Frequency Specific Optogenetic Stimulation of the Locus Coeruleus Induces Task-Relevant Plasticity in the Motor Cortex.

The locus ceruleus (LC) is the primary source of neocortical noradrenaline, which is known to be involved in diverse brain functions including sensory perception, attention, and learning. Previous studies have shown that LC stimulation paired with sensory experience can induce task-dependent plasticity in the sensory neocortex and in the hippocampus. However, it remains unknown whether LC activation similarly impacts neural representations in the agranular motor cortical regions that are responsible for movement planning and production. In this study, we test whether optogenetic stimulation of the LC paired with motor performance is sufficient to induce task-relevant plasticity in the somatotopic cortical motor map. Male and female TH-Cre + rats were trained on a skilled reaching lever-pressing task emphasizing the use of the proximal forelimb musculature, and a viral approach was used to selectively express ChR2 in noradrenergic LC neurons. Once animals reached criterial behavioral performance, they received five training sessions in which correct task performance was paired with optogenetic stimulation of the LC delivered at 3, 10, or 30 Hz. After the last stimulation session, motor cortical mapping was performed using intracortical microstimulation. Our results show that lever pressing paired with LC stimulation at 10 Hz, but not at 3 or 30 Hz, drove the expansion of the motor map representation of the task-relevant proximal FL musculature. These findings demonstrate that phasic, training-paired activation of the LC is sufficient to induce experience-dependent plasticity in the agranular motor cortex and that this LC-driven plasticity is highly dependent on the temporal dynamics of LC activation.

Longitudinal assessment of skilled forelimb motor impairments in DJ-1 knockout rats.

BACKGROUND: DJ-1 knockout (DJ-1 KO) rats exhibit a moderate parkinsonian phenotype, with gross motor deficits and ca. 50% loss of midbrain dopaminergic neurons appearing around 6-8 months of age. Fine motor impairments are often observed in Parkinson's disease (PD), but skilled motor function in recently developed transgenic rat models of PD is not well characterized. OBJECTIVES: To assess the longitudinal performance of DJ-1 KO rats on a skilled forelimb reaching task. METHODS: DJ-1 KO and wild-type (WT) rats were trained from 2 to 10 months of age on an isometric pullbar task designed to test forelimb strength and coordination. After 36 consecutive weeks of training (ca. 10 months old), task difficulty was then increased to challenge the motor capabilities of the DJ-1 KO rats. Throughout the study, subjects also received weekly assessments of gross locomotor activity in an open field. RESULTS: Pull-task performance of the DJ-1 KO rats was impaired compared to WT, with deficits reaching significance around 7-9 months of age. When challenged, DJ-1 KO rats were able to exert increased force on the pullbar but continued to exhibit deficits compared to WT rats. Throughout the study, no differences in distance traveled or rearing frequency were observed in the open field, but DJ-1 KO rats were found to spend significantly more time in the center of the open field than WT rats. CONCLUSIONS: Using a sensitive, automated assay of forelimb strength and coordination, we find that skilled forelimb motor performance is impaired in DJ-1 KO rats.

Local activation of α2 adrenergic receptors is required for vagus nerve stimulation induced motor cortical plasticity.

Vagus nerve stimulation (VNS) paired with rehabilitation training is emerging as a potential treatment for improving recovery of motor function following stroke. In rats, VNS paired with skilled forelimb training results in significant reorganization of the somatotopic cortical motor map; however, the mechanisms underlying this form of VNS-dependent plasticity remain unclear. Recent studies have shown that VNS-driven cortical plasticity is dependent on noradrenergic innervation of the neocortex. In the central nervous system, noradrenergic α2 receptors (α2-ARs) are widely expressed in the motor cortex and have been critically implicated in synaptic communication and plasticity. In current study, we examined whether activation of cortical α2-ARs is necessary for VNS-driven motor cortical reorganization to occur. Consistent with previous studies, we found that VNS paired with motor training enlarges the map representation of task-relevant musculature in the motor cortex. Infusion of α2-AR antagonists into M1 blocked VNS-driven motor map reorganization from occurring. Our results suggest that local α2-AR activation is required for VNS-induced cortical reorganization to occur, providing insight into the mechanisms that may underlie the neuroplastic effects of VNS therapy.

Vagus Nerve Stimulation Induced Motor Map Plasticity Does Not Require Cortical Dopamine.

