Tyrosine phosphorylation is involved in every stage of neuronal development

Neuronal survival and differentiation are promoted by neurotrophins and the Trk family of RPTKs. NGF promotes the survival of neurons during a period of programmed cell death in embryonic and early postnatal developmental stages. It is a target-derived neurotrophic factor that modulates the functions of the innervating axon terminals as well as gene expression in the distant cell body (see Chap. 19).

NGF was discovered over 40 years ago. Its action on neuronal survival and differentiation has long been recognized. However, its signal-transduction mechanism had remained unknown until the identification of a 140-kDa protein, TrkA RPTK, as the NGFR in 1991 [13]. Since then, other members of the Trk family have been discovered as receptors for other neurotrophins. For example, brain-derived neurotrophic factor (BDNF) has the highest affinity toward TrkB, while neutrophin-3 (NT-3) is the preferred ligand for TrkC. A 75-kDa transmembrane protein, p75, also termed the low-affinity NGFR, has been identified as a neurotrophin receptor. All neurotrophins can bind to p75 with a low affinity. Although not a PTK itself, p75 can interact with other Trk proteins and may modify the ligand-binding affinity, dose responsiveness and PTK activity. For instance, it increases the affinity of TrkA for NGF while decreasing its response to NT-3. The tyrosine-kinase activity of TrkA is enhanced by p75. Transgenic mice that do not express p75 survive through adult life but with significant loss of certain sensory and sympathetic neurons, suggesting a significant role for p75 in neuronal survival in the peripheral nervous system.

NGF induces differentiation of rat pheo-chromocytoma PC12 cells into cells with many characteristics of mature sympathetic neurons (Fig. 25-9). This is a well-characterized model system that has been used extensively to study mechanisms of the action of NGF. Studies with PC12 cells show that NGF induces autophosphorylation of at least four different tyrosine residues on human TrkA. These are Y490 in the juxtamembrane region, Y674, Y675 in the catalytic domain and Y785 in the C-terminal tail. Each phosphotyrosine serves a different function in the NGF signaling pathway (Fig. 25-8). Y674 and Y675 are the first tyrosine residues phosphorylated and are important for subsequent activation of the intrinsic kinase activity. Phosphorylation of Y490 and Y785 creates SH2-binding sites for SHC and PLC-γ1, respectively. Recruitment of PI-3 kinase to TrkA possibly involves an adaptor molecule. These three signaling molecules are in turn activated by tyrosine phosphorylation by TrkA, eventually leading to the biological effects of NGF. Independent of any of the known signaling pathways used by TrkA, a novel protein, suc-associated neurotrophic factor-induced tyrosine-phosphorylated target (SNT), is tyrosine-phosphorylated upon NGF treatment of PC12 cells. SNT is specifically activated in response to differentiation but not in response to mitogenic factors. This suggests the existence of yet another unknown signaling pathway used by TrkA to induce differentiation of neurons.

Neurotrophins acting on the same Trk tyrosine kinase can induce either cellular proliferation or differentiation depending on the cell type, suggesting that cell context determines the response to Trk activation. This may partly be related to the duration of MAPK activation. Transient activation of the MAPK pathway is associated with mitogenic response while prolonged activation induces differentiation of PC12 cells. While many aspects of TrkA signaling have been discovered, it remains unclear how TrkA promotes survival and differentiation of neurons.

Axon guidance. In many cases, the growth cone at the leading end of an axon navigates a trajectory over a long distance before it finds its target. Various extracellular cues help the growth cone navigate through different terrains to reach its destination. Growth cones often travel in segments, such that the whole journey is subdivided into shorter stretches which the growth cone will travel one at a time, detecting cues along the way until it finds its final target. Furthermore, axons may stick together and form fascicles, which can serve as a “highway” for future traveling growth cones. Extracellular cues that influence axon guidance can be attractive or repulsive and diffusible or immobilized to the cell surface or the extracellular matrix (see Chaps. 7, 27 and 28).

One of the best studied model systems of axon guidance in vertebrates is the retinotectal system. Anterior and posterior retinal axons project to the posterior and anterior tectum, respectively. Similarly, the dorsal and ventral retinal axons innervate the ventral and dorsal tectum, respectively. This reversal of projection is important for the rectification of the upside-down and back-to-front retinal image. Using membrane stripe and growth cone collapse assays, Bonhoeffer and colleagues have identified a 25-kDa protein called repulsive axon guidance signal (RAGS) as an important axon guidance molecule in this system [14]. It is expressed most highly in the posterior tectum and repels the posterior retinal axons from this region.

