Synaptic strengthening induced by brain-derived neurotrophic factor (BDNF) is associated with learning and is coupled to transcriptional activation. However, identification of the spectrum of genes associated with BDNF-induced synaptic plasticity and the correlation of expression with learning paradigms in vivo has not yet been studied. Transcriptional analysis of BDNF-induced synaptic strengthening in cultured hippocampal neurons revealed increased expression of the immediate early genes (IEGs), c-fos, early growth response gene 1 (EGR1), activity-regulated cytoskeletal-associated protein (Arc) at 20 min, and the secreted peptide VGF (non-acronymic) protein precursor at 3 hr. The induced genes served as prototypes to decipher mechanisms of both BDNF-induced transcription and plasticity. BDNF-mediated gene expression was tyrosine kinase B and mitogen-activated protein kinase-dependent, as demonstrated by pharmacological studies. Single-cell transcriptional analysis of Arc after whole-cell patch-clamp recordings indicated that increased gene expression correlated with enhancement of synaptic transmission by BDNF. Increased expression in vitro predicted elevations in vivo: VGF and the IEGs increased after trace eyeblink conditioning, a hippocampal-dependent learning paradigm. VGF protein was also upregulated by BDNF treatment and was expressed in a punctate manner in dissociated hippocampal neurons. Collectively, these findings suggested that the VGF neuropeptides may regulate synaptic function. We found a novel function for VGF by applying VGF peptides to neurons. C-terminal VGF peptides acutely increased synaptic charge in a dose-dependent manner, whereas N-terminal peptide had no effect. These observations indicate that gene profiling in vitro can reveal new mechanisms of synaptic strengthening associated with learning and memory.

Brain-derived neurotrophic factor (BDNF) is upregulated in the hippocampus by antidepressant treatments, and BDNF produces antidepressant-like effects in behavioral models of depression. In our previous work, we identified genes induced by BDNF and defined their specific roles in hippocampal neuronal development and plasticity. To identify genes downstream of BDNF that may play roles in psychiatric disorders, we examined a subset of BDNF-induced genes also regulated by 5-HT (serotonin), which includes the neuropeptide VGF (nonacronymic). To explore the function of VGF in depression, we first investigated the expression of the neuropeptide in animal models of depression. VGF was downregulated in the hippocampus after both the learned helplessness and forced swim test (FST) paradigms. Conversely, VGF infusion in the hippocampus of mice subjected to FST reduced the time spent immobile for up to 6 d, thus demonstrating a novel role for VGF as an antidepressant-like agent. Recent evidence indicates that chronic treatment of rodents with antidepressants increases neurogenesis in the adult dentate gyrus and that neurogenesis is required for the behavioral effects of antidepressants. Our studies using

Brain-derived neurotrophic factor (BDNF) modulates synaptic strength in hippocampal neurons, in addition to promoting survival and differentiation. To identify genes involved in trophic regulation of synaptic plasticity, we have used a multidisciplinary approach of differential display and family-specific slot blots in combination with whole-cell patch-clamp recordings of dissociated hippocampal neurons. Three hour exposure to BDNF elicited a 2.6-fold increase in synaptic charge and a concomitant induction of 11 genes as revealed by differential display, including the small GTP-binding vesicular trafficking protein Rab3A and the enzyme guanylate cyclase (GC). Slot blot analysis on a population of neurons confirmed an average of 3.1-fold induction of these clones. In contrast, individual pyramidal-like neurons that were first characterized electrophysiologically in the presence of BDNF and subjected to transcriptional analysis displayed more robust increases (4.8-fold), emphasizing the neuronal heterogeneity. Transcriptional changes of Rab3A and GC were accompanied by translational regulation, shown by Western blot analysis. Furthermore, a number of GC-associated and Rab3A effector molecules were induced by BDNF at either the gene or protein levels. The functional role of Rab3A in BDNF-induced synaptic plasticity was assessed using cells derived from Rab3A knock-out mice. These neurons failed to show an increase in synaptic charge in response to BDNF at 10 min; however a late response to BDNF was detected at 20 min. This late response was similar in time course to that induced by postsynaptic activation of glutamate receptors. Our results demonstrate a requirement for Rab3A and may reveal a temporal distinction between presynaptic and postsynaptic mechanisms of BDNF-induced synaptic plasticity associated with learning and memory.