Background: Vagus nerve stimulation (VNS) paired with motor rehabilitation is an emerging therapeutic strategy to enhance functional recovery after neural injuries such as stroke. Training-paired VNS drives significant neuroplasticity within the motor cortex (M1), which is thought to underlie the therapeutic effects of VNS. Though the mechanisms are not fully understood, VNS-induced cortical plasticity is known to depend on intact signaling from multiple neuromodulatory nuclei that innervate M1. Cortical dopamine (DA) plays a key role in mediating M1 synaptic plasticity and is critical for motor skill acquisition, but whether cortical DA contributes to VNS efficacy has not been tested. Objective: To determine the impact of cortical DA depletion on VNS-induced cortical plasticity. Methods: Rats were trained on a skilled reaching lever press task prior to implantation of VNS electrodes and 6-hydroxydopamine (6-OHDA) mediated DA depletion in M1. Rats then underwent training-paired VNS treatment, followed by cortical motor mapping and lesion validation. Results: In both intact and DA-depleted rats, VNS significantly increased the motor map representation of task-relevant proximal forelimb musculature and reduced task-irrelevant distal forelimb representations. VNS also significantly increased tyrosine hydroxylase (TH+) fiber density in intact M1, but this effect was not observed in lesioned hemispheres. Conclusion: Our results reveal that though VNS likely upregulates catecholaminergic signaling in intact motor cortices, DA itself is not required for VNS-induced plasticity to occur. As DA is known to critically support M1 plasticity during skill acquisition, our findings suggest that VNS may engage a unique set of neuromodulatory signaling pathways to promote neocortical plasticity.

Self-Administration of Right Vagus Nerve Stimulation Activates Midbrain Dopaminergic Nuclei.

Background: Left cervical vagus nerve stimulation (l-VNS) is an FDA-approved treatment for neurological disorders including epilepsy, major depressive disorder, and stroke, and l-VNS is increasingly under investigation for a range of other neurological indications. Traditional l-VNS is thought to induce therapeutic neuroplasticity in part through the coordinated activation of multiple broadly projecting neuromodulatory systems in the brain. Recently, it has been reported that striking lateralization exists in the anatomical and functional connectivity between the vagus nerves and the dopaminergic midbrain. These emerging findings suggest that VNS-driven activation of this important plasticity-promoting neuromodulatory system may be preferentially driven by targeting the right, rather than the left, cervical nerve. Objective: To compare the effects of right cervical VNS (r-VNS) vs. traditional l-VNS on self-administration behavior and midbrain dopaminergic activation in rats. Methods: Rats were implanted with a stimulating cuff electrode targeting either the right or left cervical vagus nerve. After surgical recovery, rats underwent a VNS self-administration assay in which lever pressing was paired with r-VNS or l-VNS delivery. Self-administration was followed by extinction, cue-only reinstatement, and stimulation reinstatement sessions. Rats were sacrificed 90 min after completion of behavioral training, and brains were removed for immunohistochemical analysis of c-Fos expression in the dopaminergic ventral tegmental area (VTA) and substantia nigra pars compacta (SNc), as well as in the noradrenergic locus coeruleus (LC). Results: Rats in the r-VNS cohort performed significantly more lever presses throughout self-administration and reinstatement sessions than did rats in the l-VNS cohort. Moreover, this appetitive behavioral responding was associated with significantly greater c-Fos expression among neuronal populations within the VTA, SNc, and LC. Differential c-Fos expression following r-VNS vs. l-VNS was particularly prominent within dopaminergic midbrain neurons. Conclusion: Our results support the existence of strong lateralization within vagal-mesencephalic signaling pathways, and suggest that VNS targeted to the right, rather than left, cervical nerve preferentially activates the midbrain dopaminergic system. These findings raise the possibility that r-VNS could provide a promising strategy for enhancing dopamine-dependent neuroplasticity, opening broad avenues for future research into the efficacy and safety of r-VNS in the treatment of neurological disease.

A Highly Selective MNK Inhibitor Rescues Deficits Associated with Fragile X Syndrome in Mice.