The receptor for RAGS is the Eph RPTK, discovered in the human erythropoietin-producing hepatocellular carcinoma cell line [15]. The ligands for Eph family RPTK are either glycophosphatidylinositol (GPI)-anchored or transmembrane proteins (see Box 3-1). Their biological activity is lost if they are detached from the membrane. This implies that Eph-mediated axon guidance is limited to short-range contact-dependent activity. Accordingly, the Eph family of receptors can be divided into those interacting with GPI-linked ligands, including the epithelial cell kinase (Eck) subgroup, and transmembrane ligands, including the Eph-like kinase (Elk) subgroup. Activated, the Eck family binds PI-3-kinase, whereas the Elk family binds Src, Yes and p120-GTPase-activating protein (GAP). The Elk family ligands are also tyrosine phosphorylated upon binding their receptors, suggesting a bidirectional flow of information. However, the exact signal-transduction mechanism leading to the repulsion of growth cones is largely unknown. It is known that repulsion of growth cones from the signal requires a local depolymerization of F-actin, while steering of the growth cone toward the signal requires an accumulation of F-actin, leading to the recruitment of microtubules. There is evidence that small G proteins, Rho and Rac, may play a role in axon guidance by regulating actin polymerization (see Chaps. 8, 27 and 29).

The Eph receptor family may have a role in the fasciculation of axons of cortical neurons growing on astrocytes in culture. The interacting proteins in this case are the Eph receptor Rek7, expressed on the axons, and the Eph ligand AL-1, found on the astrocyte surface. It is postulated that the axons clump together in a bundle because of the repulsive interaction with the astrocytes.

Another RPTK found in Drosophila, derailed, also plays a role in axon guidance as a small subset of embryonic interneurons fail to find their targets in the derailed mutant. Basic fibroblast growth factor (bFGF) present in the optic tract may activate FGFR in the growth cone of retinal ganglion cells. Addition of bFGF to the developing retinotectal tract impairs the correct innervation of tectum from the retinal ganglion cells. Expression of dominant negative FGFR in retinal ganglion cells also prevents axons from entering their target. NRPTKs appear to have a role in axon guidance. For instance, although deletion of the Abl gene in Drosophila does not yield gross morphological defects, a double mutant of Abl and fasciclin I causes major defects in axon guidance and fasciculation. Several RPTPs are expressed predominantly in the growth cones and axons in Drosophila and are likely to regulate the fasciculation of axons. For instance, in Drosophila mutants that lack Drosophila leukocyte antigen-related proteins (DLAR) or Drosophila protein-tyrosine-phosphate phosphohydrolase (DPTP), motor neurons fail to defasciculate and consequently bypass their targets.

Synapse formation. Not only is tyrosine phosphorylation involved in guiding axons to their target, once the growth cone reaches its target, tyrosine phosphorylation also plays a role in the formation of a proper synaptic structure. For instance, the acetylcholine receptor (AChR) is concentrated at the postsynaptic membrane of the neuromuscular junction at a density of 10,000 receptors/μm², which is about three orders of magnitude higher than that of the extrasynaptic region. The high concentration of AChR at the neuromuscular junction allows rapid, reliable and efficient synaptic transmission. In fact, transmission across a synapse is so rapid that the idea of a chemical synapse was not readily accepted at the turn of the century. The strategic positioning of the AChR right on the crest of junctional folds at the neuromuscular synapse reduces the distance (30 to 50 nm) for the neurotransmitter to travel before reaching its receptor. Furthermore, the high concentration of AChR at the neuromuscular junction makes it more likely that a depolarization is higher than the threshold to trigger muscular contraction. The significance of this is demonstrated by the pathological condition myasthenia gravis, in which there is muscle weakness due to a reduction of AChR present at the neuromuscular junction. Two mechanisms contribute to the enrichment of AChR at the neuromuscular junction: (i) clustering of pre-existing, diffusely distributed surface AChR and (ii) local synthesis of the receptor by subsynaptic nuclei. Both mechanisms are mediated by tyrosine phosphorylation, as discussed below.

