Neuronal transfection using particle-mediated gene transfer.

This unit describes the use of particle-mediated gene transfer (also known as biolistics) for the transfection of neuronal cell lines and brain slices. Like nuclear microinjection of DNA, biolistics results in the direct introduction of DNA into the nucleus; it is perhaps for this reason that biolistics works as well in mitotic cells as in postmitotic cells such as skeletal muscle, skin, liver, and neurons. The basic principle of biolistics is to accelerate micron-sized gold particles coated with DNA towards target cells or tissue. Cells penetrated by these particles have a high likelihood of being transfected by the DNA thus introduced. The motive force for particle acceleration can come from a variety of sources, the most widely used is described in this unit and is a supersonic shock wave generated by the rupture of a kapton membrane induced by high-pressure helium. Another option included in this unit is to propel the gold particles by gas jet entrainment.

Brain-derived neurotrophic factor differentially regulates excitatory and inhibitory synaptic transmission in hippocampal cultures.

Brain-derived neurotrophic factor (BDNF) has been postulated to be a key signaling molecule in regulating synaptic strength and overall circuit activity. In this context, we have found that BDNF dramatically increases the frequency of spontaneously initiated action potentials in hippocampal neurons in dissociated culture. Using analysis of unitary synaptic transmission and immunocytochemical methods, we determined that chronic treatment with BDNF potentiates both excitatory and inhibitory transmission, but that it does so via different mechanisms. BDNF strengthens excitation primarily by augmenting the amplitude of AMPA receptor-mediated miniature EPSCs (mEPSCs) but enhances inhibition by increasing the frequency of mIPSC and increasing the size of GABAergic synaptic terminals. In contrast to observations in other systems, BDNF-mediated increases in AMPA-receptor mediated mEPSC amplitudes did not require activity, because blocking action potentials with tetrodotoxin for the entire duration of BDNF treatment had no effect on the magnitude of this enhancement. These forms of synaptic regulations appear to be a selective action of BDNF because intrinsic excitability, synapse number, and neuronal survival are not affected in these cultures. Thus, although BDNF induces a net increase in overall circuit activity, this results from potentiation of both excitatory and inhibitory synaptic drive through distinct and selective physiological mechanisms.

Truncated and full-length TrkB receptors regulate distinct modes of dendritic growth.

Neurotrophin regulation of neuronal morphology is complex and may involve differential action of alternative Trk receptor isoforms. We transfected ferret visual cortical slices with full-length and truncated TrkB receptors to examine their roles in regulating cortical dendrite development. These TrkB isoforms had differential effects on dendritic arborization: whereas full-length TrkB increased proximal dendritic branching, truncated TrkB promoted net elongation of distal dendrites. The morphological effects of each receptor isoform were distinct, yet their actions inhibited one another. Actions of the truncated TrkB receptor did not involve unmasking of endogenous TrkC signaling. These results suggest that TrkB receptors do not regulate dendritic growth per se but, rather, the mode of such growth.

Induction of electrical excitability by NGF requires autocrine action of a CNTF-like factor.

The overlapping expression of neurotrophin and neural cytokine receptors indicates that most neuronal populations are responsive to both classes of factors, yet relatively little is known about how these two trophic signaling systems interact to regulate neuronal phenotype. We report here that one hallmark of NGF's effects on target cells, the induction of membrane electrical excitability, requires the intermediary action of a CNTF-like factor. We found that NGF's regulation of voltage-gated potassium channels, unlike its regulation of voltage-gated sodium and calcium channels, involves a CNTF-like autocrine/paracrine loop. We showed that NGF induces secretion of a soluble factor that mimics the action of exogenous CNTF in regulating voltage-gated potassium channels and that NGF's ability to regulate this potassium channel is blocked by three independent reagents that inhibit the signaling of CNTF and/or related factors. The identity of this autocrine factor does not appear to be CNTF itself. Thus, a CNTF-like autocrine/paracrine factor is both necessary and sufficient for the regulation of potassium channels by NGF and is a key determinant of the type of electrical excitability that NGF induces in target cells.

Long-term enhancement of central synaptic transmission by chronic brain-derived neurotrophic factor treatment.

Acute effects of neurotrophins on synaptic plasticity have recently received much attention, but the roles of these factors in regulating long-lasting changes in synaptic function remain unclear. To address this issue we studied the long-term (days to weeks) and short-term (minutes to hours) effects of brain-derived neurotrophic factor (BDNF) on excitatory synaptic transmission in autaptic cultures of hippocampal CA1 neurons. We found that BDNF induced long-term enhancement of the strength of non-NMDA receptor-mediated glutamatergic transmission. This upregulation of EPSC amplitude occurred via an increase in the size of unitary synaptic currents, with no significant contribution from other aspects of neuronal electrical and synaptic function including cell size, voltage-gated sodium and potassium current levels, the number and size of synaptic contacts, and the frequency of spontaneous neurotransmitter release. Chronic BDNF treatment also decreased the degree of synaptic depression measured in response to paired stimuli. Thus, BDNF induced long-term synaptic enhancement of both basal and use-dependent synaptic transmission via specific changes to the synapse rather than through generalized potentiation of neuronal growth and differentiation. Finally, we showed that the long-term effects of BDNF are functionally and mechanistically distinct from its acute effects on synaptic transmission, suggesting that, in vivo, BDNF activation of Trk receptors can have different functional effects depending on the time course of its action.

