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.

Gray matter volume of functionally relevant primary motor cortex is causally related to learning a hand motor task.

Variability in brain structure is associated with the capacity for behavioral change. However, a causal link between specific brain areas and behavioral change (such as motor learning) has not been demonstrated. We hypothesized that greater gray matter volume of a primary motor cortex (M1) area active during a hand motor learning task is positively correlated with subsequent learning of the task, and that the disruption of this area blocks learning of the task. Healthy participants underwent structural MRI before learning a skilled hand motor task. Next, participants performed this learning task during fMRI to determine M1 areas functionally active during this task. This functional ROI was anatomically constrained with M1 boundaries to create a group-level "Active-M1" ROI used to measure gray matter volume in each participant. Greater gray matter volume in the left hemisphere Active-M1 ROI was related to greater motor learning in the corresponding right hand. When M1 hand area was disrupted with repetitive transcranial stimulation (rTMS), learning of the motor task was blocked, confirming its causal link to motor learning. Our combined imaging and rTMS approach revealed greater cortical volume in a task-relevant M1 area is causally related to learning of a hand motor task in healthy humans.

Spared Premotor Areas Undergo Rapid Nonlinear Changes in Functional Organization Following a Focal Ischemic Infarct in Primary Motor Cortex of Squirrel Monkeys.

Recovery of motor function after stroke is accompanied by reorganization of movement representations in spared cortical motor regions. It is widely assumed that map reorganization parallels recovery, suggesting a causal relationship. We examined this assumption by measuring changes in motor representations in eight male and six female squirrel monkeys in the first few weeks after injury, a time when motor recovery is most rapid. Maps of movement representations were derived using intracortical microstimulation techniques in primary motor cortex (M1), ventral premotor cortex (PMv), and dorsal premotor cortex (PMd) in 14 adult squirrel monkeys before and after a focal infarct in the M1 distal forelimb area. Maps were derived at baseline and at either 2 (n = 7) or 3 weeks (n = 7) postinfarct. In PMv the forelimb maps remained unchanged at 2 weeks but contracted significantly (-42.4%) at 3 weeks. In PMd the forelimb maps expanded significantly (+110.6%) at 2 weeks but contracted significantly (-57.4%) at 3 weeks. Motor deficits were equivalent at both time points. These results highlight two features of plasticity after M1 lesions. First, significant contraction of distal forelimb motor maps in both PMv and PMd is evident by 3 weeks. Second, an unpredictable nonlinear pattern of reorganization occurs in the distal forelimb representation in PMd, first expanding at 2 weeks, and then contracting at 3 weeks postinjury. Together with previous results demonstrating reliable map expansions in PMv several weeks to months after M1 injury, the subacute time period may represent a critical window for the timing of therapeutic interventions.SIGNIFICANCE STATEMENT The relationship between motor recovery and motor map reorganization after cortical injury has rarely been examined in acute/subacute periods. In nonhuman primates, premotor maps were examined at 2 and 3 weeks after injury to primary motor cortex. Although maps are known to expand late after injury, the present study demonstrates early map expansion at 2 weeks (dorsal premotor cortex) followed by contraction at 3 weeks (dorsal and ventral premotor cortex). This nonlinear map reorganization during a time of gradual behavioral recovery suggests that the relationship between map plasticity and motor recovery is much more complex than previously thought. It also suggests that rehabilitative motor training may have its most potent effects during this early dynamic phase of map reorganization.

Chronic Stimulation Improves Motor Performance in an Ambulatory Rat Model of Spinal Cord Injury.