Agrin induces clustering of the AChR at the neuromuscular junction by activating the muscle-specific kinase (MuSK) RPTK. The clustering of pre-existing AChR at the postsynaptic membrane is triggered by a neuronally derived extracellular matrix protein discovered by McMahan's [16] laboratory in the 1980s called agrin, from the Greek word ageirein meaning “to assemble.” Since then, the agrin hypothesis [16] has been supported by a number of experiments from various laboratories and confirmed by the agrin knockout mutant mouse [17]. These mice are stillborn and never move, probably due to lack of neuronal transmission across the neuromuscular junction. Microscopic analysis reveals the absence of AChR clusters at the neuromuscular junction.

The molar ratio of agrin to AChR at the neuromuscular junction is 1:50 to 100, suggesting that agrin induces AChR clustering through some intracellular signal-transduction mechanism rather than via any structural constraints. Tyrosine phosphorylation of the AChR may play a role in its clustering at the neuromuscular junction. First, agrin induces tyrosine phosphorylation of the AChR prior to clustering of the receptor in muscle [18]. All agents which induce AChR clustering in muscle cells in culture also induce tyrosine phosphorylation of the AChR. These include agrin, rapsyn, electrical fields and latex beads. However, PTK inhibitors which inhibit AChR tyrosine phosphorylation also inhibit AChR clustering. Although tyrosine phosphorylation appears to be important to the action of agrin on muscle, a causal relationship between AChR tyrosine phosphorylation and clustering has not been established.

The putative receptor for agrin is an RPTK known as MuSK [19]. The extracellular domain of MuSK resembles that of the ROR family RPTKs, while the kinase domain is similar to that of the Trk neurotrophic receptor (Fig. 25-6). MuSK is expressed at low concentrations in proliferating myoblasts and is induced upon differentiation and fusion. It is downregulated dramatically in mature muscle except at the neuromuscular junction. These properties are consistent with the role of MuSK in muscle development and the function of neuromuscular junction.

MuSK knockout mice show disruption of AChR clusters at the neuromuscular junction [19] similar to that seen with the agrin knockout mice [17]. Furthermore, muscle cells in culture from these mutant mice do not respond to agrin. Expression of MuSK in immature myotubes stimulates agrin binding. These data strongly indicate that MuSK is necessary for the response to agrin. It is postulated that MuSK is part of the agrin receptor, and a myotube-associated specificity component (MASC) is required to form the agrin receptor. This is similar to the CNTF receptor, in which more than one component is required for the assembly of the intact receptor.

Synapses in the central nervous system share a similar structural architecture with the neuromuscular junction. Proteins important for the organization of the neuromuscular junction are also found in the central nervous system. For instance, agrin is expressed in the brain during periods of intense synaptogenesis. Therefore, it is possible that similar mechanisms are employed for clustering neurotransmitter receptors in the central nervous system.

Acetylcholine receptor-inducing activity (ARIA) increases the expression of AChR subunits by subsynaptic nuclei through activation of the erbB RPTKs. In addition to clustering of pre-existing AChRs, neuronal innervation increases the transcription of AChR subunits in nuclei near the synapse despite the downregulation of AChR synthesis in extrasynaptic regions. This transcriptional regulation results in a local concentration of AChR subunit mRNAs in the subsynaptic cytoplasm. Innervation also switches the AChR subunit composition from α₂βγδ to α₂βϵδ since transcription of the ϵ subunit is most sensitive to neuronal input. The switchover from γ to ϵ in the AChR complex increases the conductance of the channel but decreases the duration of its opening.

Transcriptional control of the AChR is regulated by ARIA, which was first identified in the late 1970s by Fischbach's laboratory as the trophic factor for the expression of AChR in muscle present in chick brain and spinal cord [20]. It is a member of the neuregulin family, which also includes neu differentiation factor, heregulin and glial growth factor. The precursor of ARIA is a 67-kDa transmembrane glycoprotein which is proteolysed to the 42-kDa mature ARIA before its release from the motor neuron. Similar to agrin, the released ARIA is deposited on the basal lamina in the synaptic cleft. The interaction is so stable that even after denervation the ARIA deposited on the basal lamina can be recognized by regenerated muscle fiber as the site for active synthesis of AChR, even in the absence of neuronal innervation. Besides its action on the AChR, ARIA can increase the synthesis of voltage-gated Na⁺ channels, which are concentrated at the depths of junctional folds of the neuromuscular junction at a concentration of 5,000/μm². The synthesis of two other synaptic proteins, AChE and rapsyn, is unaffected by ARIA.

