Signaling from Neural Impulses to Genes

Abstract

Nerve impulses regulate expression of genes that control receptors, channels, enzymes, and structural proteins. This activity-dependent feedback allows adaptation to changing requirements and environmental conditions. The signal transduction mechanisms carrying information from the cell membrane to the nucleus are becoming well characterized, but a more dynamic view of intracellular signaling is emerging to explain cellular responses to specific patterns of neural impulses. This review analyzes this interface between electrophysiology and molecular cell biology to examine the signals, substrates, and processes that enable the nervous system to regulate its structure and function as a consequence of its own operation.

Prior to synaptogenesis, spontaneous impulse activity affects intracellular signaling systems that control cytoskeletal proteins and regulates expression of cell adhesion molecules. These responses influence migration of postmitotic neurons (1), outgrowth of neurites (2), fasciculation of axons, and axonal-glial associations (3). Gene regulation is often sensitive to the precise pattern or frequency of neural stimulation. Expression of high voltage activated (HVA) and low voltage activated (LVA) calcium channels in mouse dorsal root ganglion (DRG) neurons are regulated differentially by different patterns of stimulation (4), and maturation of potassium channels in developing frog spinal neurons is regulated by precise frequencies of calcium spikes (5). After synaptogenesis, synaptic strength and remodeling of synapses at the neuromuscular junction (6) and visual cortex (7) are critically dependent on impulse activity at appropriate times in development (8). Gene expression and new protein synthesis are essential for long-term change in synaptic strength in hippocampal long-term potentiation (LTP) of adult brain (9). To influence these varied processes, neural impulses must access many different signaling systems to activate a diverse array of specific genes.

Activity-dependent Gene Expression

The immediate early (IE) gene c-fos has become an important experimental model for studics of stimulus-transcription coupling because it is activated within minutes by many different stimuli (10). Moreover, the Fos protein coded by this gene translocates back into the nucleus to form homo- and heterodimers that bind to AP-1 recognition sites in the promoter region of other genes (11). Thus, c-fos codes a transcription factor that can regulate long-term structural and functional properties of neurons in response to relatively brief physiological stimulation (12). By inducing adaptive responses in neurons, c-fos participates in responses to brain injury, sensory stimulation, activation of neurotransmitter receptors, stress, circadian rhythms, and long-term changes in synaptic strength (13).

A large variety of IE genes are activated by impulse activity. Electrical stimulation of hippocampal neurons induces transcription of c-fos, c-jun, junB, zif268 (krox-24), krox-20, and others, but it is significant that different IE genes are activated by different patterns of stimulation (13–15). Determining which intracellular signaling systems convey information from neural impulses to the nucleus is a complex task because of the many routes through which action potentials influence intracellular signaling reactions (Fig. 1). Pharmacological studies show that transcription of genes is stimulated by several different intracellular signaling pathways, including those involving protein kinase C (PKC), mitogen-activated protein kinase (MAPK), Calcium/calmodulin protein kinase (CaMK), and protein kinase A (PKA). How the multiple second messenger pathways operate as a system to control expression of c-fos in response to specific modes of pharmacological stimulation and different patterns of neural impulse activity remains a difficult and important problem. It is necessary to begin at the neuronal membrane and examine how action potentials can influence biochemical signaling reactions within the neuron. Next, the intnnuclear events that regulate gene transcription will be considered. Some key molecules in intracellular signaling from action potentials are presented in Table 1. The brief survey that follows considers a range of mechanisms transducing action potentials into intracellular signaling reactions, but only a limited subset of these would be operational in an individual cell type.

Fig. 1.

Action potentials interact with a broad array of signaling molecules, through direct electrical effects, calcium and noncalcium ion flux, and secretion of signaling molecules. Multiple points of interaction between signaling pathways are evident.

Table 1.