BACKGROUND: The purpose of this proof-of-concept feasibility study was to determine if spike-triggered intraspinal microstimulation (ISMS), a form of activity dependent stimulation (ADS), results in improved motor performance in an ambulatory rat model of spinal cord injury (SCI). METHODS: Experiments were carried out in adult male Sprague Dawley rats with moderate thoracic contusion injury. Rats were assigned to one of two groups: Control or ADS therapy. Four weeks post-SCI, all rats were implanted with a recording microelectrode in the left hindlimb motor cortex and a fine-wire stimulating electrode in the contralateral lumbar spinal cord. ADS was administered for 4 hours/day, 4 days/week, for 4 weeks. During therapy sessions, single-unit spikes were discriminated in real time in the hindlimb motor cortex and used to trigger stimulation in the spinal cord ventral horn. Control rats were similarly implanted with electrodes but did not receive stimulation therapy. RESULTS: Motor performances of each rat were evaluated before SCI contusion, once a week post-SCI for four weeks (prior to electrode implantation), and once a week post-conditioning for four weeks. Basso, Beattie, and Bresnahan (BBB) locomotor scores were significantly improved in ADS rats compared to Control rats at 1 and 2 weeks after initiation of therapy. Foot fault scores on the Horizontal Ladder were significantly improved in ADS rats compared to pre-therapy ADS and Control rats after 1 week of therapy and recovered to near pre-injury scores after 3 weeks of therapy. The Ledged Beam test showed deficits after SCI in both ADS and Control rats but there were no significant differences between groups after 4 weeks of ADS therapy. CONCLUSIONS: These results show that chronic stimulation after spinal cord injury using a methodology of spike-triggered ISMS enhances behavioral recovery of locomotor function as measured by the BBB score and the Horizontal Ladder task. However, it is still uncertain if the behavioral improvements seen were dependent on spike-triggered ISMS.

Entrainment of Network Activity by Closed-Loop Microstimulation in Healthy Ambulatory Rats.

As our understanding of volitional motor function increases, it is clear that complex movements are the result of the interactions of multiple cortical regions rather than just the output properties of primary motor cortex. However, our understanding of the interactions among these regions is limited. In this study, we used the activity-dependent stimulation (ADS) technique to determine the short/long-term effects on network activity and neuroplasticity of intracortical connections. ADS uses the intrinsic neural activity of one region to trigger stimulations in a separate region of the brain and can manipulate neuronal connectivity in vivo. Our aim was to compare single-unit neuronal activity within premotor cortex (rostral forelimb area, [RFA] in rats) in response to ADS (triggered from RFA) and randomly-generated stimulation in the somatosensory area (S1) within single sessions and across 21 consecutive days of stimulation. We examined firing rate and correlation between spikes and stimuli in chronically-implanted healthy ambulatory rats during spontaneous and evoked activity. At the end of the treatment, we evaluated changes of synaptophysin expression. Our results demonstrated the ability of ADS to modulate RFA firing properties and to promote synaptogenesis in S1, strengthening the idea that this Hebbian-inspired protocol can be used to modulate cortical connectivity.

Differential Effects of Open- and Closed-Loop Intracortical Microstimulation on Firing Patterns of Neurons in Distant Cortical Areas.

Intracortical microstimulation can be used successfully to modulate neuronal activity. Activity-dependent stimulation (ADS), in which action potentials recorded extracellularly from a single neuron are used to trigger stimulation at another cortical location (closed-loop), is an effective treatment for behavioral recovery after brain lesion, but the related neurophysiological changes are still not clear. Here, we investigated the ability of ADS and random stimulation (RS) to alter firing patterns of distant cortical locations. We recorded 591 neuronal units from 23 Long-Evan healthy anesthetized rats. Stimulation was delivered to either forelimb or barrel field somatosensory cortex, using either RS or ADS triggered from spikes recorded in the rostral forelimb area (RFA). Both RS and ADS stimulation protocols rapidly altered spike firing within RFA compared with no stimulation. We observed increase in firing rates and change of spike patterns. ADS was more effective than RS in increasing evoked spikes during the stimulation periods, by producing a reliable, progressive increase in stimulus-related activity over time and an increased coupling of the trigger channel with the network. These results are critical for understanding the efficacy of closed-loop electrical microstimulation protocols in altering activity patterns in interconnected brain networks, thus modulating cortical state and functional connectivity.

Manifestations of Apprehension and Anxiety in a Sprague Dawley Cranial Defect Model.

