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The ability to culture and maintain postnatal mouse hippocampal and cortical neurons is highly advantageous, particularly for studies on genetically engineered mouse models. Here we present a protocol to isolate and culture pyramidal neurons from the early postnatal (P0-P1) mouse hippocampus and cortex. These low-density dissociated cultures are grown on poly- L -lysine–coated glass substrates without feeder layers. Cultured neurons survive well, develop extensive axonal and dendritic arbors, express neuronal and synaptic markers, and form functional synaptic connections. Further, they are highly amenable to low- and high-efficiency transfection and time-lapse imaging. This optimized cell culture technique can be used to culture and maintain neurons for a variety of applications including immunocytochemistry, biochemical studies, shRNA-mediated knockdown and live imaging studies. The preparation of the glass substrate must begin 5 d before the culture. The dissection and plating out of neurons takes 3–4 h and neurons can be maintained in culture for up to 4 weeks.

Half of human spinal cord injuries lead to chronic paralysis. Here, we introduce an electrochemical neuroprosthesis and a robotic postural interface designed to encourage supraspinally mediated movements in rats with paralyzing lesions. Despite the interruption of direct supraspinal pathways, the cortex regained the capacity to transform contextual information into task-specific commands to execute refined locomotion. This recovery relied on the extensive remodeling of cortical projections, including the formation of brainstem and intraspinal relays that restored qualitative control over electrochemically enabled lumbosacral circuitries. Automated treadmill-restricted training, which did not engage cortical neurons, failed to promote translesional plasticity and recovery. By encouraging active participation under functional states, our training paradigm triggered a cortex-dependent recovery that may improve function after similar injuries in humans.

Axon regeneration in the central nervous system normally fails, in part because of a developmental decline in the intrinsic ability of CNS projection neurons to extend axons. Members of the KLF family of transcription factors regulate regenerative potential in developing CNS neurons. Expression of one family member, KLF7, is down-regulated developmentally, and overexpression of KLF7 in cortical neurons in vitro promotes axonal growth. To circumvent difficulties in achieving high neuronal expression of exogenous KLF7, we created a chimera with the VP16 transactivation domain, which displayed enhanced neuronal expression compared with the native protein while maintaining transcriptional activation and growth promotion in vitro. Overexpression of VP16-KLF7 overcame the developmental loss of regenerative ability in cortical slice cultures. Adult corticospinal tract (CST) neurons failed to up-regulate KLF7 in response to axon injury, and overexpression of VP16-KLF7 in vivo promoted both sprouting and regenerative axon growth in the CST of adult mice. These findings identify a unique means of promoting CST axon regeneration in vivo by reengineering a developmentally down-regulated, growth-promoting transcription factor.

The striatum shows general topographic organization and regional differences in behavioral functions. How corticostriatal topography differs across cortical areas and cell types to support these distinct functions is unclear. This study contrasted corticostriatal projections from two layer 5 cell types, intratelencephalic (IT-type) and pyramidal tract (PT-type) neurons, using viral vectors expressing fluorescent reporters in Cre-driver mice. Corticostriatal projections from sensory and motor cortex are somatotopic, with a decreasing topographic specificity as injection sites move from sensory to motor and frontal areas. Topographic organization differs between IT-type and PT-type neurons, including injections in the same site, with IT-type neurons having higher topographic stereotypy than PT-type neurons. Furthermore, IT-type projections from interconnected cortical areas have stronger correlations in corticostriatal targeting than PT-type projections do. As predicted by a longstanding model, corticostriatal projections of interconnected cortical areas form parallel circuits in the basal ganglia. How corticostriatal connections of different pyramidal cell types are organized, particularly in convergent circuits, has not been evaluated in detail. Here, cell type-specific Cre-driver mice reveal that pyramidal tract-type corticostriatal projections, though broadly similar to intratelencephalic-type projections from the same cortical region, are generally more restricted and variable in their topographic termination patterns.

The sensorimotor cortex is crucial for learning and executing new movements with precision. It selectively modulates sensory information flow and represents motor information in a spatially organized manner. The pyramidal system is made up of layer 5 pyramidal tract neurons (PTNs), which are organized into populations with distinct morphological, genetic and functional properties. These subpopulations project to different subcortical structures in a segregated manner. To understand whether PTNs projecting to different structures play distinct functional roles in motor control, we characterized two types of layer 5 neurons in the motor cortex: corticorubral (CR) neurons, which project to the red nucleus, and corticospinal (CS) neurons, which project to the spinal cord. To analyze movement performance in rats, we compared the selective optogenetic inhibition of motor cortex CS or CR neurons during lever movement execution in response to a light stimulus. As the animals progressed through the training sessions, the variability of lever trajectories decreased, and the movements became more stereotyped. Photoinhibition of CS or CR neurons increased the performance variability of learned movements but differentially affected kinematic parameters. CR neuron inhibition affected amplitude, duration, reaction times, speed, and acceleration of the movement. In contrast, the inhibition of CS neurons mainly altered the duration and acceleration of the movement. The results indicate that the same motor order would be expected to have different causal effects when sent to different brain regions.