Selected Key Molecules in lntracellular Signaling from Action Potentials

Symbol	Molecule	Description	Actions	Reference
	Effector Enzymes
AC	Adenylate cyclase	Stimulated by G-linked receptors and calcium	Generates cAMP	16, 17
PLC	Phospholipase C	Stimulated by neurotransmitter receptors	Converts PIP2 to DAG, IP3,binds RTK	18
NOS	Nitric oxide synthase	Stimulated by calcium	Generates NO from arginine	19
PLA2	Phospholipase A	Stimulated by calcium, PKC, G-protein, Tyr K	Releases arachidonic acid	20
	Second Messengers
cAMP	3',5'-cAMP	Cytoplasmic second messenger	Activates PKA	16, 17
DAG	1,2-diacylglycerol	Membrane associated messenger	Activates PKC	21
IP3	lnositol 1,4,5-trisphosphate	Cytoplasmic second messenger	Stimulates Ca2+ release from ser	22, 23
NO	Nitric oxide	Non-ploar gas, transcellular diffusion	Activates GTPase	24
Ca2+	Calcium	Cytoplasmic and nuclear messenger	Bound by calmodulin	25–27
cADP ribose	Generated from NOS	Regulates release of calcium from internal stores		28
	Protein Kinases
CaM KII	Calcium-calmodulin kinase II	Activated by calcium/calmodulin	Multiple substrates, nucleus, and cytoplasm	29–31
CaM KIV	Calcium-calmodulin kinase IV	Activated by calcium/calmodulin	Phosphorylates Ser 133 of CREB	32
PKC	Protein kinase C	Serine/threonine protein kinases	Activated by calcium and DAG	33, 34
PKA	Protein kinase A	Activated by cAMP	Phosphorylates CREB	35
RTK	Receptor tyrosine kinase	Activated by ligand binding to cell-surface receptors	Intrinsic tyrosine kinase activity	36, 37
Raf		Serine/threonine-specific . protein kinase	Binds and phosphorylates MEK	38
MEK	MAP kinase kinase	Tyrosine/threonine specific protein kinase	Phosphorylates MAPK	39
MAPK	Mitogen-activated protein kinase	Serine/threonine-specific protein kinase	Phosphorylates SRF	40
	Protein Phosphatases
Calcineurin	Protein phosphatase-2B	Calcium/calmodulin-dependent protein phosphatase	Regulates L-type. Calcium channels and other PKA substrates	41
PP1	Protein phosphatase 1	Serine/threonine phosphatase, inhibited by 11 & 2	Dephosphorylates CREB Ser-133	42
	G Protein
Ras	GTP binding protein	Activated by binding GTP, activates MAPK cascade	Binds N-terminal of Raf, relays signals from tyrosine kinase to serine/threonine kinase	43, 44
	(GEF)	Guanine-nucleotide exchange factor
Sos 1	Son of sevenless	Converts Ras to the active GTP-bound form	Proline sequences bind SH3 domains of GRB2 to trigger MAPK cascade	37, 45
	Adaptor Proteins
GRB2	Growth factor receptor-bound protein 2	Associates with phosphorylated growth factors via one SH2 domain to phosphotyrosine of Shc or growth factor, and binds Sos via 2 SH3 domains	Links GRB2-Sos in a complex with phosphotyrosine residues of growth factor receptors, activates Sos	44, 46
Shc	SH2 domain-containing adaptor protein	Binds phosphotyrosine of growth factors and is, itself, inducibly phosphorylated on tyrosine	Links tyrosine kinase receptors to Ras by complexing with GRB2 and growth factors	47
	Transcription Factor
Elk-1	p62TCF	Ets related protein	Binds as a ternary complex with SRF	48
SRF	Serum response factor	Activated by growth factor and PKC pathways	Binds SRE (CCATATTAGG)	49, 50
CREB	cAMP response element binding protein	Activated by calcium and cAMP pathways	Binds Ca/CRE (GTGACGTA)	51
CBP	CREB binding protein	Co-activator binds to phosphorylated CREB	Enhances transcription	52
AP-1	Activator protein 1	Jun/Fos heterodimer or Jun homodimer	Binds FAP in c-fos gene (TGCGTCA)	11, 53
SIF	sis-Inducible element factor	Activated by growth factors	Binds SIE in c-fos gene (TTCCGTCAA)	54

PIP₂, phosphatidylinositol4,5-bisphosphate.

Action Potentials and Intracellular Signaling

Direct Electrical Effects

Direct electrical effects constitute a minor form of signaling from action potentials, but electrical fields can induce voltage-dependent conformational changes in enzymes or their membrane-associated substrates, and cause lateral diffusion of receptors or signaling molecules in the membrane to activate intracellular signaling cascades through protein-protein interactions (Fig. 1). Electrical fields have been shown to induce aggregation of acetylcholine receptors at synaptic junctions (55), influence neurite outgrowth (56, 57), modulate activity of CaM KII (58), PKA (59) and adenylyl cyclase (60), and induce calcium influx by lateral diffusion of membrane proteins in neural crest cells, which, in turn, activates PKC (61). Lateral diffusion of membrane proteins in an electric field stimulates tyrosine phosphorylation in muscle cells to induce acetylcholine receptor clustering (62).