BACKGROUND: Syndrome of the trephined is a neurologic condition that commonly arises in patients who undergo craniectomy and have a prolonged cranial defect. Symptoms of this condition include headache, difficulties concentrating, diminished fine motor/dexterity skills, mood changes, and anxiety/apprehension. The authors hypothesize that an animal model demonstrating anxiety/apprehension in rats who undergo craniectomy is feasible utilizing standardized animal behavioral testing. METHODS: Sprague Dawley rats were the stratified to 1 of 2 groups for comparison of neurobehavioral outcomes. Group #1 (closed cranial group) had their cranial trephination immediately closed with acrylic to restore normal cranial anatomy and Group #2 (open cranial group) had their cranial trephination enlarged to represent a decompressive hemicraniectomy immediately. Anxiety/apprehension was studied using a standardized rodent open field test. Statistical comparison of differences among the 2 groups was performed. RESULTS: Ten rats were studied with 5 rats in each group. Standard rodent open field testing of anxiety demonstrated no difference among the 2 groups at 1 week. Rats in the "Open cranial group" demonstrated progressively more anxiety over the following 3-month period. Rats in the "Open cranial group" demonstrated increasing anxiety levels as compared with rats in the "Closed cranial group." At week 16, the "Open cranial group" anxiety levels were significantly greater than week 4 (t = 2.24, P = 0.04) demonstrating a significant linear trend over time (R = 0.99; P = 0.002). The "Closed cranial group" did not show this trend (R = 07; P = 0.74). CONCLUSION: Our study demonstrates that anxiety and apprehension are more prevalent in rats with an open, prolonged cranial defect in comparison to those with a closed cranium. This correlates with similar finds in humans with syndrome of the trephined.

Dose response of task-specific upper limb training in people at least 6 months poststroke: A phase II, single-blind, randomized, controlled trial.

OBJECTIVE: The objectives of this work were to (1) determine whether higher doses of motor therapy in chronic poststroke hemiparesis result in better outcomes, compared to lower doses, and (2) evaluate potential modifiers of the dose-response relationship. METHODS: Eighty-five adults with upper extremity paresis ≥6 months poststroke were randomized to one of four dose groups in this single-blind, parallel, randomized, control trial. The dosing parameter manipulated was amount of task-specific training, as indexed by the number of task repetitions. Groups received 3,200, 6,400, 9,600, or individualized maximum (IM) repetitions, during 1-hour sessions, 4 days/week for 8 weeks. The intervention was an individualized, progressive, task-specific upper-limb training program designed to improve upper-limb functional motor capacity. The primary outcome was the slope of the Action Research Arm Test (ARAT) during the intervention. Effects of dose and potential modifiers of the dose-response relationship were evaluated with hierarchical linear models. RESULTS: ARAT scores for the 3,200, 9,600, and IM groups improved over time as indicated by slopes (ΔARAT/week, mean ± standard errors) of 0.40 ± 0.15, 0.31 ± 0.16, and 0.66 ± 0.14, respectively (p < 0.05). The slope of the 6,400 group was smaller (-0.05 ± 0.15) and significantly different from the 3,200 and IM groups (p < 0.001). Initial motor capacity, neglect, and other tested characteristics did not modify the dose-response relationship. INTERPRETATION: Overall, treatment effects were small. There was no evidence of a dose-response effect of task-specific training on functional capacity in people with long-standing upper-limb paresis poststroke. Ann Neurol 2016;80:342-354.

Restoration of function after brain damage using a neural prosthesis.

Neural interface systems are becoming increasingly more feasible for brain repair strategies. This paper tests the hypothesis that recovery after brain injury can be facilitated by a neural prosthesis serving as a communication link between distant locations in the cerebral cortex. The primary motor area in the cerebral cortex was injured in a rat model of focal brain injury, disrupting communication between motor and somatosensory areas and resulting in impaired reaching and grasping abilities. After implantation of microelectrodes in cerebral cortex, a neural prosthesis discriminated action potentials (spikes) in premotor cortex that triggered electrical stimulation in somatosensory cortex continuously over subsequent weeks. Within 1 wk, while receiving spike-triggered stimulation, rats showed substantially improved reaching and grasping functions that were indistinguishable from prelesion levels by 2 wk. Post hoc analysis of the spikes evoked by the stimulation provides compelling evidence that the neural prosthesis enhanced functional connectivity between the two target areas. This proof-of-concept study demonstrates that neural interface systems can be used effectively to bridge damaged neural pathways functionally and promote recovery after brain injury.

Reversibility of Murine Motor Deficits Following Hemi-Craniectomy and Cranioplasty.

