Non-invasive High Frequency Repetitive Transcranial Magnetic Stimulation (hfrTMS) Robustly Activates Molecular Pathways Implicated in Neuronal Growth and Synaptic Plasticity in Select Populations of Neurons.

Patterns of neuronal activity that induce synaptic plasticity and memory storage activate kinase cascades in neurons that are thought to be part of the mechanism for synaptic modification. One such cascade involves induction of phosphorylation of ribosomal protein S6 in neurons due to synaptic activation of AKT/mTOR and via a different pathway, activation of MAP kinase/ERK1/2. Here, we show that phosphorylation of ribosomal protein S6 can also be strongly activated by high frequency repetitive transcranial magnetic stimulation (hfrTMS). HfrTMS was delivered to lightly anesthetized rats using a stimulation protocol that is a standard for inducing LTP in the perforant path in vivo (trains of 8 pulses at 400 Hz repeated at intervals of 1/10 s). Stimulation produced stimulus-locked motor responses but did not elicit behavioral seizures either during or after stimulation. After as little as 10 min of hfrTMS, immunostaining using phospho-specific antibodies for the phosphorylated form of ribosomal protein S6 (rpS6) revealed robust induction of rpS6 phosphorylation in large numbers of neurons in the cortex, especially the piriform cortex, and also in thalamic relay nuclei. Quantification revealed that the extent of the increased immunostaining depended on the number of trains and stimulus intensity. Of note, immunostaining for the immediate early genes Arc and c-fos revealed strong induction of IEG expression in many of the same populations of neurons throughout the cortex, but not the thalamus. These results indicate that hfrTMS can robustly activate molecular pathways critical for plasticity, which may contribute to the beneficial effects of TMS on recovery following brain and spinal cord injury and symptom amelioration in human psychiatric disorders. These molecular processes may be a useful surrogate marker to allow optimization of TMS parameters for maximal therapeutic benefit

Non-invasive High Frequency Repetitive Transcranial Magnetic Stimulation (hfrTMS) Robustly Activates Molecular Pathways Implicated in Neuronal Growth and Synaptic Plasticity in Select Populations of Neurons.

Patterns of neuronal activity that induce synaptic plasticity and memory storage activate kinase cascades in neurons that are thought to be part of the mechanism for synaptic modification. One such cascade involves induction of phosphorylation of ribosomal protein S6 in neurons due to synaptic activation of AKT/mTOR and via a different pathway, activation of MAP kinase/ERK1/2. Here, we show that phosphorylation of ribosomal protein S6 can also be strongly activated by high frequency repetitive transcranial magnetic stimulation (hfrTMS). HfrTMS was delivered to lightly anesthetized rats using a stimulation protocol that is a standard for inducing LTP in the perforant path in vivo (trains of 8 pulses at 400 Hz repeated at intervals of 1/10 s). Stimulation produced stimulus-locked motor responses but did not elicit behavioral seizures either during or after stimulation. After as little as 10 min of hfrTMS, immunostaining using phospho-specific antibodies for the phosphorylated form of ribosomal protein S6 (rpS6) revealed robust induction of rpS6 phosphorylation in large numbers of neurons in the cortex, especially the piriform cortex, and also in thalamic relay nuclei. Quantification revealed that the extent of the increased immunostaining depended on the number of trains and stimulus intensity. Of note, immunostaining for the immediate early genes Arc and c-fos revealed strong induction of IEG expression in many of the same populations of neurons throughout the cortex, but not the thalamus. These results indicate that hfrTMS can robustly activate molecular pathways critical for plasticity, which may contribute to the beneficial effects of TMS on recovery following brain and spinal cord injury and symptom amelioration in human psychiatric disorders. These molecular processes may be a useful surrogate marker to allow optimization of TMS parameters for maximal therapeutic benefit

A re-assessment of long distance growth and connectivity of neural stem cells after severe spinal cord injury

As part of the NIH "Facilities of Research Excellence-Spinal Cord Injury" project to support independent replication, we repeated key parts of a study reporting robust engraftment of neural stem cells (NSCs) treated with growth factors after complete spinal cord transection in rats. Rats (n=20) received complete transections at thoracic level 3 (T3) and 2weeks later received NSC transplants in a fibrin matrix with a growth factor cocktail using 2 different transplantation methods (with and without removal of scar tissue). Control rats (n=9) received transections only. Hindlimb locomotor function was assessed with the BBB scale. Nine weeks post injury, reticulospinal tract axons were traced in 6 rats by injecting BDA into the reticular formation. Transplants grew to fill the lesion cavity in most rats although grafts made with scar tissue removal had large central cavities. Grafts blended extensively with host tissue obliterating the astroglial boundary at the cut ends, but in most cases there was a well-defined partition within the graft that separated rostral and caudal parts of the graft. In some cases, the partition contained non-neuronal scar tissue. There was extensive outgrowth of GFP labeled axons from the graft, but there was minimal ingrowth of host axons into the graft revealed by tract tracing and immunocytochemistry for 5HT. There were no statistically significant differences between transplant and control groups in the degree of locomotor recovery. Our results confirm the previous report that NSC transplants can fill lesion cavities and robustly extend axons, but reveal that most grafts do not create a continuous bridge of neural tissue between rostral and caudal segments