Ion Flux

Influx of calcium is the principal means of coupling action potentials to intracellular signaling pathways, but other ion fluxes can be involved. These ions act indirectly on calcium ion fluxes or operate through undefined intracellular signaling reactions. Sodium, chloride, and potassium ions can affect cytoplasmic calcium levels through interactions that alter the membrane potential or regulate calcium-permeable channels. The sodium/calcium exchanger reverses direction to admit calcium ions into the cell when the intracellular concentration of sodium ions is elevated by prolonged or intense action potential activity (63). The induction of long-term depression in the cerebellum (64) and activity-dependent internalization of sodium channels (65) are driven by sodium influx. Oligodendrocytc progenitor cell proliferation and differentiation are largely genetically regulated, yet these processes are inhibited by a sodium-dependent block of the delayed rectifier K⁺ channel, which can be induced by glutamate activation of the AMPA receptor (66).

Membrane Receptors and Channels

Activity-dependent secretion of extracellular signaling molecules, including peptides, neurotransmitters, neurotrophins, and retrograde messengers, opens another dimension for coupling action potentials to intracellular signaling reactions. These extracellular messengers bind receptors to activate calcium-permeable neurotransmitter channels, such as the acetylcholine (67), AMPA (68), or N-methyl-d-aspartate (NMDA) receptors (69), or to stimulate receptors that are independent of calcium. Neuronal-glial signaling (70) is another important mode of activity-dependent modification. Proliferation of oligodendrocytc precursor cells in developing rat optic nerve depends on clectrical activity in axons to induce secretion of a growth factor (71).

Brain-derived neurotrophic factor (BDNF) is induced as an IE gene, i.e., without the requirement for protein synthesis, in hippocampal neurons following NMDA receptor stimulation (72), and expression of BDNF induced by activation of voltage-sensitive calcium channels (VSCC) and NMDA channels can act as an endogenously produced neurotrophic factor for cortical neurons in culture (73). The induction of LTP in the hippocampus increases BDNF and NGF mRNA but decreases NT-3 mRNA (74), and the activity-dependent formation of ocular dominance columns in kitten visual cortex is blocked by infusion of BDNF or NT-4/5 (75).

Neural impulses also regulate expression of cell receptors, including cell adhesion molecules (3), neurotrophin receptors (76), neurotnnsmitter receptors, and ion channels (4, 65). These channels and receptors are some of the major signal transducing molcculcs in neuronal membrane.

Calcium Signaling and Activation of Protein Kinases

Calcium enters electrically active neurons through various voltage-gated calcium channels that have distinct and widely ranging electrophysiological properties (77). The increased concentration of cytoplasmic calcium can, in turn, regulate receptors on the smooth endoplasmic membrane to control release of calcium from intracellular stores (22, 23). Calcium ions interact with many signaling cascades to control gene expression (25). Calcium exerts effects primarily through binding to specific calcium-binding proteins. The calcium-binding protein calmodulin is a focal point for cross-talk between numerous signaling pathways and for intracellular calcium concentration. Calmodulin binds calcium ions and modulates the activity of protein kinases, phosphatases, and adenylyl and guanylyl cyclases as a function of the intracellular calcium concentration (26). Voltage-gated calcium influx triggers c-fos transcription in PC12 cells, and this response is blocked by inhibitors of calmodulin (78).

The multifunctional protein kinase, Ca⁺⁺/calmodulin-dependent kinase II (CaM KII), is a cytosolic enzyme activated by stimuli that elevate [Ca⁺⁺]_i, CaM kinase II is an oligomeric protein containing 10–12 subunits of about 55 kDa in size (29). CaM kinase II has been implicated in synaptic plasticity (29, 30), and CaM kinase II and IV are present in the nucleus where they are involved in transcriptional activation of c-fos in response to membrane depolarization (78–80).

