Src, a molecular switch governing gain control of synaptic transmission mediated by N-methyl-d-aspartate receptors

The N-methyl-d-aspartate (NMDA) receptor is a principal subtype of glutamate receptor mediating fast excitatory transmission at synapses in the dorsal horn of the spinal cord and other regions of the central nervous system. NMDA receptors are crucial for the lasting enhancement of synaptic transmission that occurs both physiologically and in pathological conditions such as chronic pain. Over the past several years, evidence has accumulated indicating that the activity of NMDA receptors is regulated by the protein tyrosine kinase, Src. Recently it has been discovered that, by means of up-regulating NMDA receptor function, activation of Src mediates the induction of the lasting enhancement of excitatory transmission known as long-term potentiation in the CA1 region of the hippocampus. Also, Src has been found to amplify the up-regulation of NMDA receptor function that is produced by raising the intracellular concentration of sodium. Sodium concentration increases in neuronal dendrites during high levels of firing activity, which is precisely when Src becomes activated. Therefore, we propose that the boost in NMDA receptor function produced by the coincidence of activating Src and raising intracellular sodium may be important in physiological and pathophysiological enhancement of excitatory transmission in the dorsal horn of the spinal cord and elsewhere in the central nervous system.

Transcriptional and posttranslational plasticity and the generation of inflammatory pain

Inflammatory pain manifests as spontaneous pain and pain hypersensitivity. Spontaneous pain reflects direct activation of specific receptors on nociceptor terminals by inflammatory mediators. Pain hypersensitivity is the consequence of early posttranslational changes, both in the peripheral terminals of the nociceptor and in dorsal horn neurons, as well as later transcription-dependent changes in effector genes, again in primary sensory and dorsal horn neurons. This inflammatory neuroplasticity is the consequence of a combination of activity-dependent changes in the neurons and specific signal molecules initiating particular signal-transduction pathways. These pathways phosphorylate membrane proteins, changing their function, and activate transcription factors, altering gene expression. Two distinct aspects of sensory neuron function are changed as a result of these processes, basal sensitivity, or the capacity of peripheral stimuli to evoke pain, and stimulus-evoked hypersensitivity, the capacity of certain inputs to generate prolonged alterations in the sensitivity of the system. Posttranslational changes largely alter basal sensitivity. Transcriptional changes both potentiate the system and alter neuronal phenotype. Potentiation occurs as a result of the up-regulation in the dorsal root ganglion of centrally acting neuromodulators and simultaneously in the dorsal horn of their receptors. This means that the response to subsequent inputs is augmented, particularly those that induce stimulus-induced hypersensitivity. Alterations in phenotype includes the acquisition by A fibers of neurochemical features typical of C fibers, enabling these fibers to induce stimulus-evoked hypersensitivity, something only C fiber inputs normally can do. Elucidation of the molecular mechanisms responsible provides new opportunities for therapeutic approaches to managing inflammatory pain.

Rapidly induced auditory plasticity: The ventriloquism aftereffect

Cortical representational plasticity has been well documented after peripheral and central injuries or improvements in perceptual and motor abilities. This has led to inferences that the changes in cortical representations parallel and account for the improvement in performance during the period of skill acquisition. There have also been several examples of rapidly induced changes in cortical neuronal response properties, for example, by intracortical microstimulation or by classical conditioning paradigms. This report describes similar rapidly induced changes in a cortically mediated perception in human subjects, the ventriloquism aftereffect, which presumably reflects a corresponding change in the cortical representation of acoustic space. The ventriloquism aftereffect describes an enduring shift in the perception of the spatial location of acoustic stimuli after a period of exposure of spatially disparate and simultaneously presented acoustic and visual stimuli. Exposure of a mismatch of 8° for 20–30 min is sufficient to shift the perception of acoustic space by approximately the same amount across subjects and acoustic frequencies. Given that the cerebral cortex is necessary for the perception of acoustic space, it is likely that the ventriloquism aftereffect reflects a change in the cortical representation of acoustic space. Comparisons between the responses of single cortical neurons in the behaving macaque monkey and the stimulus parameters that give rise to the ventriloquism aftereffect suggest that the changes in the cortical representation of acoustic space may begin as early as the primary auditory cortex.