Fragile X syndrome (FXS) is the most common inherited source of intellectual disability in humans. FXS is caused by mutations that trigger epigenetic silencing of the Fmr1 gene. Loss of Fmr1 results in increased activity of the mitogen-activated protein kinase (MAPK) pathway. An important downstream consequence is activation of the mitogen-activated protein kinase interacting protein kinase (MNK). MNK phosphorylates the mRNA cap-binding protein, eukaryotic initiation factor 4E (eIF4E). Excessive phosphorylation of eIF4E has been directly implicated in the cognitive and behavioral deficits associated with FXS. Pharmacological reduction of eIF4E phosphorylation is one potential strategy for FXS treatment. We demonstrate that systemic dosing of a highly specific, orally available MNK inhibitor, eFT508, attenuates numerous deficits associated with loss of Fmr1 in mice. eFT508 resolves a range of phenotypic abnormalities associated with FXS including macroorchidism, aberrant spinogenesis, and alterations in synaptic plasticity. Key behavioral deficits related to anxiety, social interaction, obsessive and repetitive activities, and object recognition are ameliorated by eFT508. Collectively, this work establishes eFT508 as a potential means to reverse deficits associated with FXS.

Vagus nerve stimulation promotes cortical reorganization and reduces task-dependent calorie intake in male and female rats.

Numerous preclinical studies demonstrate that vagus nerve stimulation (VNS) paired with motor rehabilitation improves functional recovery after neural injuries such as stroke, traumatic brain injury, and spinal cord injury, in part by driving neural plasticity within the motor cortex. To date, these studies have been performed almost exclusively in female rats, however, the risk for neural injuries of all types is significantly higher among males than females. We therefore sought to determine whether VNS was equally effective at driving motor cortical plasticity in both sexes. Male and female rats were trained on a skilled lever press task prior to VNS electrode implantation. After recovery, rats received ten training sessions in which VNS, or sham stimulation, was paired with correct motor performance. At the completion of these treatment sessions, somatotopic mapping of motor cortex was performed. We found that performance on the lever task was similar between male and female rats, though on average, males performed more trials per training session, consistent with their larger size and higher caloric need. Training-paired VNS effectively induced cortical motor map reorganization in both male and female rats. Notably, we also found that VNS reduced lever-press associated caloric intake during treatment in both sexes. These VNS-driven effects were robust to behavioral and biological differences between male and female subjects. Taken together, our results suggest that, in both male and female rats, VNS simultaneously engages both pro-plasticity neuromodulation within the neocortex and satiety or reward-related networks that reduce task-associated caloric intake.

Preparation of Peripheral Nerve Stimulation Electrodes for Chronic Implantation in Rats.

Peripheral nerve cuff electrodes have long been used in the neurosciences and related fields for stimulation of, for example, vagus or sciatic nerves. Several recent studies have demonstrated the effectiveness of chronic VNS in enhancing central nervous system plasticity to improve motor rehabilitation, extinction learning, and sensory discrimination. Construction of chronically implantable devices for use in such studies is challenging due to rats' small size, and typical protocols require extensive training of personnel and time-consuming microfabrication methods. Alternatively, commercially available implantable cuff electrodes can be purchased at a significantly higher cost. In this protocol, we present a simple, low-cost method for construction of small, chronically implantable peripheral nerve cuff electrodes for use in rats. We validate the short and long-term reliability of our cuff electrodes by demonstrating that VNS in ketamine/xylazine anesthetized rats produces decreases in breathing rate consistent with activation of the Hering-Breuer reflex, both at the time of implantation and up to 10 weeks after device implantation. We further demonstrate the suitability of the cuff electrodes for use in chronic stimulation studies by pairing VNS with skilled lever press performance to induce motor cortical map plasticity.

Striatal, Hippocampal, and Cortical Networks Are Differentially Responsive to the M4- and M1-Muscarinic Acetylcholine Receptor Mediated Effects of Xanomeline.

Preclinical and clinical data suggest that muscarinic acetylcholine receptor activation may be therapeutically beneficial for the treatment of schizophrenia and Alzheimer's diseases. This is best exemplified by clinical observations with xanomeline, the efficacy of which is thought to be mediated through co-activation of the M1 and M4 muscarinic acetylcholine receptors (mAChRs). Here we examined the impact of treatment with xanomeline and compared it to the actions of selective M1 and M4 mAChR activators on in vivo intracellular signaling cascades in mice, including 3'-5'-cyclic adenosine monophosphate response element binding protein (CREB) phosphorylation and inositol phosphate-1 (IP1) accumulation in the striatum, hippocampus, and prefrontal cortex. We additionally assessed the effects of xanomeline on hippocampal electrophysiological signatures in rats using ex vivo recordings from CA1 (Cornu Ammonis 1) as well as in vivo hippocampal theta. As expected, xanomeline's effects across these readouts were consistent with activation of both M1 and M4 mAChRs; however, differences were observed across different brain regions, suggesting non-uniform activation of these receptor subtypes in the central nervous system. Interestingly, despite having nearly equal in vitro potency at the M1 and the M4 mAChRs, during in vivo assays xanomeline produced M4-like effects at significantly lower brain exposures than those at which M1-like effects were observed. Our results raise the possibility that clinical efficacy observed with xanomeline was driven, in part, through its non-uniform activation of mAChR subtypes in the central nervous system and, at lower doses, through preferential agonism of the M4 mAChR.