Calcium can influence cAMP levels by activating CaM-dependent adenylyl cyclases (16, 17). cAMP-dependent protein kinase (PKA) is composed of two subunits, catalytic and regulatory, which dissociate to activate the enzyme upon binding cAMP. The gill withdrawal reflex of Aplysia, a simple model of associative learning, requires a Ca⁺⁺/CaM-dependent adenylyl cyclase stimulated by neurotransmitter activation of a G protein (81). The catalytic activity of PKA phosphorylates and inhibits a potassium channel, but long-term changes in synaptic strength are dependent on gene expression (82, 83). Learning and memory in Drosoplrila (84), and LTP in the mossy fiber synapses of the hippocampus (85) are de-pendent on Ca⁺⁺/CaM-activated adenylyl cyclase, activated by neural impulses. Learning and memory in Drosophila are disrupted either by mutations of the rut gene, which causes deficient Ca⁺⁺/CaM-activated adenylyl cyclase activity, or the dunce gene, which causes deficient cyclic nucleotide phosphodiesterase activity (86). Numerous genes, including the somatostatin gene (87, 88) and c-fos gene are activated by CAMP (89, 90).

Neural stimulation can lead to activation of PKC. At least nine isozymes of this serine/threonine kinase are known, and they are differentially activated by calcium, phosphatidyl serine, and 1,2-diacylglycerol (DAG) (33). The gamma isoform is specific to neural tissue, and a rise in intracellular calcium causes the catalytically inactive PKC in the cytosol to bind to the plasma membrane, where it can be activated by DAG. The second messenger DAG is generated by the catalytic activity of the plasma membrane enzyme phospholipase C, which cleaves PIP₂, to generate DAG and the cytosolic second messenger IP₃. At least four types of IP₃, receptors, localized to the endoplasmic reticulum and the nuclear membrane, release calcium in response to IP₃, (22, 23). The phospholipase C pathway is activated by muscarinic, adrenergic, histaminergic, serotonergjc, and glutamatergic stimulation (21).

Stimulation of PKC induces c-fos transcription (49, 91, 92), and depletion of PKC activity (93) or The PKC inhibitor 2-aminopurine blocks c-fos induction in 3T3 cells (94). PKC is involved in regulation of cell growth through phosphorylation of proteins controlling the translocation of transcription factors, such as NF-κB, from the cytoplasm to the nucleus (95). In cerebellar granule cells, synaptic activation of glutamatergic synapses activates this transcription factor by increasing intracellular calcium through NMDA receptors at early stages of development and through non-NMDA receptors at later stages (96).

Interactions between Tyrosine Kinases and Calcium Signaling

Growth factors and cell adhesion molecules have a powerful influence on neuronal structure, function, and survival during development and postnatal life. Action potentials influence this pathway by: 1) stimulating the secretion of extracellular signaling molecules that activate these receptors, 2) modulating the expression of receptors for these molecules, 3) interacting with downstream elements in the signaling cascades. Transcription of c-fos is stimulated by growth factors and tyrosine kinase-dependent signaling pathways (97–99).

Neurotrophins activate Src and Ras proteins that stimulate the serine/threonine kinase Raf. Raf kinases are signal-integrating enzymes that can switch tyrosine kinase signaling to serine/threonine phosphorylation and connect growth factor receptors with transcription factors (38). Receptors that regulate Raf kinase include members of the serpentine family containing seven transmembrane helices, transmembrane tyrosine kinase receptors, and cytokine receptors that regulate intracellular tyrosine kinases. These receptors stimulate Raf kinases through a process that involves the small GTP-binding protein Ras. Ras is connected to the activated receptors by cytosolic adapter proteins, such as Grb2, which recruit the guanine nucleotide exchange factor Sos, to increase levels of Ras-GTP (Table 1). The SH2 domain in Grb2 binds to a specific phosphotyrosine residue in the receptor tyrosine kinase and activates Sos by binding to it through two SH3 domains. In its active form, Sos functions as a guananine nucleotide exchange factor to convert Ras to the active GTP bound form. This then triggers a protein kinase cascade leading to changes in gene expression. The only known substrate for Raf is a serine/threonine kinase MAP/ERK kinase (MEK). This stimulates another serine/threonine kinase, mitogen-activated protein kinase (MAPK or ERK) to build an amplification step in the cytoplasmic kinase cascade to phosphorylate transcription factors, cytoskeletal elements, and other substrates (39).

