Maternal immune activation causes age- and region-specific changes in brain cytokines in offspring throughout development.

Maternal infection is a risk factor for autism spectrum disorder (ASD) and schizophrenia (SZ). Indeed, modeling this risk factor in mice through maternal immune activation (MIA) causes ASD- and SZ-like neuropathologies and behaviors in the offspring. Although MIA upregulates pro-inflammatory cytokines in the fetal brain, whether MIA leads to long-lasting changes in brain cytokines during postnatal development remains unknown. Here, we tested this possibility by measuring protein levels of 23 cytokines in the blood and three brain regions from offspring of poly(I:C)- and saline-injected mice at five postnatal ages using multiplex arrays. Most cytokines examined are present in sera and brains throughout development. MIA induces changes in the levels of many cytokines in the brains and sera of offspring in a region- and age-specific manner. These MIA-induced changes follow a few, unexpected and distinct patterns. In frontal and cingulate cortices, several, mostly pro-inflammatory, cytokines are elevated at birth, followed by decreases during periods of synaptogenesis and plasticity, and increases again in the adult. Cytokines are also altered in postnatal hippocampus, but in a pattern distinct from the other regions. The MIA-induced changes in brain cytokines do not correlate with changes in serum cytokines from the same animals. Finally, these MIA-induced cytokine changes are not accompanied by breaches in the blood-brain barrier, immune cell infiltration or increases in microglial density. Together, these data indicate that MIA leads to long-lasting, region-specific changes in brain cytokines in offspring-similar to those reported for ASD and SZ-that may alter CNS development and behavior.

Neurotrophins and neuronal differentiation in the central nervous system.

The central nervous system requires the proper formation of exquisitely precise circuits to function properly. These neuronal circuits are assembled during development by the formation of synaptic connections between hundreds of thousands of differentiating neurons. For these circuits to form correctly, neurons must elaborate precisely patterned axonal and dendritic arbors. Although the cellular and molecular mechanisms that guide neuronal differentiation and formation of connections remain mostly unknown, the neurotrophins have emerged recently as attractive candidates for regulating neuronal differentiation in the developing brain. The experiments reviewed here provide strong support for a bifunctional role for the neurotrophins in axonal and dendritic growth and are consistent with the exciting possibility that the neurotrophins might mediate activity-dependent synaptic plasticity.

Cellular and molecular mechanisms of dendrite growth.

Proper growth and branching of dendrites are crucial for nervous system function; patterns of dendritic arborization determine the nature and amount of innervation that a neuron receives and specific dendritic membrane properties define its computational capabilities. Until recently, there was relatively little known about the cellular and molecular mechanisms of dendritic growth, perhaps because dendrites were historically considered to be intrinsically determined, passive elements in the formation of connections in the nervous system. In the last few years, however, overwhelming evidence has accumulated indicating that dendritic growth is remarkably dynamic and responsive to environmental signals, including guidance molecules and levels and patterns of activity. This manuscript reviews our current understanding of the cellular and molecular mechanisms of dendritic growth, the influence of activity in sculpting specific patterns of dendritic arbors, and a potential integral role for dendrites in activity-dependent development of circuits in the nervous system.

Nonsaturation of AMPA and NMDA receptors at hippocampal synapses.

An important issue in synaptic physiology is the extent to which postsynaptic receptors are saturated by the neurotransmitter released from a single synaptic vesicle. Although the bulk of evidence supports receptor saturation, recent studies have started to reveal that alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and N-methyl-D-aspartate (NMDA) receptors may not be saturated by a single vesicle of glutamate. Here, we address this question through a study of putative single synapses, made by hippocampal neurons in culture, that are identified by FM1-43 staining. An analysis of the sources of variability in the amplitudes of miniature excitatory postsynaptic currents at single synapses reveals that this variability must arise presynaptically, from variations in the quantity of agonist released. Thus, glutamate receptors at hippocampal synapses are not generally saturated by quantal release.

Biolistic transfection of neurons.

One method used to study gene function is through the manipulation of gene expression by transfecting cells with DNA constructs designed to overexpress or knock out particular proteins. Unfortunately, transfection of cells and tissues remains a rate-limiting step for molecular studies in many fields, especially neurobiology. Conventional transfection techniques are of limited effectiveness, particularly in intact tissue. This protocol describes an alternative method for transfecting cells, called biolistics. Biolistics is a physical method of transfection in which target tissue is bombarded with DNA-coated gold particles using a "gene gun," produced by Bio-Rad Laboratories. Cells penetrated by gold particles have a high likelihood of becoming transfected. Because biolistic transfection relies only on the physical penetration of a cell's membrane, it is possible to use biolistics to transfect cells that are resistant to transfection by other methods, such as neurons in primary culture and organotypic slice cultures. This protocol provides information on optimizing the biolistic parameters for transfecting neurons in both of these preparations. Once optimized, biolistic transfection is a reliable and efficient method for studying gene function in many cell types, especially postmitotic neurons.

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.

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.

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.

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.

Neuronal transfection in brain slices using particle-mediated gene transfer.

Difficulties in neuronal transfection continue to restrict the applicability of molecular approaches to neurobiology. Conventional transfection techniques have been of limited effectiveness, particularly in intact neural tissues. Viral vectors effectively transfect neurons both in vitro and in vivo but are labor intensive to construct, difficult to control, and often compromise cell viability. We describe here an alternative strategy using particle-mediated gene transfer for the transfection of neurons and glia in intact brain slices. This approach is efficient, reliable, and does not require advanced molecular biological facilities for its application.