Plasticity in the neural coding of auditory space in the mammalian brain

Sound localization relies on the neural processing of monaural and binaural spatial cues that arise from the way sounds interact with the head and external ears. Neurophysiological studies of animals raised with abnormal sensory inputs show that the map of auditory space in the superior colliculus is shaped during development by both auditory and visual experience. An example of this plasticity is provided by monaural occlusion during infancy, which leads to compensatory changes in auditory spatial tuning that tend to preserve the alignment between the neural representations of visual and auditory space. Adaptive changes also take place in sound localization behavior, as demonstrated by the fact that ferrets raised and tested with one ear plugged learn to localize as accurately as control animals. In both cases, these adjustments may involve greater use of monaural spectral cues provided by the other ear. Although plasticity in the auditory space map seems to be restricted to development, adult ferrets show some recovery of sound localization behavior after long-term monaural occlusion. The capacity for behavioral adaptation is, however, task dependent, because auditory spatial acuity and binaural unmasking (a measure of the spatial contribution to the “cocktail party effect”) are permanently impaired by chronically plugging one ear, both in infancy but especially in adulthood. Experience-induced plasticity allows the neural circuitry underlying sound localization to be customized to individual characteristics, such as the size and shape of the head and ears, and to compensate for natural conductive hearing losses, including those associated with middle ear disease in infancy.

Synaptic pattern formation during cellular recognition

Cell–cell recognition often requires the formation of a highly organized pattern of receptor proteins (a synapse) in the intercellular junction. Recent experiments [e.g., Monks, C. R. F., Freiberg, B. A., Kupfer, H., Sciaky, N. & Kupfer, A. (1998) Nature (London) 395, 82–86; Grakoui, A., Bromley, S. K., Sumen, C., Davis, M. M., Shaw, A. S., Allen, P. M. & Dustin, M. L. (1999) Science 285, 221–227; and Davis, D. M., Chiu, I., Fassett, M., Cohen, G. B., Mandelboim, O. & Strominger, J. L. (1999) Proc. Natl. Acad. Sci. USA 96, 15062–15067] vividly demonstrate a complex evolution of cell shape and spatial receptor–ligand patterns (several microns in size) in the intercellular junction during immunological synapse formation. The current view is that this dynamic rearrangement of proteins into organized supramolecular activation clusters is driven primarily by active cytoskeletal processes [e.g., Dustin, M. L. & Cooper, J. A. (2000) Nat. Immunol. 1, 23–29; and Wulfing, C. & Davis, M. M. (1998) Science 282, 2266–2269]. Here, aided by a quantitative analysis of the relevant physico-chemical processes, we demonstrate that the essential characteristics of synaptic patterns observed in living cells can result from spontaneous self-assembly processes. Active cellular interventions are superimposed on these self-organizing tendencies and may also serve to regulate the spontaneous processes. We find that the protein binding/dissociation characteristics, protein mobilities, and membrane constraints measured in the cellular environment are delicately balanced such that the length and time scales of spontaneously evolving patterns are in near-quantitative agreement with observations for synapse formation between T cells and supported membranes [Grakoui, A., Bromley, S. K., Sumen, C., Davis, M. M., Shaw, A. S., Allen, P. M. & Dustin, M. L. (1999) Science 285, 221–227]. The model we present provides a common way of analyzing immunological synapse formation in disparate systems (e.g., T cell/antigen-presenting cell junctions with different MHC-peptides, natural killer cells, etc.).

Genetic programs of epithelial cell plasticity directed by transforming growth factor-β

Epithelial–mesenchymal transitions (EMTs) are an essential manifestation of epithelial cell plasticity during morphogenesis, wound healing, and tumor progression. Transforming growth factor-β (TGF-β) modulates epithelial plasticity in these physiological contexts by inducing EMT. Here we report a transcriptome screen of genetic programs of TGF-β-induced EMT in human keratinocytes and propose functional roles for extracellular response kinase (ERK) mitogen-activated protein kinase signaling in cell motility and disruption of adherens junctions. We used DNA arrays of 16,580 human cDNAs to identify 728 known genes regulated by TGF-β within 4 hours after treatment. TGF-β-stimulated ERK signaling mediated regulation of 80 target genes not previously associated with this pathway. This subset is enriched for genes with defined roles in cell–matrix interactions, cell motility, and endocytosis. ERK-independent genetic programs underlying the onset of EMT involve key pathways and regulators of epithelial dedifferentiation, undifferentiated transitional and mesenchymal progenitor phenotypes, and mediators of cytoskeletal reorganization. The gene expression profiling approach delineates complex context-dependent signaling pathways and transcriptional events that determine epithelial cell plasticity controlled by TGF-β. Investigation of the identified pathways and genes will advance the understanding of molecular mechanisms that underlie tumor invasiveness and metastasis.