Neural impulse activity interacts with the intracellular cascade from growth factor receptors by increasing the concentration of intracellular calcium (Fig. 2) (100, 101). Recent research shows that calcium influx through L-type VSCC in PC12 neurons leads to tyrosine phosphorylation of the adapter protein Shc and its association with the adapter protein Grb2, which is bound to the guanine nucleotide exchange factor Sos1. In response to calcium influx, Shc, Grb2, and Sosl associate with epidermal growth factor receptor (EGFR), inducing tyrosine phosphorylation of the EGFR and activating the MAPK signaling pathway (47). Thus, action potentials can influence biochemical mechanisms controlled by growth factor receptor signal transduction pathways.

Fig. 2.

The immediate early gene c-fos is an important experimental model for studies of stimulus-transcription coupling. Action potentials stimulate membrane receptors and ion channels to activate second messenger molecules. These stimulate protein kinases to modify activity of DNA binding proteins that regulate gene transcription. Calcium ions and cAMP activate c-fos transcription primarily by phosphorylating the transcription factor CREB, which binds to the Ca/CRE. Growth factors and PKC activate c-fos transcription primarily through the MAP kinase cascade, which phosphorylates Elk-1 and SRF, which bind to the SRE. The FAP site provides negative feedback regulation of c-fos transcription by binding of homo- and heterodimers of the Fos protein. The SIE site is activated by growth factor stimulation. Multiple points of convergence and interaction between these basic pathways are known. Calcium ions can stimulate growth factor receptors in some cells by stimulating the GTP-binding protein Ras, and all four regulatory elements interact through DNA binding proteins that form an interdependent transcriptional complex. After transcription is initiated, elongation of c-fos transcripts can be regulated by both calcium and cAMP-dependent mechanisms. See Addendum.

Regulation of c-fos Transcription in Neurons

The promoter region of the c-fos gene contains four regulatory sequences that bind different DNA binding proteins (Fig. 2). Stimulation that activates the MAP kinase pathway phosphorylates the transcription factor, serum response factor (SRF) (49), and the ternary complex factor Elk-1. Elk-1 binds together with SRF to the serum response element (SRE) in the promoter region of the c-fos gene to increase transcriptional activity. As discussed above, stimulation of MAPK occurs in response to activity-dependent calcium influx through a mechanism involving activation of the small guanine nucleotide binding protein Ras (102) or through stimulation of growth factor receptors.

Membrane receptors that stimulate PKA activity stimulate transcription of c-fos by phosphorylating serine 133 on the transcription factor CREB bound to the Ca/CRE element in the promoter region (88, 103). In some neurons, calcium influx can regulate transcription of c-fos by activating CaM kinase II (80) to phosphorylate serine 133 of CREB. CaM kinase IV can also phosphorylate CREB at serine 133, and recent evidence Suggests that phosphorylation of CREB at serine 142 by CaM KII can inhibit transcriptional activation (32). In a humbling example of evolutionary conservation, learning and memory in Drosophila (104), Aplysia (81, 82), and mouse hippocampus (105) are all dependent on regulation of the CREB family of transcription factors in response to neural impulses.

Two other less-well-characterized regulatory regions have been identified in the c-fos promoter. The sis-inducible element (SIE) activates c-fos transcription in response to growth factors (54), and the c-fos AP-1 binding element (FAP) (53) provides negative feedback regulation by binding to the Fos protein. However, the FAP is also required for serum inducibility of c-fos expression.

Interactions between signaling cascades provide a mechanism for integrating responses from multiple stimuli. Calcium-dependent signaling pathways and cAMP-dependent pathways converge by phosphorylating the transcription factor CREB (Fig. 2). Signaling from growth factors through the MAP kinase pathway and PKC-dependent signaling converge on the SRF.The individual sequence elements in the promoter region of the c-fos gene are generally thought to operate independently, but this view is being modified by recent experiments on transgenic animals (106). Transcription of the c-fos gene is submaximal in response to any stimulus (growth factors, depolarization, cAMP stimulation) in mice with point mutations in any one of the four regulatory sequences. For example, a mutation of the SRE (thought to be largely calcium independent) has the same negative effect as mutation of the Ca/CRE in response to calcium channel activation. This suggests that all four of the regulatory elements are required in concert to regulate the c-fos promoter (106). which implies assembly of multiple transcription factors into a large interdependent transcriptional complex. This would require extensive remodeling of the DNA structure to permit interaction among the transcription factors bound to separate sites on the DNA. This type of interaction may be lost in transient transfection assays because of differences in chromatin organization between transfected and chromosomal DNA.