BACKGROUND: Hemi-craniectomy is a common surgical procedure which allows the brain to swell and herniate and is often utilized to treat traumatic brain injury. When left untreated the scalp skin typically sinks on the side of the craniectomy creating a phenotype termed "sinking skin flap syndrome." In addition, these same patients often develop long-term neurocognitive deficits termed "syndrome of the trephined" as a result of their craniectomy which reverse when the cranial skull is replaced. The authors hypothesize that a mouse animal model can be developed demonstrating long-term neurologic deficits attributed to hemi-craniectomy skull defects similar to humans with syndrome of the trephined. METHODS: Thirty C57 mice were randomized among 3 groups: Group 1 = control group (sham surgery), Group 2 = hemi-craniectomy only, and Group 3 = hemi-craniectomy with immediate cranioplasty. Motor deficits were studied using a beam walk test. Statistical comparison of differences among the 3 groups was performed. RESULTS: Beam walk test results demonstrated the craniectomy group had a statistically higher contralateral footfault slip/step ratio when compared with the control group (P <0.05). Comparison of the control group and the cranioplasty group demonstrated contralateral footfault slip/step ratio that was statistically different for 7 days postoperative but no statistical differences thereafter. Comparison of the craniectomy group and the cranioplasty group demonstrated statistically significant differences for 14 days; however, motor deficits were not statistically different than baseline thereafter. No ipsilateral footfault deficits were detected in this study. CONCLUSION: Motor deficits that are attributed to hemi-craniectomy bone defects alone are demonstrated in a mouse animal model. These motor deficits resemble some symptoms associated with human syndrome of the trephined.

Effects of postinfarct myelin-associated glycoprotein antibody treatment on motor recovery and motor map plasticity in squirrel monkeys.

BACKGROUND AND PURPOSE: New insights into the brain's ability to reorganize after injury are beginning to suggest novel restorative therapy targets. Potential therapies include pharmacological agents designed to promote axonal growth. The purpose of this study was to test the efficacy of one such drug, GSK249320, a monoclonal antibody that blocks the axon outgrowth inhibition molecule, myelin-associated glycoprotein, to facilitate recovery of motor skills in a nonhuman primate model of ischemic cortical damage. METHODS: Using a between-groups repeated-measures design, squirrel monkeys were randomized to 1 of 2 groups: an experimental group received intravenous GSK249320 beginning 24 hours after an ischemic infarct in motor cortex with repeated dosages given at 1-week intervals for 6 weeks and a control group received only the vehicle at matched time periods. The primary end point was a motor performance index based on a distal forelimb reach-and-retrieval task. Neurophysiological mapping techniques were used to determine changes in spared motor representations. RESULTS: All monkeys recovered to baseline motor performance levels by postinfarct day 16. Functional recovery in the experimental group was significantly facilitated on the primary end point, albeit using slower movements. At 7 weeks post infarct, motor maps in the spared ventral premotor cortex in the experimental group decreased in area compared with the control group. CONCLUSIONS: GSK249320, initiated 24 hours after a focal cortical ischemic infarct, facilitated functional recovery. Together with the neurophysiological data, these results suggest that GSK249320 has a substantial biological effect on spared cortical tissue. However, its mechanisms of action may be widespread and not strictly limited to peri-infarct cortex and nearby premotor areas.

Management of patients with stroke: is it time to expand treatment options?

Approximately 700,000 people in the United States have an ischemic stroke annually. Substantial research has tested therapies for the very early treatment of ischemic stroke but, to date, only intravenous thrombolysis and intra-arterial measures to restore perfusion have shown success. Despite a 15-year effort to increase the use of these therapies, only approximately 5% of patients with stroke are currently being treated. Although most patients with stroke have some neurological recovery, more than half of stroke survivors have residual impairments that lead to disability or long-term institutionalized care. Laboratory research has demonstrated several mechanisms that help the brain to recover after a stroke. New pharmacological and cell-based approaches that are known to promote brain plasticity are emerging from laboratory studies and may soon expand the window for stroke treatment to restore function. It is time to build on this knowledge and to translate the understanding of recovery after stroke into the clinical setting. Measures that might augment recovery should become a major focus of clinical research in stroke in the 21st century.

Recovery of precision grasping after motor cortex lesion does not require forced use of the impaired hand in Macaca mulatta.