Neurotrophins and synaptic plasticity.

Despite considerable evidence that neuronal activity influences the organization and function of circuits in the developing and adult brain, the molecular signals that translate activity into structural and functional changes in connections remain largely obscure. This review discusses the evidence implicating neurotrophins as molecular mediators of synaptic and morphological plasticity. Neurotrophins are attractive candidates for these roles because they and their receptors are expressed in areas of the brain that undergo plasticity, activity can regulate their levels and secretion, and they regulate both synaptic transmission and neuronal growth. Although numerous experiments show demonstrable effects of neurotrophins on synaptic plasticity, the rules and mechanisms by which they exert their effects remain intriguingly elusive.

Instructive roles of neurotrophins in synaptic plasticity.

Together, these experiments show that neurotrophins regulate neuronal function in a factor-dependent fashion, both in terms of membrane excitability and dendritic growth, and indicate that neurotrophins can play instructive roles in regulating neuronal phenotype and function. Given that neurons generally receive neurotrophic inputs from many different cellular sources, both regional and distant (e.g., via axonal and dendritic projections), instructive actions of neurotrophic factors may contribute to matching the morphology and electrical function of neurons to the neural circuits of which they are part. Moreover, during ongoing development and function of the nervous system, these same or similar neurotrophic factors may be key instructive signals in regulating synaptic plasticity of neural circuits.

Neurotrophin regulation of ionic currents and cell size depends on cell context.

Trk receptor activation by neurotrophins is often considered to have a defined set of actions on target neurons, including supporting neuronal survival, inducing morphological differentiation, and regulating a host of target genes that specify neuronal phenotype. It is not known if all such regulatory effects are obligatory, or if some may vary depending on the cell context in which the receptors are expressed. We have examined this issue by comparing neurotrophin effects on the regulation of electrical excitability and morphological differentiation in two strains of PC12 cells. We found that while neurotrophins induced neurite extension and increased calcium currents in both PC12 cell types, sodium current levels were regulated in only one of these strains. Moreover, we found little correlation between calcium current levels and the extent of morphological differentiation when compared in individual cells of a single strain. Thus, the regulatory effects of neurotrophins on cell phenotype are not fully determined by the Trk receptors that they activate; rather, they can vary with differences in cell context that arise not only between different cell lineages, but also between individual cells of clonal relation.

Opposing roles for endogenous BDNF and NT-3 in regulating cortical dendritic growth.

Neurons within each layer of cerebral cortex express multiple members of the neurotrophin family and their corresponding receptors. This multiplicity could provide functional redundancy; alternatively, different neurotrophins may direct distinct aspects of cortical neuronal growth and differentiation. By neutralizing endogenous neurotrophins in organotypic slices of developing cortex with Trk receptor bodies (Trk-IgGs), we found that BDNF and NT-3 oppose one another in regulating the dendritic growth of pyramidal neurons. In layer 4, both endogenous and exogenous NT-3 inhibited the dendritic growth stimulated by BDNF. In contrast, in layer 6 both endogenous and exogenous BDNF inhibited dendritic growth stimulated by NT-3. These antagonistic actions of endogenous BDNF and NT-3 provide a mechanism by which dendritic growth and retraction can be dynamically regulated during cortical development, and suggest that the multiple neurotrophins expressed in developing cortex represent distinct components of an extracellular signaling system for regulating dendritic growth.

c-myc-Dependent hepatoma cell apoptosis results from oxidative stress and not a deficiency of growth factors.

Expression of c-myc regulates apoptotic cell death in the human hepatoma cell line HuH-7 during culture in serum-free medium (SFM) plus zinc. To understand the mechanism of this c-myc effect, the ability of various serum-contained factors to prevent apoptosis was determined. Apoptosis was not inhibited by growth factors and was even accelerated by supplementation with insulin-like growth factor I or insulin. Cell death was prevented by SFM supplementation with the amino acid glutamine but not serine or asparagine. Improved cell survival with glutamine was associated with increased levels of glutathione (GSH). In HuH-7 cells cultured in SFM plus zinc, c-myc expression led to decreased levels of GSH, and elevated intracellular levels of hydrogen peroxide (H2O2). Cell death induced by c-myc expression was inhibited by the addition of catalase or dimethyl sulfoxide, a hydroxyl radical scavenger, or by increased intracellular expression of catalase. In contrast to findings in fibroblasts, c-myc-dependent apoptosis during serum deprivation in HuH-7 hepatoma cells was unrelated to a loss of growth factors. Apoptosis resulted from H2O2-mediated oxidative stress with associated glutamine dependent intracellular GSH depletion.