The three-dimensional chromatin structure may be an important factor in calcium-dependent regulation of elongation of c-fos transcripts after transcription is initiated. Elongation of transcripts is the principal control mechanism over the kinetics and accumulation of c-fos mRNA in 2B4 cells (107). Without stimulation that elevates [Ca⁺⁺]_i, most transcripts that are initiated at the c-fos promoter do not reach the end of the gene and fail to generate mature mRNA molecules. RNA polymerase activity is probably not responsive to Ca⁺⁺ directly, but sensitivity may be provided through a transiently activated protein kinase that phosphorylates RNA polymerase, or phosphorylates a polymerase-associated elongation factor or DNA regulatory protein. This calcium-dependent effect on elongation is not observed in transfected genes, suggesting that the chromatin structure or topological constraints on the transcriptional template may play a role in regulating elongation (107). Recent experiments in PC12 cells show that phosphorylation of CREB at ser133 is necessary for initiation of transcrjption but not sufficient for c-fos expression (108). In these experiments, a cAMP-dependent phosphorylation of CREB at a site distinct from ser133 or phosphorylation of an accessory binding protein, such as CREB binding protein (CBP) (52), are presumably required to permit elongation of the full-length c-fos transcript.

Signal Integration and Segregation

How specificity between stimulus and response is maintained within an increasingly complicated web of interactive signaling reactions is a fundamental and largely unresolved problem in cell biology, but in neurobiology an additional complexity arises. Information in the nervous system is coded in the temporal pattern of impulse activity; yet how signaling reactions extract and transmit information from temporally varying stimulation has received relatively less attention. It is assumed that concentration thresholds of second messengers produced by different patterns of impulse activity would control the responses to temporally varying stimulation, but a more dynamic and systems-level view of this process is beginning to evolve.

Studies of c-fos expression in DRG neurons in response to different patterns of neural impulses show that neither a large increase in intracellular calcium nor a prolonged increase in residual calcium are required to activate gene transcription (Fig. 3). Indeed, stimulus bursts producing larger increases in calcium can result in less c-fos transcription than smaller, but more closely spaced bursts. Moreover, stimulation with only 1 action potential every 10 seconds is sufficient to induce c-fos transcription, but this stimulus produces only a small, very transient calcium spike of 20 nM [Ca⁺⁺], accompanying each action potential (15). This suggests that the time-dependent concentration dynamics of intracellular signaling reactions, not simply the steady-state or peak concentrations, are critical in understanding activation of specific genes in response to different temporal patterns of stimulation. Late-onset genes also show similar sensitivity to specific frequencies or patterns of impulse activity. Expression of the preprotachykinin neurotransmitter precursor gene (109), LVA and HVA voltage-sensitive calcium channels (4), and the neural cell adhesion molecule L1 (3) are regulated by different patterns of neural impulse activity. Stimulation with 1 action potential every 10 seconds downregulates L1 expression in DRG neurons, but 1 action potential every second or constant dcpolarization with KCl has no effect (Fig. 4) (3). Neurite outgrowth and maturation of the potassium channel in developing frog spinal neurons are differentially regulated by the frequency of calcium spikes, rather than the concentration differences (9,and sustained activation of MAP kinase is required for differentiation of PC12 cells (110).

Fig. 3.

Activation of c-fos transcription in DRG neurons is sensitive to different patterns of neural impulses. Stimulation with short bursts (6 action potentials at 10 Hz) (b) every minute is more effective in activating c-fos transcription than the same number of action potentials delivered in bursts of twice this duration (12 action potentials at 10 Hz) every 2 minutes (c).Extremely low frequency stimulation (0.1 Hz) produces a significant increase in c-fos expression (a). The magnitude or duration of the electrically evoked intracellular calcium transients, measured with fura-2 ratio fluorescence, show that neither high levels of intracellular calcium nor prolonged elevation of intracellular calcium concentration are necessary to activate c-fos transcription. These results suggeet that the temporal dynamics of second messenger generation, not only peak or Steady-State concentration levels, are critical in understanding how pulses of neural impulses activate transcription of genes. Data are adapted from (15).

Fig. 4.

