of forelimb muscle electromyographic (EMG) activity was used to investigate individual forelimb muscle representation within the primary motor cortex (M1) of rhesus macaques with the objective of determining the extent of intra-areal somatotopic organization. Two monkeys were trained to perform a reach-to-grasp task requiring multijoint coordination of the forelimb. EMG activity was simultaneously recorded from 24 forelimb muscles including 5 shoulder, 7 elbow, 5 wrist, 5 digit, and 2 intrinsic hand muscles. Microstimulation (15 µA at 15 Hz) was delivered throughout the movement task and individual stimuli were used as triggers for generating StTAs of EMG activity. StTAs were used to map the cortical representations of individual forelimb muscles. As reported previously (Park et al. 2001), cortical maps revealed a central core of distal muscle (wrist, digit, and intrinsic hand) representation surrounded by a horseshoe-shaped proximal (shoulder and elbow) muscle representation. In the present study, we found that shoulder and elbow flexor muscles were predominantly represented in the lateral branch of the horseshoe whereas extensors were predominantly represented in the medial branch. Distal muscles were represented within the core distal forelimb representation and showed extensive overlap. For the first time, we also show maps of inhibitory output from motor cortex, which follow many of the same organizational features as the maps of excitatory output.NEW & NOTEWORTHY While the orderly representation of major body parts along the precentral gyrus has been known for decades, questions have been raised about the possible existence of additional more detailed aspects of somatotopy. In this study, we have investigated this question with respect to muscles of the arm and show consistent features of within-arm (intra-areal) somatotopic organization. For the first time we also show maps of how inhibitory output from motor cortex is organized.

Cortical Effects on Ipsilateral Hindlimb Muscles Revealed with Stimulus-Triggered Averaging of EMG Activity.

While a large body of evidence supports the view that ipsilateral motor cortex may make an important contribution to normal movements and to recovery of function following cortical injury (Chollet et al. 1991; Fisher 1992; Caramia et al. 2000; Feydy et al. 2002), relatively little is known about the properties of output from motor cortex to ipsilateral muscles. Our aim in this study was to characterize the organization of output effects on hindlimb muscles from ipsilateral motor cortex using stimulus-triggered averaging of EMG activity. Stimulus-triggered averages of EMG activity were computed from microstimuli applied at 60-120 µA to sites in both contralateral and ipsilateral M1 of macaque monkeys during the performance of a hindlimb push-pull task. Although the poststimulus effects (PStEs) from ipsilateral M1 were fewer in number and substantially weaker, clear and consistent effects were obtained at an intensity of 120 µA. The mean onset latency of ipsilateral poststimulus facilitation was longer than contralateral effects by an average of 0.7 ms. However, the shortest latency effects in ipsilateral muscles were as short as the shortest latency effects in the corresponding contralateral muscles suggesting a minimal synaptic linkage that is equally direct in both cases.

Subject-specific computational modeling of DBS in the PPTg area.

Deep brain stimulation (DBS) in the pedunculopontine tegmental nucleus (PPTg) has been proposed to alleviate medically intractable gait difficulties associated with Parkinson's disease. Clinical trials have shown somewhat variable outcomes, stemming in part from surgical targeting variability, modulating fiber pathways implicated in side effects, and a general lack of mechanistic understanding of DBS in this brain region. Subject-specific computational models of DBS are a promising tool to investigate the underlying therapy and side effects. In this study, a parkinsonian rhesus macaque was implanted unilaterally with an 8-contact DBS lead in the PPTg region. Fiber tracts adjacent to PPTg, including the oculomotor nerve, central tegmental tract, and superior cerebellar peduncle, were reconstructed from a combination of pre-implant 7T MRI, post-implant CT, and post-mortem histology. These structures were populated with axon models and coupled with a finite element model simulating the voltage distribution in the surrounding neural tissue during stimulation. This study introduces two empirical approaches to evaluate model parameters. First, incremental monopolar cathodic stimulation (20 Hz, 90 µs pulse width) was evaluated for each electrode, during which a right eyelid flutter was observed at the proximal four contacts (-1.0 to -1.4 mA). These current amplitudes followed closely with model predicted activation of the oculomotor nerve when assuming an anisotropic conduction medium. Second, PET imaging was collected OFF-DBS and twice during DBS (two different contacts), which supported the model predicted activation of the central tegmental tract and superior cerebellar peduncle. Together, subject-specific models provide a framework to more precisely predict pathways modulated by DBS.