Neurotrophins differentially regulate voltage-gated ion channels.

Neurotrophic factors profoundly affect neuronal differentiation, but whether they influence neuronal phenotype in instructive ways remains unclear: do different neurotrophic factors always trigger identical programs of differentiation or can each impose distinct functional properties even when acting upon the same population of target neurons? We addressed this issue by examining the regulatory effects of the four neurotrophins on the molecular components of electrical excitability, voltage-gated ion channels, within a single cellular context. Using patch clamp methods, we studied neurotrophin regulation of voltage-gated sodium, calcium, and potassium currents in SK-N-SH neuroblastoma cells. We found that each neurotrophin induced a unique pattern of expression of ionic currents despite similar activation of initial signal transduction events. Thus, each neurotrophin imposed a different excitable phenotype even when acting upon the same target cells.

Focal delivery of neurotrophins into the central nervous system using fluorescent latex microspheres.

A new technique for in vivo and in vitro delivery of neurotrophins is described. The method is based upon the ability of certain fluorescent latex microspheres to adsorb large quantities of neurotrophin and then release the factor for at least three to four days. Injection of such neurotrophin-coated microspheres in vivo provides focal delivery of neurotrophins to distinct populations of neurons. The fluorescence of the microspheres accurately identifies the region of neurotrophin treatment in vivo; two independent methods indicate that microsphere-born proteins do not diffuse significantly beyond the site of injection. In addition to providing focal delivery and labeling of the site of application, labeled microspheres are retrogradely transported by neurons; thus, neurons whose distant terminals are exposed to neurotrophin can be identified and analyzed. This approach provides a powerful method for the introduction of neuroactive proteins into defined and localized regions of the central nervous system.

Neurotrophin regulation of cortical dendritic growth requires activity.

Neurotrophins have been proposed to mediate several forms of activity-dependent competition in the central nervous system. A key element of such hypotheses is that neurotrophins act preferentially on active neurons; however, little direct evidence supports this postulate. We therefore examined, in ferret cortical brain slices, the interactions between activity and neurotrophins in regulating dendritic growth of layer 4 pyramidal neurons. Inhibition of spontaneous electrical activity, synaptic transmission, or L-type calcium channels each prevented the otherwise dramatic increase in dendritic arborizations elicited by brain-derived neurotrophic factor. In developing cortex, this requirement for conjoint neurotrophin signaling and activity provides a mechanism for selectively enhancing the growth and connectivity of active neurons.

NT-4-mediated rescue of lateral geniculate neurons from effects of monocular deprivation.

Altering the balance of activity between the two eyes during the critical period for visual-system development profoundly affects competitive interactions among neurons in the lateral geniculate nucleus and primary visual cortex. Neurons in the lateral geniculate nucleus that are deprived of activity by closing or silencing one eye atrophy as a result of competition with non-deprived neurons for some critical factor(s) presumed to be present in the cortex. Based on their actions in the developing visual system, neurotrophins are attractive candidates for such factors. We tested whether neurotrophins mediate intracortical competition of afferents from the lateral geniculate nucleus by using monocular deprivation and a new method for highly localized, in vivo delivery of neurotrophins. This method allowed unambiguous identification of neurons that were exposed to neurotrophin. Here we report that only one neurotrophin, the TrkB ligand NT-4, rescued neurons in the lateral geniculate nucleus from the dystrophic effects of monocular deprivation.

Neurotrophins regulate dendritic growth in developing visual cortex.

Although dendritic growth and differentiation are critical for the proper development and function of neocortex, the molecular signals that regulate these processes are largely unknown. The potential role of neurotrophins was tested by treating slices of developing visual cortex with NGF, BDNF, NT-3, or NT-4 and by subsequently visualizing the dendrites of pyramidal neurons using particle-mediated gene transfer. Specific neurotrophins increased the length and complexity of dendrites of defined cell populations. Basal dendrites of neurons in each cortical layer responded most strongly to a single neurotrophin: neurons in layer 4 to BDNF and neurons in layers 5 and 6 to NT-4. In contrast, apical dendrites responded to a range of neurotrophins. On both apical and basal dendrites, the effects of the TrkB receptor ligands, BDNF and NT-4, were distinct. The spectrum of neurotrophic actions and the laminar specificity of these actions implicate endogenous neurotrophins as regulatory signals in the development of specific dendritic patterns in mammalian neocortex.