The effect of ARIA on gene expression is dependent on tyrosine phosphorylation, as evidenced by its inhibition by PTK inhibitors and potentiation by a PTP inhibitor. In fact, ARIA induces tyrosine phosphorylation of 185-kDa proteins in the muscle [21]. The 185-kDa proteins turn out to be the components of the ARIA receptor, which are members of the EGFR tyrosine kinase family. This family contains four known members: erbB1 (EGFR), erbB2, erbB3 and erbB4. The last three members are found in the muscle and can respond to ARIA. However, erbB3 does not possess intrinsic tyrosine kinase activity, so it has to couple with another erbB protein to respond to ARIA. erbB3 and erbB4 are present at the neuromuscular junction and can interact directly with ARIA [22]. Like many other RPTK pathways, activation of AChR gene expression depends on the MAPK signaling pathway.

Tyrosine phosphorylation plays an important role in synaptic transmission

A number of synaptic molecules, such as neurotransmitter receptors, voltage-gated ion channels, enzymes and proteins involved in neurotransmitter release, are tyrosine-phosphorylated. The importance of tyrosine phosphorylation on animal behavior has been demonstrated using genetic techniques. For example, mutant mice missing PTKs show diminished long-term potentiation (LTP) and/or learning and memory [23] (see Chap. 50).

Tyrosine phosphorylation of neurotransmitter receptors, including the AChR, N-methyl-d-aspartate (NMDA) receptor and GABA_AR, plays a pivotal role in modulating synaptic efficacy and plasticity. Several neurotransmitter receptors and voltage-gated ion channels in both the peripheral and central nervous systems are tyrosine-phosphorylated (Table 25-2). Furthermore, tyrosine phosphorylation of these surface signal-transducing molecules significantly modulates their electrophysiological properties, producing a prominent effect on neuronal signal propagation.

Table 25-2

Regulation of Ion Channel Function by Tyrosine Phosphorylation.

Acetylcholine receptors. Due to its enrichment and easy access in the Torpedo electric organ, the AChR is one of the best studied neurotransmitter receptors (see Chap. 11). It sits on the post-synaptic side of the mammalian neuromuscular junction. Upon binding acetylcholine, this ligand-gated ion channel depolarizes the sarcolemma and triggers muscle contraction. The AChR is composed of five subunits, α₂βγδ (embryonic) or α₂βϵδ (adult). Each subunit has four transmembrane domains, with both the N- and C-termini in the extracellular space. Between the third and fourth transmembrane domains is a large cytoplasmic region in which there are a number of consensus phosphorylation sites (Fig. 25-12). A single conserved tyrosine residue in the cytoplasmic loop of each of the β, γ and δ subunits is phosphorylated by PTKs in the postsynaptic membrane [24]. Tyrosine phosphorylation, like serine phosphorylation, of the AChR regulates its rate of desensitization [25].

Figure 25-12

Transmembrane topology of three ligand-gated ion channel subunits and their potential domains for tyrosine phosphorylation. Both the acetylcholine receptor (AChR) and GABA_A receptor (GABA_AR) subunits have four transmembrane domains with both the N- and (more...)

The AChR is highly tyrosine-phosphorylated in intact electric organ and muscle, in contrast to its low tyrosine phosphorylation in cultured rat myotubes, <0.001 mol phosphate/mol subunit. This raises the possibility that innervation increases the tyrosine phosphorylation of the AChR. In fact, tyrosine phosphorylation of the AChR increases as muscle is innervated during development or in culture [18]. However, denervation results in loss of phosphotyrosine on the AChR. In light of the similar subunit and site specificity of agrin- and neuron-induced tyrosine phosphorylation of the AChR, agrin probably mediates the regulation of AChR tyrosine phosphorylation by the motor neuron.

The physiological PTK responsible for phosphorylating the AChR is unknown. Since MuSK RPTK is the putative agrin receptor, it is a candidate kinase for phosphorylating the AChR. Indeed, coexpression of MuSK and AChR together with rapsyn in quail fibroblasts induces tyrosine phosphorylation of the AChR. The AChR also has been shown to bind to Fyn, Fyk and Src NRPTKs and can be phosphorylated by these enzymes. Although agrin regulates tyrosine phosphorylation of the AChR, the exact physiological role of this phosphorylation remains to be elucidated.