Properties of primary motor cortex output to hindlimb muscles in the macaque monkey.

The cortical control of forelimb motor function has been studied extensively, especially in the primate. In contrast, cortical control of the hindlimb has been relatively neglected. This study assessed the output properties of the primary motor cortex (M1) hindlimb representation in terms of the sign, latency, magnitude, and distribution of effects in stimulus-triggered averages (StTAs) of electromyography (EMG) activity recorded from 19 muscles, including hip, knee, ankle, digit, and intrinsic foot muscles, during a push-pull task compared with data reported previously on the forelimb. StTAs (15, 30, and 60 µA at 15 Hz) of EMG activity were computed at 317 putative layer V sites in two rhesus macaques. Poststimulus facilitation (PStF) was distributed equally between distal and proximal muscles, whereas poststimulus suppression (PStS) was more common in distal muscles than proximal muscles (51/49%, respectively, for PStF; 72/28%, respectively, for PStS) at 30 µA. Mean PStF and PStS onset latency generally increased the more distal the joint of a muscle's action. Most significantly, the average magnitude of hindlimb poststimulus effects was considerably weaker than the average magnitude of effects from forelimb M1. In addition, forelimb PStF magnitude increased consistently from proximal to distal joints, whereas hindlimb PStF magnitude was similar at all joints except the intrinsic foot muscles, which had a magnitude of approximately double that of all of the other muscles. The results suggest a greater monosynaptic input to forelimb compared with hindlimb motoneurons, as well as a more direct synaptic linkage for the intrinsic foot muscles compared with the other hindlimb muscles.

EMG activation patterns associated with high frequency, long-duration intracortical microstimulation of primary motor cortex.

The delivery of high-frequency, long-duration intracortical microstimulation (HFLD-ICMS) to primary motor cortex (M1) in primates produces hand movements to a common final end-point regardless of the starting hand position (Graziano et al., 2002). We have confirmed this general conclusion. We further investigated the extent to which the (1) temporal pattern, (2) magnitude, and (3) latency of electromyographic (EMG) activation associated with HFLD-ICMS-evoked movements are dependent on task conditions, including limb posture. HFLD-ICMS was applied to layer V sites in M1 cortex. EMG activation with HFLD-ICMS was evaluated while two male rhesus macaques performed a number of tasks in which the starting position of the hand could be varied throughout the workspace. HFLD-ICMS-evoked EMG activity was largely stable across all parameters tested independent of starting hand position. The most common temporal pattern of HFLD-ICMS-evoked EMG activity (58% of responses) was a sharp rise to a plateau. The plateau level was maintained essentially constant for the entire duration of the stimulus train. The plateau pattern is qualitatively different from the largely bell-shaped patterns typical of EMG activity associated with natural goal directed movements (Brown and Cooke, 1990; Hoffman and Strick, 1999). HFLD-ICMS produces relatively fixed parameters of muscle activation independent of limb position. We conclude that joint movement associated with HFLD-ICMS occurs as a function of the length-tension properties of stimulus-activated muscles until an equilibrium between agonist and antagonist muscle force is achieved.

Cortical output to fast and slow muscles of the ankle in the rhesus macaque.

The cortical control of fast and slow muscles of the ankle has been the subject of numerous reports yielding conflicting results. Although it is generally agreed that cortical stimulation yields short latency facilitation of fast muscles, the effects on the slow muscle, soleus, remain controversial. Some studies have shown predominant facilitation of soleus from the cortex while others have provided evidence of differential control in which soleus is predominantly inhibited from the cortex. The objective of this study was to investigate the cortical control of fast and slow muscles of the ankle using stimulus triggered averaging (StTA) of EMG activity, which is a sensitive method of detecting output effects on muscle activity. This method also has relatively high spatial resolution and can be applied in awake, behaving subjects. Two rhesus macaques were trained to perform a hindlimb push-pull task. Stimulus triggered averages (StTAs) of EMG activity (15, 30, and 60 µA at 15 Hz) were computed for four muscles of the ankle [tibialis anterior (TA), medial gastrocnemius (MG), lateral gastrocnemius (LG), and soleus] as the monkeys performed the task. Poststimulus facilitation (PStF) was observed in both the fast muscles (TA, MG, and LG) as well as the slow muscle (soleus) and was as common and as strong in soleus as in the fast muscles. However, while poststimulus suppression (PStS) was observed in all muscles, it was more common in the slow muscle compared to the fast muscles and was as common as facilitation at low stimulus intensities. Overall, our results demonstrate that cortical facilitation of soleus has an organization that is very similar to that of the fast ankle muscles. However, cortical inhibition is organized differently allowing for more prominent suppression of soleus motoneurons.

