The general interest of the laboratory lies in understanding the molecular mechanisms that regulate neuronal and muscle plasticity during development. Until recently, a commonly held view in neurobiology was that neural inputs (particularly neuronal activity) are instructive to the functional properties of postsynaptic targets. Recently, a different picture has begun to emerge. Early in development and independently of innervation, lineage plays a fundamental role in the pre-patterning of targets. Later, during the maturation of neural connections, activity can modify the plastic properties of postsynaptic targets. Moreover, the view that electrical activity exerts its effects predominantly by regulating the levels of intracellular calcium is undergoing revision because of the realization that the action of growth/differentiation factors on synaptic function is activity-dependent and can occur in the time scale of minutes. We are using two experimental models to investigate how neural factors and activity regulate neuronal and muscle plasticity during development. In the first project, we are investigating how neuregulins acutely regulate synaptic transmission at interneuronal synapses. In the second, we are studying how lineage and motoneuron activity contribute to the emergence and plasticity of different muscle types.

Neuregulin Effects on Synaptic Plasticity: Possible Role in Schizophrenia
Buonanno
Neuregulins (NRG 1-3) are growth/differentiation factors that signal by means of a family of receptor tyrosine kinases known as ErbB 1-4. Present knowledge of NRGs mostly originates from studies on NRG-1. The pro-NRG-1 is synthesized as a transmembrane precursor in the soma, then transported down axons, and proteolytically released in its active form in an activity-dependent fashion. Earlier work by our group and others showed that long-term exposure (more than two days) of neurons to NRG-1 elicits changes in the composition of neurotransmitter receptors for glutamate (NMDA subtype), GABA, and acetylcholine by selectively regulating the expression of distinct receptor subunits. We found that co-activation of both glutamate and ErbB receptors is necessary for NRG-1 to induce NMDA receptor expression, suggesting a cross-talk between these signaling pathways. The subsequent demonstration that ErbB4 and NMDA receptors co-localize at glutamatergic synapses with PSD-95, a PDZ protein coupling postsynaptic receptors to signaling complexes, led us to hypothesize that the NRG/ErbB signaling pathway may acutely modify synaptic properties (Garcia et al., 2000).

FIGURE 20

NRG and ErbB Receptor Expression in the Developing Central Nervous System
Longart, Liu, Vasudevan, Karavanova, Buonanno; in collaboration with Carroll
We investigated the regional and temporal expression of NRGs and their receptors because such information is essential for understanding how the NRG/ErbB pathway contributes to neuronal function. We used multiple approaches in our studies, including in situ hybridization, Western blots, and immunofluorescence histochemistry. The patterns of NRG 1-3 mRNA expression differ markedly during development (Fig. 20). In general, NRG-1 expression is highest early in development and becomes restricted postnatally. NRG-2 mRNA levels in embryonic and newborn mouse brains are low compared with those of NRG-3. Seven days after birth, the highest levels of NRG-2 are found in the dentate gyrus, olfactory bulb, and cerebellum. In contrast, NRG-3 is the most highly expressed NRG in the brain and shows the least regional or developmental regulation. To study the cellular and subcellular distribution of these proteins in brain, we generated and characterized antibodies against NRG-2 and NRG-3. Preliminary studies indicate that NRG-2 and NRG-3 accumulate in neurons, although expression in glia cannot be ruled out. Interestingly, the subcellular distributions of NRG 1-3 differ, suggesting that NRGs may perform distinct functions during neuronal development and maturation.

Although ErbB receptors are critical for neural development, their regional distributions during development and in the adult were unknown. ErbB receptors are differentially expressed in neurons and glia. ErbB2 is expressed in most cells, ErbB3 is predominantly found in glia and subpopulations of neurons, and ErbB4 is mostly restricted to neurons and oligodendrocytes. The punctate labeling with ErbB2 and ErbB4 antibodies is highest in synapse-rich regions, which is consistent with the previous finding that ErbB receptors accumulate at postsynaptic densities (Garcia et al., 2000). ErbB4 receptors are mostly restricted to the dendrites and cell bodies of GABAergic neurons, where they co-localize at excitatory synapses with NMDA receptors and PSD-95. The existence of complexes of these proteins at postsynaptic densities suggests that the NRG/ErbB pathway may function to regulate synaptic function acutely.

NRG-1 Acutely Modifies Synaptic Transmission in Cultured Hippocampal Neurons
Liu, Longart, Buonanno; in collaboration with Vicini
To test the aforementioned hypothesis, we analyzed the acute affects of NRG on synaptic transmission. A brief application of 5nM NRG1b1 (about two minutes), which causes the phosphorylation of ErbB2 and ErbB4, induces a persistent and significant increase in the firing patterns of cultured hippocampal neurons. These effects are specific because they are not observed with the NRG-a2 splice variant that differs slightly with NRG1-b1 and is a weaker activator of ErbB receptors. The findings suggest that NRGs may play important roles in synchronizing synaptic or network activity of hippocampal circuitry. Experiments are in progress to understand the underlying mechanisms regulating synaptic plasticity in response to NRG in dissociated hippocampal fresh slices and cultures.

The implications of these findings for basic and clinical science may be extremely important in light of a recent study associating NRG-1 mutations with schizophrenia in Icelandic families. Moreover, mutant mice with decreased levels of NRG-1 and ErbB receptors have fewer NMDA receptors and manifest behavioral deficits reported to be consistent with schizophrenia. Pharmacological agents used to treat schizophrenia reverse these behavioral changes in mutant mice.

Contribution of Developmental History and Neural Activity to the Fiber-Type Specificity of Troponin I Genes
Buonanno
The developmental and neuronal regulation of skeletal muscle fiber types provides an excellent model in which to study how patterned activity regulates plasticity of postsynaptic targets. Our long-term objectives are to identify transcription factors that both regulate the emergence of slow- and fast-twitch fibers during development and intraconvert their contractile properties in response to specific patterns of motoneuron activity. The troponin I slow (TnIs) and fast (TnIf) genes serve as our experimental paradigm because their expression is fiber-type–specific and regulated by selective patterns of electrical impulses that mimic motoneuron activity. We have determined that the General Transcription Factor 3 (GTF3), expressed in numerous tissues including muscle and brain, contributes to the establishment of fiber types during perinatal development (Calvo et al., 2001).

Transcription Factor GTF3 Contributes to the Slow Muscle Program
Vullhorst, Karavanova, Buonanno
The TnIs is activated during terminal myogenic differentiation in all skeletal muscles regardless of their future fiber type and is later confined to prospective slow fibers during fetal development. The SURE element (for slow upstream regulatory enhancer), which confers slow fiber specificity to TnIs expression, requires interactions between multiple transcription factors. The bicoid-like motif (BLM) in SURE is bound by GTF3 and is required for slow fiber-specific expression. GTF3 is expressed at its highest levels during fetal development in numerous tissues, and its expression in muscles is repressed after birth. Experiments in adult regenerating muscles (which re-express GTF3) transfected with GTF3 by using electroporation, as well as analysis of GTF3 mutant mice, support the role of GTF3 in regulating the slow-twitch muscle program.