Role of Rab3 GDP/GTP Exchange Protein in Synaptic Vesicle Trafficking at the Mouse Neuromuscular Junction

The Rab3 small G protein family consists of four members, Rab3A, -3B, -3C, and -3D. Of these members, Rab3A regulates Ca2+-dependent neurotransmitter release. These small G proteins are activated by Rab3 GDP/GTP exchange protein (Rab3 GEP). To determine the function of Rab3 GEP during neurotransmitter release, we have knocked out Rab3 GEP in mice. Rab3 GEP−/− mice developed normally but died immediately after birth. Embryos at E18.5 showed no evoked action potentials of the diaphragm and gastrocnemius muscles in response to electrical stimulation of the phrenic and sciatic nerves, respectively. In contrast, axonal conduction of the spinal cord and the phrenic nerve was not impaired. Total numbers of synaptic vesicles, especially those docked at the presynaptic plasma membrane, were reduced at the neuromuscular junction ∼10-fold compared with controls, whereas postsynaptic structures and functions appeared normal. Thus, Rab3 GEP is essential for neurotransmitter release and probably for formation and trafficking of the synaptic vesicles.

Cytoskeletal microdifferentiation: A mechanism for organizing morphological plasticity in dendrites

Experimental evidence suggests that microfilaments and microtubules play contrasting roles in regulating the balance between motility and stability in neuronal structures. Actin-containing microfilaments are associated with structural plasticity, both during development when their dynamic activity drives the exploratory activity of growth cones and after circuit formation when the actin-rich dendritic spines of excitatory synapses retain a capacity for rapid changes in morphology. By contrast, microtubules predominate in axonal and dendritic processes, which appear to be morphologically relatively more stable. To compare the cytoplasmic distributions and dynamics of microfilaments and microtubules we made time-lapse recordings of actin or the microtubule-associated protein 2 tagged with green fluorescent protein in neurons growing in dispersed culture or in tissue slices from transgenic mice. The results complement existing evidence indicating that the high concentrations of actin present in dendritic spines is a specialization for morphological plasticity. By contrast, microtubule-associated protein 2 is limited to the shafts of dendrites where time-lapse recordings show little evidence for dynamic activity. A parallel exists between the partitioning of microfilaments and microtubules in motile and stable domains of growing processes during development and between dendrite shafts and spines at excitatory synapses in established neuronal circuits. These data thus suggest a mechanism, conserved through development and adulthood, in which the differential dynamics of actin and microtubules determine the plasticity of neuronal structures.

Synaptic regulation of protein synthesis and the fragile X protein

Protein synthesis occurs in neuronal dendrites, often near synapses. Polyribosomal aggregates often appear in dendritic spines, particularly during development. Polyribosomal aggregates in spines increase during experience-dependent synaptogenesis, e.g., in rats in a complex environment. Some protein synthesis appears to be regulated directly by synaptic activity. We use “synaptoneurosomes,” a preparation highly enriched in pinched-off, resealed presynaptic processes attached to resealed postsynaptic processes that retain normal functions of neurotransmitter release, receptor activation, and various postsynaptic responses including signaling pathways and protein synthesis. We have found that, when synaptoneurosomes are stimulated with glutamate or group I metabotropic glutamate receptor agonists such as dihydroxyphenylglycine, mRNA is rapidly taken up into polyribosomal aggregates, and labeled methionine is incorporated into protein. One of the proteins synthesized is FMRP, the protein that is reduced or absent in fragile X mental retardation syndrome. FMRP has three RNA-binding domains and reportedly binds to a significant number of mRNAs. We have found that dihydroxyphenylglycine-activated protein synthesis in synaptoneurosomes is dramatically reduced in a knockout mouse model of fragile X syndrome, which cannot produce full-length FMRP, suggesting that FMRP is involved in or required for this process. Studies of autopsy samples from patients with fragile X syndrome have indicated that dendritic spines may fail to assume a normal mature size and shape and that there are more spines per unit dendrite length in the patient samples. Similar findings on spine size and shape have come from studies of the knockout mouse. Study of the development of the somatosensory cortical region containing the barrel-like cell arrangements that process whisker information suggests that normal dendritic regression is impaired in the knockout mouse. This finding suggests that FMRP may be required for the normal processes of maturation and elimination to occur in cerebral cortical development.

Synaptic sprouting increases the uptake capacities of motoneurons in amyotrophic lateral sclerosis mice

Using adenoviruses encoding reporter genes as retrograde tracers, we assessed the capacity of motoneurons to take up and retrogradely transport adenoviral particles injected into the muscles of transgenic mice expressing the G93A human superoxide dismutase mutation, a model of amyotrophic lateral sclerosis. Surprisingly, transgene expression in the motoneurons was significantly higher in symptomatic mice than in control or presymptomatic mice. Using botulinum toxin to induce nerve sprouting at neuromuscular junctions, we showed that the unexpectedly high level of motoneurons retrograde transduction results, at least in part, from newly acquired uptake properties of the sprouts. These findings demonstrate the remarkable uptake properties of amyotrophic lateral sclerosis motoneurons in response to denervation and the rationale of using intramuscular injections of adenoviruses to overexpress therapeutic proteins in motor neuron diseases.