N-Methyl-d-aspartate receptors. Glutamate is the major excitatory neurotransmitter in the central nervous system (see Chap. 15). Its receptors can be divided into three types: AMPA/ kainate, NMDA and metabotropic receptors. The first two types are ligand-gated ion channels, while the metabotropic receptors are G protein-linked. The AMPA/kainate receptors mediate fast excitatory synaptic transmission, while the NMDA receptors play a critical role in the induction of synaptic plasticity and excitotoxicity. The NMDA receptors are composed of two different types of subunit: NR1 and NR2.

The activity of NMDA receptors is enhanced by tyrosine phosphorylation. In spinal cord dorsal horn neurons, PTK inhibitors diminish NMDA receptor activity. However, intracellular perfusion of Src or a PTP inhibitor potentiates the NMDA current. Although it is still unknown whether direct tyrosine phosphorylation of the NMDA receptor regulates its activity, accumulating evidence has suggested that tyrosine phosphorylation of the NR2 subunits regulates NMDA function. For instance, the NR2A and 2B subunits are tyrosine-phosphorylated in vivo [4,26]. Heterologous cells expressing NMDA receptors composed of the NR1 and NR2A subunits exhibited increased NMDA-induced current in the presence of v-Src, while NMDA receptors consisting of NR1 with other NR2 subunits are not potentiated by v-Src. Furthermore, NMDA receptor conductance in excised patches can be potentiated by tyrosine phosphorylation, suggesting that the NMDA receptor itself or some tightly associated tyrosine-phosphorylated proteins are mediating the increase. In fact, Src has been suggested to interact with the NMDA receptor in vivo via its unique N-terminal domain [27]. Another NRPTK, Fyn, phosphorylates the NR2 subunits in vitro. Identification of tyrosine-phosphorylation sites and analysis of their effect on the electrophysiology of the NMDA receptor will be the next step in the elucidation of the direct involvement of NMDA receptors in regulation by tyrosine phosphorylation. The NR2B subunit from the dentate gyrus has increased tyrosine phosphorylation during the maintenance phase of LTP, suggesting a role for tyrosine phosphorylation in synaptic plasticity. However, the NMDA receptor also can be inhibited indirectly by PDGFR in cultured hippocampal neurons. The PDGFR activates PLC-γ1 to produce IP₃, a calcium-elevating agent. The increase in intracellular calcium in turn inhibits NMDA receptor activity. Therefore, tyrosine phosphorylation directly or indirectly influences the NMDA receptors in both the positive and negative directions.

GABA receptors. GABA is one of the major inhibitory neurotransmitters in the central nervous system (see Chap. 16). The subunits from the GABA_A receptor (GABA_AR) include α, β, γ and δ. The GABA_AR complex is believed to be pentameric and similar in structure to the AChR, with each subunit spanning the membrane four times. Similar to the AChR, the intracellular region between the third and fourth transmembrane domains contains a number of consensus protein-phosphorylation sites (Fig. 25-12). Coexpression of GABA_AR subunits α1, β1 and γ2L with v-Src induces tyrosine phosphorylation on both the β and γ subunits. GABA-mediated whole-cell current is also potentiated [28]. Mutation of a tyrosine residue on the γ subunit abolished the potentiating effect of v-Src, suggesting that direct tyrosine phosphorylation of GABA_AR increases its activity.

Activation of the PDGFR inhibits GABA_AR in hippocampal neurons. Similar to its effect on the NMDA receptor, the PDGFR can activate PLC-γ1, generate IP₃ and increase intracellular calcium, which inhibits the GABA_AR. GABAergic transmission is inhibited by NT-3, the ligand for the RPTK TrkC. Therefore, direct tyrosine phosphorylation of the GABA_AR potentiates its activity, while release of intracellular calcium from RPTK activation appears to inhibit its function.

Voltage-gated ion channels. Besides the neurotransmitter-gated ion channels, some voltage-gated ion channels are targets of PTKs. These include a delayed rectifier potassium channel (RAK), hKv1.5 and a voltage-gated cation channel in Aplysia bag cell neurons. Tyrosine phosphorylation inhibits these voltage-gated ion channels.