Although transcranial magnetic stimulation (TMS) activates a number of different neuron types in the cortex, the final output elicited in corticospinal neurones is surprisingly stereotyped. A single TMS pulse evokes a series of descending corticospinal volleys that are separated from each other by about 1.5 ms (i.e., ~670 Hz). This evoked descending corticospinal activity can be directly recorded by an epidural electrode placed over the high cervical cord. The earliest wave is thought to originate from the direct activation of the axons of fast-conducting pyramidal tract neurones (PTN) and is therefore termed \"D\" wave. The later waves are thought to originate from indirect, trans-synaptic activation of PTNs and are termed \"I\" waves. The anatomical and computational characteristics of a canonical microcircuit model of cerebral cortex composed of layer II and III and layer V excitatory pyramidal cells, inhibitory interneurons, and cortico-cortical and thalamo-cortical inputs can account for the main characteristics of the corticospinal activity evoked by TMS including its regular and rhythmic nature, the stimulus intensity-dependence and its pharmacological modulation. In this review we summarize present knowledge of the physiological basis of the effects of TMS of the human motor cortex describing possible interactions between TMS and simple canonical microcircuits of neocortex. According to the canonical model, a TMS pulse induces strong depolarization of the excitatory cells in the superficial layers of the circuit. This leads to highly synchronized recruitment of clusters of excitatory neurons, including layer V PTNs, and of inhibitory interneurons producing a high frequency (~670 Hz) repetitive discharge of the corticospinal axons. The role of the inhibitory circuits is crucial to entrain the firing of the excitatory networks to produce a high-frequency discharge and to control the number and magnitude of evoked excitatory discharge in layer V PTNs. In summary, simple canonical microcircuits of neocortex can explain activation of corticospinal neurons in human motor cortex by TMS.

ObjectivesNeurochemical markers of amyotrophic lateral sclerosis (ALS) that reflect underlying disease mechanisms might help in diagnosis, staging and prediction of outcome. We aimed at determining the origin and differential diagnostic and prognostic potential of the putative marker of microglial activation chitotriosidase (CHIT1).MethodsAltogether 316 patients were included, comprising patients with sporadic ALS, ALS mimics (disease controls (DCo)), frontotemporal lobar degeneration (FTLD), Creutzfeldt-Jakob disease (CJD), Alzheimer’s disease (AD), Parkinson’s disease (PD) and healthy controls (Con). CHIT1 and neurofilament levels were determined in cerebrospinal fluid (CSF) and blood and analysed with regard to diagnostic sensitivity and specificity and prognostic performance. Additionally, postmortem tissue was analysed for CHIT1 expression.ResultsIn ALS, CHIT1 CSF levels were higher compared with Con (p<0.0001), DCo (p<0.05) and neurodegenerative diseases (AD p<0.05, PD p<0.01, FTLD p<0.0001) except CJD. CHIT1 concentrations were correlated with ALS disease progression and severity but not with the survival time, as did neurofilaments. Serum CHIT1 levels were not different in ALS compared with any other study group. In the spinal cord of patients with ALS, but not Con, AD or CJD cases, CHIT1 was expressed in the corticospinal tract and CHIT1 staining colocalised with markers of microglia (IBA1) and macrophages (CD68).ConclusionsCHIT1 concentrations in the CSF of patients with ALS may reflect the extent of microglia/macrophage activation in the white matter of the spinal cord. CHIT1 could be a potentially useful marker for differential diagnosis and prediction of disease progression in ALS and, therefore, seems suitable as a supplemental marker for patient stratification in therapeutic trials.

Prior anatomical studies have suggested that intratelencephalic (IT) and pyramidal tract (PT) cortical neurons project to different populations of striatal spiny projection neurons (SPNs). Here, the authors find using optogenetic stimulation that both IT and PT neurons project to both direct and indirect pathway SPNs. Anatomical studies have led to the assertion that intratelencephalic and pyramidal tract cortical neurons innervate different striatal projection neurons. To test this hypothesis, we measured the responses of mouse striatal neurons to optogenetic activation of intratelencephalic and pyramidal tract axons. Contrary to expectation, direct and indirect pathway striatal spiny projection neurons responded to both intratelencephalic and pyramidal tract activation, arguing that these cortical networks innervate both striatal projection neurons.

Grafts of spinal-cord-derived neural progenitor cells (NPCs) enable the robust regeneration of corticospinal axons and restore forelimb function after spinal cord injury 1 ; however, the molecular mechanisms that underlie this regeneration are unknown. Here we perform translational profiling specifically of corticospinal tract (CST) motor neurons in mice, to identify their ‘regenerative transcriptome’ after spinal cord injury and NPC grafting. Notably, both injury alone and injury combined with NPC grafts elicit virtually identical early transcriptomic responses in host CST neurons. However, in mice with injury alone this regenerative transcriptome is downregulated after two weeks, whereas in NPC-grafted mice this transcriptome is sustained. The regenerative transcriptome represents a reversion to an embryonic transcriptional state of the CST neuron. The huntingtin gene ( Htt ) is a central hub in the regeneration transcriptome; deletion of Htt significantly attenuates regeneration, which shows that Htt has a key role in neural plasticity after injury. In mouse models of central nervous system injury, Htt is shown to be a key component of the regulatory program associated with reversion of the neuronal transcriptome to a less-mature state.

The primary motor cortex (M1) is a central hub for motor learning and execution. M1 is composed of heterogeneous cell types with varying relationships to movement. Here, we tagged active neurons at different stages of motor task performance in mice and characterized cell type composition. We identified corticothalamic neurons (M1 CT ) as consistently enriched with training progression. Using two-photon calcium imaging, we found that M1 CT activity is largely suppressed during movement, and this negative correlation augments with training. Increasing M1 CT activity through closed-loop optogenetic manipulations during forelimb movement significantly hinders execution, an effect that became stronger with training. Similar manipulations, however, had little effect on locomotion. In contrast, M1 corticospinal neurons positively correlate with movement, with an increase during training. We uncovered that M1 CT neurons suppress corticospinal activity via feedforward inhibition, also scaling with training. These results identify a permissive role of corticothalamic neurons in movement execution through disinhibition of corticospinal neurons. How different cell types in primary motor cortex contribute to movement during learning and/or execution is not fully understood. Here authors show that corticothalamic neurons of the primary motor cortex are silent during movement and that this activity signature is critical for proficient movement generation.