Delayed Degradation and Impaired Dendritic Delivery of Intron-Lacking EGFP-Arc/Arg3.1 mRNA in EGFP-Arc Transgenic Mice

Arc is a unique immediate early gene (IEG) whose expression is induced as synapses are modified during learning. Newly-synthesized Arc mRNA is rapidly transported throughout dendrites and localizes near recently activated synapses. Arc mRNA levels are regulated by rapid degradation, which is accelerated by synaptic activity in a translation-dependent process. One possible mechanism is nonsense-mediated mRNA decay (NMD), which depends on the presence of a splice junction in the 3′UTR. Here, we test this hypothesis using transgenic mice that express EGFP-Arc. Because the transgene was constructed from Arc cDNA, it lacks intron structures in the 3′UTR that are present in the endogenous Arc gene. NMD depends on the presence of proteins of the exon junction complex (EJC) downstream of a stop codon, so EGFP-Arc mRNA should not undergo NMD. Assessment of Arc mRNA rundown in the presence of the transcription inhibitor actinomycin-D confirmed delayed degradation of EGFP-Arc mRNA. EGFP-Arc mRNA and protein are expressed at much higher levels in transgenic mice under basal and activated conditions but EGFP-Arc mRNA does not enter dendrites efficiently. In a physiological assay in which cycloheximide (CHX) was infused after induction of Arc by seizures, there were increases in endogenous Arc mRNA levels consistent with translation-dependent Arc mRNA decay but this was not seen with EGFP-Arc mRNA. Taken together, our results indicate: (1) Arc mRNA degradation occurs via a mechanism with characteristics of NMD; (2) rapid dendritic delivery of newly synthesized Arc mRNA after induction may depend in part on prior splicing of the 3′UTR

Characterization of Ectopic Colonies That Form in Widespread Areas of the Nervous System with Neural Stem Cell Transplants into the Site of a Severe Spinal Cord Injury

We reported previously the formation of ectopic colonies in widespread areas of the nervous system after transplantation of fetal neural stem cells (NSCs) into spinal cord transection sites. Here, we characterize the incidence, distribution, and cellular composition of the colonies. NSCs harvested from E14 spinal cords from rats that express GFP were treated with a growth factor cocktail and grafted into the site of a complete spinal cord transection. Two months after transplant, spinal cord and brain tissue were analyzed histologically. Ectopic colonies were found at long distances from the transplant in the central canal of the spinal cord, the surface of the brainstem and spinal cord, and in the fourth ventricle. Colonies were present in 50% of the rats, and most rats had multiple colonies. Axons extended from the colonies into the host CNS. Colonies were strongly positive for nestin, a marker for neural precursors, and contained NeuN-positive cells with processes resembling dendrites, GFAP-positive astrocytes, APC/CC1-positive oligodendrocytes, and Ki-67-positive cells, indicating ongoing proliferation. Stereological analyses revealed an estimated 21,818 cells in a colony in the fourth ventricle, of which 1005 (5%) were Ki-67 positive. Immunostaining for synaptic markers (synaptophysin and VGluT-1) revealed large numbers of synaptophysin-positive puncta within the colonies but fewer VGluT-1 puncta. Continuing expansion of NSC-derived cell masses in confined spaces in the spinal cord and brain could produce symptoms attributable to compression of nearby tissue. It remains to be determined whether other cell types with self-renewing potential can also form colonies

Delayed Degradation and Impaired Dendritic Delivery of Intron-Lacking EGFP-Arc/Arg3.1 mRNA in EGFP-Arc Transgenic Mice.

Arc is a unique immediate early gene (IEG) whose expression is induced as synapses are modified during learning. Newly-synthesized Arc mRNA is rapidly transported throughout dendrites and localizes near recently activated synapses. Arc mRNA levels are regulated by rapid degradation, which is accelerated by synaptic activity in a translation-dependent process. One possible mechanism is nonsense-mediated mRNA decay (NMD), which depends on the presence of a splice junction in the 3'UTR. Here, we test this hypothesis using transgenic mice that express EGFP-Arc. Because the transgene was constructed from Arc cDNA, it lacks intron structures in the 3'UTR that are present in the endogenous Arc gene. NMD depends on the presence of proteins of the exon junction complex (EJC) downstream of a stop codon, so EGFP-Arc mRNA should not undergo NMD. Assessment of Arc mRNA rundown in the presence of the transcription inhibitor actinomycin-D confirmed delayed degradation of EGFP-Arc mRNA. EGFP-Arc mRNA and protein are expressed at much higher levels in transgenic mice under basal and activated conditions but EGFP-Arc mRNA does not enter dendrites efficiently. In a physiological assay in which cycloheximide (CHX) was infused after induction of Arc by seizures, there were increases in endogenous Arc mRNA levels consistent with translation-dependent Arc mRNA decay but this was not seen with EGFP-Arc mRNA. Taken together, our results indicate: (1) Arc mRNA degradation occurs via a mechanism with characteristics of NMD; (2) rapid dendritic delivery of newly synthesized Arc mRNA after induction may depend in part on prior splicing of the 3'UTR