Design and Synthesis of γ- and δ-Lactam M1 Positive Allosteric Modulators (PAMs): Convulsion and Cholinergic Toxicity of an M1-Selective PAM with Weak Agonist Activity.

Recent data demonstrated that activation of the muscarinic M1 receptor by a subtype-selective positive allosteric modulator (PAM) contributes to the gastrointestinal (GI) and cardiovascular (CV) cholinergic adverse events (AEs) previously attributed to M2 and M3 activation. These studies were conducted using PAMs that also exhibited allosteric agonist activity, leaving open the possibility that direct activation by allosteric agonism, rather than allosteric modulation, could be responsible for the adverse effects. This article describes the design and synthesis of lactam-derived M1 PAMs that address this hypothesis. The lead molecule from this series, compound 1 (PF-06827443), is a potent, low-clearance, orally bioavailable, and CNS-penetrant M1-selective PAM with minimal agonist activity. Compound 1 was tested in dose escalation studies in rats and dogs and was found to induce cholinergic AEs and convulsion at therapeutic indices similar to previous compounds with more agonist activity. These findings provide preliminary evidence that positive allosteric modulation of M1 is sufficient to elicit cholinergic AEs.

Effects of M1 and M4 activation on excitatory synaptic transmission in CA1.

Hippocampal networks are particularly susceptible to dysfunction in many neurodegenerative diseases and neuropsychiatric disorders including Alzheimer's disease, Lewy body dementia, and schizophrenia. CA1, a major output region of the hippocampus, receives glutamatergic input from both hippocampal CA3 and entorhinal cortex, via the Schaffer collateral (SC) and temporoammonic (TA) pathways, respectively. SC and TA inputs to CA1 are thought to be differentially involved in the retrieval of previously stored memories versus the encoding of novel information, and switching between these two crucial hippocampal functions is thought to critically depend on acetylcholine (ACh) acting at muscarinic receptors. In this study, we aimed to determine the roles of specific subtypes of muscarinic receptors in mediating the neuromodulatory effects of ACh on glutamatergic synaptic transmission in the SC and TA pathways of CA1. Using selective pharmacological activation of M1 or M4 receptors along with extracellular and intracellular electrophysiology recordings from adult rat hippocampal slices, we demonstrate that activation of M1 receptors increases spontaneous spike rates of neuronal ensembles in CA1 and increases the intrinsic excitability of pyramidal neurons and interneurons. Selective activation of M4 receptors inhibits glutamate release in the SC pathway, while leaving synaptic transmission in the TA pathway comparatively intact. These results suggest specific mechanisms by which M1 and M4 activation may normalize CA1 circuit activity following disruptions of signaling that accompany neurodegenerative dementias or neuropsychiatric disorders. These findings are of particular interest in light of clinical findings that xanomeline, an M1/M4 preferring agonist, was able to improve cognitive and behavioral symptoms in patients with Alzheimer's disease or schizophrenia.

Neural mechanisms of transient neocortical beta rhythms: Converging evidence from humans, computational modeling, monkeys, and mice.

Human neocortical 15-29-Hz beta oscillations are strong predictors of perceptual and motor performance. However, the mechanistic origin of beta in vivo is unknown, hindering understanding of its functional role. Combining human magnetoencephalography (MEG), computational modeling, and laminar recordings in animals, we present a new theory that accounts for the origin of spontaneous neocortical beta. In our MEG data, spontaneous beta activity from somatosensory and frontal cortex emerged as noncontinuous beta events typically lasting <150 ms with a stereotypical waveform. Computational modeling uniquely designed to infer the electrical currents underlying these signals showed that beta events could emerge from the integration of nearly synchronous bursts of excitatory synaptic drive targeting proximal and distal dendrites of pyramidal neurons, where the defining feature of a beta event was a strong distal drive that lasted one beta period (∼50 ms). This beta mechanism rigorously accounted for the beta event profiles; several other mechanisms did not. The spatial location of synaptic drive in the model to supragranular and infragranular layers was critical to the emergence of beta events and led to the prediction that beta events should be associated with a specific laminar current profile. Laminar recordings in somatosensory neocortex from anesthetized mice and awake monkeys supported these predictions, suggesting this beta mechanism is conserved across species and recording modalities. These findings make several predictions about optimal states for perceptual and motor performance and guide causal interventions to modulate beta for optimal function.