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and mortality. Based on the nature of primary injury following TBI, complex and heterogeneous secondary consequences result, which are followed by regenerative processes 1,2 . Primary injury can be induced by a direct contusion to the brain from skull fracture or from shearing and stretching of tissue causing displacement of brain due to movement 3,4 . The resulting hematomas and lacerations cause a vascular response 3,5 , and the morphological and functional damage of the white matter leads to diffuse axonal injury [6][7][8] . Additional secondary changes commonly seen in the brain are edema and increased intracranial pressure . Following TBI there are microscopic alterations in biochemical and physiological pathways involving the release of excitotoxic neurotransmitters, immune mediators and oxygen radicals [10][11][12] , which ultimately result in long-term neurological disabilities 13,14 . Thus choosing appropriate animal models of TBI that present similar cellular and molecular events in human and rodent TBI is critical for studying the mechanisms underlying injury and repair.Various experimental models of TBI have been developed to reproduce aspects of TBI observed in humans, among them three specific models are widely adapted for rodents: fluid percussion, cortical impact and weight drop/impact acceleration 1 . The fluid percussion device produces an injury through a craniectomy by applying a brief fluid pressure pulse on to the intact dura. The pulse is created by a pendulum striking the piston of a reservoir of fluid. The percussion produces brief displacement and deformation of neural tissue 1,15 . Conversely, cortical impact injury delivers mechanical energy to the intact dura via a rigid impactor under pneumatic pressure 16,17 . The weight drop/impact model is characterized by the fall of a rod with a specific mass on the closed skull 18 . Among the TBI models, LFP is the most established and commonly used model to evaluate mixed focal and diffuse brain injury 19. It is reproducible and is standardized to allow for the manipulation of injury parameters. LFP recapitulates injuries observed in humans, thus rendering it clinically relevant, and allows for exploration of novel therapeutics for clinical translation 20 .We describe the detailed protocol to perform LFP procedure in mice. The injury inflicted is mild to moderate, with brain regions such as cortex, hippocampus and corpus callosum being most vulnerable. Hippocampal and motor learning tasks are explored following LFP. Video LinkThe video component of this article can be found at https://www.jove.com/video/3063/ Protocol 1. Craniectomy 1. A sterile surgical site is prepared including a stereotaxic alignment instrument (Kopf Instruments) for mice with a mouse gas anesthesia head holder (Kopf Instruments) connected to an anesthesia machine with O 2 flush (Parkland Scientific) to allow for continuou...

Macrolides-lincosamides-streptogramin B (MLS) resistance is commonly found in Staphylococcus aureus and coagulase-negative staphylococci (22 and 45%, respectively, among isolates from three New Jersey hospitals). We have examined representative subsets of 107 MLS-resistant isolates for the molecular nature of the resistance determinant, the erm gene, by dot blot and Southern hybridization analysis. All of 35 S. aureus isolates examined and 39 of 42 coagulase-negative isolates examined were found to harbor the ermA or ermC evolutionary variant. Genes of the ermC class occurred exclusively on a small plasmid similar to or indistinguishable from one (pNE131) previously described in S. epidermidis. Genes of the ermA class occurred exclusively in the chromosome, and restriction patterns indicated that they were part of a transposon, Tn554, characteristic of the classical S. aureus ermA strain. Unlike S. aureus ermA strains examined previously, which harbor TnS54 at a single specific (primary) site, four of our S. aureus isolates had second inserts at different chromosomal sites. The majority of our coagulase-negative isolates had two or more inserts, neither of which occurred at the classical primary site and many of which differed from one another in location (as inferred from restriction patterns). Coagulase-negative staphylococci constitute a large reservoir of the ermA and ermC class of determinants, with clear potential for interspecies spread.The MLS phenotype refers to cross-resistance to three groups of antibiotics: the macrolides (exemplified by erythromycin), the lincosamides (exemplified by clindamycin), and the streptogramins B (exemplified by vernamycin Ba). The classical phenotype was described for four Staphylococcus aureus isolates, in which resistance to the macrolide spiramycin was shown to be inducible by low levels of erythromycin (2). The phenotype has since been shown to occur in a variety of bacterial genera (5), but the S. aureus system has been subjected to the most intensive scrutiny and is understood in greatest detail (see references 26 and 36 for summaries). Neither lincosamides, streptogramins, nor most macrolides are effective inducers. The induction process entails activation of an mRNA that encodes a 23S RNA methylase, the erm methylase, which in turn renders newly synthesized ribosomes resistant to the MLS agents by methylating a specific adenosine residue (equivalent to Escherichia coli 23S RNA A2058 [33]) of the rRNA component of the peptidyl transferase center. The activation involves alteration in the secondary structure of the mRNA, resulting from a stall of an erythromycin-ribosome complex on the leader region(s) encoding short polypeptides. Constitutive variants readily arise via mutational changes in these leader regions.