We investigated recovery of precision grasping of small objects between the index finger and thumb of the impaired hand without forced use after surgically placed lesions to the hand/arm areas of M1 and M1 + lateral premotor cortex in two monkeys. The unilateral lesions were contralateral to the monkey's preferred hand, which was established in prelesion testing as the hand used most often to acquire raisins in a foraging board (FB) task in which the monkey was free to use either hand to acquire treats. The lesions initially produced a clear paresis of the contralesional hand and use of only the ipsilesional hand to acquire raisins in the FB task. However, beginning about 3 weeks after the lesion both monkeys spontaneously began using the impaired contralesional hand in the FB task and increased use of that hand over the next few tests. Moreover, the monkeys clearly used precision grasp to acquire the raisins in a similar manner to prelesion performances, although grasp durations were longer. Although the monkeys used the contralesional hand more often than the ipsilesional hand in some postlesion testing sessions, they did not recover to use the hand as often as in prelesion testing when the preferred hand was used almost exclusively. These findings suggest that recovery of fine hand/digit motor function after localized damage to the lateral frontal motor areas in rhesus monkeys does not require forced use of the impaired hand.

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.

Traumatic Brain Injury Alters the Trajectory of Age-Related Mitochondrial Change.

Background: Some epidemiologic studies associate traumatic brain injury (TBI) with Alzheimer's disease (AD). Objective: To test whether a TBI-induced acceleration of age-related mitochondrial change could potentially mediate the reported TBI-AD association. Methods: We administered unilateral controlled cortical impact (CCI) or sham injuries to 5-month-old C57BL/6J and tau transgenic rTg4510 mice. In the non-transgenics, we assessed behavior (1-5 days, 1 month, and 15 months), lesion size (1 and 15 months), respiratory chain enzymes (1 and 15 months), and mitochondrial DNA copy number (mtDNAcn) (1 and 15 months) after CCI/sham. In the transgenics we quantified post-injury mtDNAcn and tangle burden. Results: In the non-transgenics CCI caused acute behavioral deficits that improved or resolved by 1-month post-injury. Protein-normalized complex I and cytochrome oxidase activities were not significantly altered at 1 or 15 months, although complex I activity in the CCI ipsilesional cortex declined during that period. Hippocampal mtDNAcn was not altered by injury at 1 month, increased with age, and rose to the greatest extent in the CCI contralesional hippocampus. In the injured then aged transgenics, the ipsilesional hippocampus contained less mtDNA and fewer tangles than the contralesional hippocampus; mtDNAcn and tangle counts did not correlate. Conclusions: As mice age their brains increase mtDNAcn as part of a compensatory response that preserves mitochondrial function, and TBI enhances this response. TBI may, therefore, increase the amount of compensation required to preserve late-life mitochondrial function. If TBI does modify AD risk, altering the trajectory or biology of aging-related mitochondrial changes could mediate the effect.

Distal forelimb representations in primary motor cortex are redistributed after forelimb restriction: a longitudinal study in adult squirrel monkeys.

Primary motor cortex (M1) movement representations reflect acquired motor skills. Representations of muscles and joints used in a skilled task expand. However, it is unknown whether motor restriction in healthy individuals results in complementary reductions in M1 representations. With the use of intracortical microstimulation techniques in squirrel monkeys, detailed maps of movement representations in M1 were derived before and up to 35 wk after restriction of the preferred distal forelimb (DFL) by use of a soft cast. Although total DFL area and movement threshold remained constant, casting resulted in a redistribution of digit and wrist/forearm representations. Digit representations progressively decreased, whereas wrist/forearm representations progressively increased in areal extent. In three of four monkeys, hand preference returned to normal by the end of the postcast recovery period, and postrecovery maps demonstrated reversal of restriction-induced changes. However, in one monkey, a chronic motor impairment occurred in the casted limb. Rehabilitation via a forced-use paradigm resulted in recovery in use and skill of the impaired limb, as well as restoration of normal motor maps. These results demonstrate that plasticity in motor representations can be induced by training or restricting movements of the limb. Physiological changes induced by restriction appear to be reversible, even in the case of adverse motor outcomes. The respective contributions of both disuse and lost motor skills are discussed. These results have relevance for clinical conditions requiring forelimb casting as well as interpreting the differential effects of injury and disuse that are necessarily intertwined after cortical injury, as occurs in stroke.

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.