Voltage-gated potassium channels maintain resting potential, regulate excitability and repolarize the membrane after an action potential. Modulation of potassium channels by tyrosine phosphorylation will influence these membrane properties. Delayed rectifier potassium channels (RAK) coexpressed with the m1 muscarinic AChR in Xenopus oocytes are inhibited by carbachol, a nonmetabolizable analog of acetylcholine [29]. Activation of the m1 muscarinic receptor releases diacylglycerol and IP₃ from the hydrolysis of PIP₂. Diacylglycerol activates PKC and IP₃ releases calcium from intracellular stores. Possibly by their action on a PYK2-related tyrosine kinase, calcium and PKC eventually lead to tyrosine phosphorylation of RAK on Y132 in the cytoplasmic domain near the N-terminus, resulting in an impaired RAK response. Similarly, the human potassium channel hKv1.5 is tyrosine-phosphorylated and suppressed when coexpressed with v-Src in transfected cells [30]. In vivo association between hKv1.5 and Src is mediated by proline-rich sequences on the potassium channel and Src SH3 domain.

The voltage-gated cation channel displays two modes of activity patterns in Aplysia bag cell neurons. In the bursting mode, the channel rapidly switches between the open and closed states for a brief period of time, followed by a longer period of inactivity. In the nonbursting mode, the channel opens and closes briefly. Treatment of the inside-out patch with a purified PTP switches the channel from the bursting to the nonbursting mode, thus increasing the channel opening time [31]. Apparently, an endogenous PTP can be activated by PKA to induce a similar mode switching in the cationic channel. However, it is unknown whether these effects are mediated by direct tyrosine dephosphorylation of the channel or some closely associated protein.

Synaptic transmission. Neurotrophins are known for their long-term effects on survival and differentiation of neuronal stem cells. However, accumulating evidence has suggested that neurotrophins also can have an acute effect on synaptic transmission in both the peripheral and central nervous systems. At the frog neuromuscular junction, BDNF and NT-3 can potentiate both the evoked synaptic transmission and spontaneous miniature synaptic response [32]. Similarly, at the hippocampal Schaffer collateral-CA1 synapse, these two neurotrophins, but not NGF, increase synaptic transmission by 200 to 300% [33].

Long-term potentiation (LTP) also is affected by neurotrophins. Mutant mice lacking BDNF show diminished LTP. Treatment with TrkB-IgG fusion protein, which adsorbs endogenous BDNF, also reduces LTP induced in the hippocampus [34]. However, addition of exogenous BDNF promotes the induction of LTP. Although both neurotrophins and LTP can potentiate synaptic transmission at the same synapse, they use at least partially distinct mechanisms since (i) unlike their effect on LTP, NMDA-receptor antagonists do not block the action of neurotrophins [33] and (ii) LTP does not completely occlude neurotrophin-mediated potentiation and vice versa [33].

Exactly how the neurotrophins enhance synaptic transmission is still unclear, but both pre- and postsynaptic mechanisms appear to be involved. At the frog neuromuscular junction and the Schaffer collateral-CA1 synapse, neurotrophin potentiation of synaptic transmission appears to increase neurotransmitter release at the presynaptic nerve terminal [32,33]. Since potassium channels are inhibited by tyrosine phosphorylation (see above, under Voltage-gated ion channel), it is possible that the action of neurotrophins can be mediated by tyrosine phosphorylation of these channels, which results in prolonged depolarization and, consequently, enhanced neurotransmitter release from the presynaptic terminal. Another protein that has the potential to alter neurotransmitter release is synaptophysin, a neurotransmitter vesicle protein that is tyrosine-phosphorylated.

A postsynaptic mechanism has been suggested at the Schaffer collateral-CA1 synapse. The high-affinity BDNF receptor TrkB has been found in the postsynaptic density. Furthermore, injection of a Trk tyrosine kinase inhibitor, K252a, into the postsynaptic neuron inhibits the facilitatory response of BDNF. Finally, the NT-3-induced increase in the frequency of neuronal impulse activity in cultured cortical neurons has been attributed to a third mechanism, such as reduction of the inhibitory GABA_AR activity.

A number of studies have shown that neuronal activity can increase neurotrophin expression. This strengthens the notion that the action of neurotrophins is closely linked to neuronal activity. For instance, induction of LTP at the perforant path-dentate granule cell synapses increases the expression of NGF and BDNF mRNA. Furthermore, LTP induction at the Schaffer collateral-CA1 synapse results in an increased expression of BDNF and NT-3.