Hijacking cortical motor output with repetitive microstimulation.

High-frequency repetitive microstimulation has been widely used as a method of investigating the properties of cortical motor output. Despite its widespread use, few studies have investigated how activity evoked by high-frequency stimulation may interact with the existing activity of cortical cells resulting from natural synaptic inputs. A reasonable assumption might be that the stimulus-evoked activity sums with the existing natural activity. However, another possibility is that the stimulus-evoked firing of cortical neurons might block and replace the natural activity. We refer to this latter possibility as "neural hijacking." Evidence from analysis of EMG activity evoked by repetitive microstimulation (200 Hz, 500 ms) of primary motor cortex in two rhesus monkeys during performance of a reach-to-grasp task strongly supports the neural hijacking hypothesis.

Methods for chronic recording of EMG activity from large numbers of hindlimb muscles in awake rhesus macaques.

Studies of the neural control of movement often rely on the ability to record EMG activity during natural behavioral tasks over long periods of time. Increasing the number of recorded muscles and the time over which recordings are made allows more rigorous answers to many questions related to the descending control of motor output. Chronic recording of EMG activity from multiple hindlimb muscles has been reported in the cat but few studies have been done in non-human primates. This paper describes two chronic EMG implant methods that are minimally invasive, relatively non-traumatic and capable of recording from large numbers of hindlimb muscles simultaneously for periods of many months to years.

Stability of output effects from motor cortex to forelimb muscles in primates.

Stimulus-triggered averaging (StTA) of electromyographic (EMG) activity is a form of intracortical microstimulation that enables documentation in awake animals of the sign, magnitude, latency, and distribution of output effects from cortical and brainstem areas to motoneurons of different muscles. In this study, we show that the properties of effects in StTAs are stable and mostly independent of task conditions. StTAs of EMG activity from 24 forelimb muscles were collected from two male rhesus monkeys while they performed three tasks: (1) an isometric step tracking wrist task, (2) an isometric whole-arm push-pull task, and (3) a reach-to-grasp task. Layer V sites in primary motor cortex were identified and microstimuli were applied (15 muA) at a low rate (15 Hz). Our results show that the sign of effects (facilitation or suppression) in StTAs of EMG activity are remarkably stable in the presence of joint angle position changes (96% stable), whole-arm posture changes (97% stable), and across fundamentally different types of tasks such as arm push-pull versus reach-to-grasp (81% stable). Furthermore, comparing effects across different phases of a task also yielded remarkable stability (range, 84-96%). At different shoulder, elbow, and wrist angles, the magnitudes of effects in individual muscles were highly correlated. Our results demonstrate that M1 output effects obtained with StTA of EMG activity are highly stable across widely varying joint angles and motor tasks. This study further validates the use of StTA for mapping and other studies of cortical motor output.

Human podocytes adhere to the KRGDS motif of the alpha3alpha4alpha5 collagen IV network.

Podocyte adhesion to the glomerular basement membrane is required for proper function of the glomerular filtration barrier. However, the mechanism whereby podocytes adhere to collagen IV networks, a major component of the glomerular basement membrane, is poorly understood. The predominant collagen IV network is composed of triple helical protomers containing the alpha3alpha4alpha5 chains. The protomers connect via the trimeric noncollagenous (NC1) domains to form hexamers at the interface. Because the NC1 domains of this network can potentially support integrin-dependent cell adhesion, it was determined whether individual NC1 monomers or alpha3alpha4alpha5 hexamers support podocyte adhesion. It was found that, although human podocytes did not adhere to NC1 domains proper, they did adhere via integrin alphavbeta3 to a KRGDS motif located adjacent to alpha3NC1 domains. Because the KRGDS motif is a site of phosphorylation, its interactions with integrin alphavbeta3 may play a critical role in cell signaling in physiologic and pathologic states.