Differential entrainment and learning-related dynamics of spike and local field potential activity in the sensorimotor and associative striatum.

Parallel cortico-basal ganglia loops are thought to have distinct but interacting functions in motor learning and habit formation. In rats, the striatal projection neuron populations (MSNs) in the dorsolateral and dorsomedial striatum, respectively corresponding to sensorimotor and associative regions of the striatum, exhibit contrasting dynamics as rats acquire T-maze tasks (Thorn et al., 2010). Here, we asked whether these patterns could be related to the activity of local interneuron populations in the striatum and to the local field potential activity recorded simultaneously in the corresponding regions. We found that dorsolateral and dorsomedial striatal fast-spiking interneurons exhibited task-specific and training-related dynamics consistent with those of corresponding MSN populations. Moreover, both MSNs and interneuron populations in both regions became entrained to theta-band (5-12 Hz) frequencies during task acquisition. However, the predominant entrainment frequencies were different for the sensorimotor and associative zones. Dorsolateral striatal neurons became entrained mid-task to oscillations centered ∼ 5 Hz, whereas simultaneously recorded neurons in the dorsomedial region became entrained to higher frequency (∼ 10 Hz) rhythms. These region-specific patterns of entrainment evolved dynamically with the development of region-specific patterns of interneuron and MSN activity, indicating that, with learning, these two striatal regions can develop different frequency-modulated circuit activities in parallel. We suggest that such differential entrainment of sensorimotor and associative neuronal populations, acquired through learning, could be critical for coordinating information flow throughout each trans-striatal network while simultaneously enabling nearby components of the separate networks to operate independently.

Temporal and mosaic Tsc1 deletion in the developing thalamus disrupts thalamocortical circuitry, neural function, and behavior.

Tuberous sclerosis is a developmental genetic disorder caused by mutations in TSC1, which results in epilepsy, autism, and intellectual disability. The cause of these neurological deficits remains unresolved. Imaging studies suggest that the thalamus may be affected in tuberous sclerosis patients, but this has not been experimentally interrogated. We hypothesized that thalamic deletion of Tsc1 at distinct stages of mouse brain development would produce differential phenotypes. We show that mosaic Tsc1 deletion within thalamic precursors at embryonic day (E) 12.5 disrupts thalamic circuitry and alters neuronal physiology. Tsc1 deletion at this early stage is unique in causing both seizures and compulsive grooming in adult mice. In contrast, only a subset of these phenotypes occurs when thalamic Tsc1 is deleted at a later embryonic stage. Our findings demonstrate that abnormalities in a discrete population of neurons can cause global brain dysfunction and that phenotype severity depends on developmental timing and degree of genetic mosaicism.

Pausing to regroup: thalamic gating of cortico-basal ganglia networks.

How the cholinergic and dopaminergic systems of the striatum interact and how these interface with the massive neocortical input to the striatum are classic questions of cardinal interest to neurology and psychiatry. In this issue of Neuron, Ding and colleagues show that a key to these puzzles lies in the thalamic inputs to the striatum targeting its cholinergic interneurons.

Differential dynamics of activity changes in dorsolateral and dorsomedial striatal loops during learning.

The basal ganglia are implicated in a remarkable range of functions influencing emotion and cognition as well as motor behavior. Current models of basal ganglia function hypothesize that parallel limbic, associative, and motor cortico-basal ganglia loops contribute to this diverse set of functions, but little is yet known about how these loops operate and how their activities evolve during learning. To address these issues, we recorded simultaneously in sensorimotor and associative regions of the striatum as rats learned different versions of a conditional T-maze task. We found highly contrasting patterns of activity in these regions during task performance and found that these different patterns of structured activity developed concurrently, but with sharply different dynamics. Based on the region-specific dynamics of these patterns across learning, we suggest a working model whereby dorsomedial associative loops can modulate the access of dorsolateral sensorimotor loops to the control of action.