Expression of the neural cell adhesion molecule L1 is regulated by Specific patterns of neural impulses₄ A₁ levels of L1 mRNA (expressed as a % of control, nonstimulated levels) are reduced significantly after 5 days of stimulation at a frequency of 0.1 HZ, but 0.3 or 1 Hz stimulation has no effect; a), nonstimulation vs. 0.1 Hz, P < 0.001; b), nonstimulation vs. 0.3 and 1.0 Hz, not significant. B, LI mRNA was compared using RT-PCR (L1 = 136bp) with reference to an internal standard (I.S. = 225 bp) In mouse DRG neurons in vitro. Data are adapted from (3).

Resonant Signal Transduction

Kinetic features of competing signaling pathways may produce resonance between specific systems of signaling reactions and specific temporal patterns of activation (111). Although relevant to signaling in all cells, the dynamic response of signaling systems would seem of even greater importance in the nervous system, where the stimulus is temporally varying. New studies of LTP in transgenic mice expressing an autophosphorylated form of CaM KII show that rather than acting as a switch, autophosphorylation regulates the frequency-response function that couples presynaptic action potential activity to the production of either LTP or long-term depression (112). Similar theoretical formulations of the importance of temporal regulation of intracellular signaling networks have been put forward recently for non-neuronal cells, using analogies to computer-based neural networks (113) and electronic circuits (114).

In addition to reaction kinetics, translocation of enzymes, diffusion and oscillations of second messenger concentration, protein-protein interactions and assembly of transcriptional complexes would introduce different time-dependent limitations in individual pathways in response to intermittent neural impulse activity. Translocation has been shown for a number of signaling proteins, including PKC, cGMP-dependent protein kinase, CaM KII, MAPK, casein kinase II, and the protein phosphatases, PPI, PP2A, PP2B, and PP2C (115).

Calcium transients evoked by action potentials propagate through the cytoplasm in a manner limited by diffusion, location of calcium permeable channels, calcium-binding proteins, calcium pumps, and electrical propagation efficiency (116). Calcium oscillations and intracellular waves have been proposed to transform amplitude-modulated signals into frequency-modulated signals (117), but the spatial/temporal dynamics of second messenger generation remains an area of considerable research and controversy. Studies of the largest subcellular compartmentalization, that between the cytoplasm and the nucleus, illustrate this difficulty. Depolarization-induced calcium transients in the nucleus of DRG neurons have been reported to exceed those of the cytoplasm by several-fold (118), to remain insulated from large cytoplasmic calcium ion changes (119), or to show no difference relative to the cytoplasm (120, 121). The latter studies conclude that either amplified or dampened nuclear responses result from measurement artifacts. New methods may be required to resolve these important questions.

Control of Gene Expression by Electrical Activity: An Overview

To provide plasticity in neuronal structure and function, expression of genes is controlled in neurons by electrical impulse activity, but the network of signaling reactions linking action potentials to genes is a complex system of multiple interactive elements. Membrane depolarization is transduced into intracellular signaling reactions primarily by controlling intracellular calcium and secreting extracellular signaling molecules, but other modes of signaling occur. Calmodulin, Ras, and several other key molecules function as transducers of information between parallel intracellular signaling cascades. This cross-talk widens the substrates available for regulation by neural impulses and allows integration among different extracellular signals. An essential question for the future is how these systems of signaling reactions are regulated to provide a specific response to a specific stimulus, particularly in response to different patterns of neural impulses. As in nonneuronal cells, subcellular heterogeneity contributes to stimulus-response specificity, but it is becoming increasingly evident from studies in neurons that the temporal dynamics of signal transduction systems are key to understanding how information is conveyed within the cell from intermittent pulses of neural impulses. Understanding how genes are regulated in neurons by specific patterns of neural input is an especially intriguing problem relevant to development, learning and plasticity of the nervous system.

Addendum

New information continues to produce an increasingly complex view of signaling pathways regulating the c-fos gene. New pathways are being described with multiple points of interaction between intracellular signaling systems that were once thought to provide discrete lines of communication. Calcium influx can stimulate c-fos transcription through the SRE in cortical neurons through phosphorylation of SRF, and also by MAPK phosphorylation of the ternary complex factor Elk-1 that binds to SRF (122). Conversely, growth factor stimulation can act on the CaCRE through a MAPK pathway acting upon ribosomal protein S6 kinase (RSK2 or pp^90RSK), which phosphorylates. CREB at ser 133 (123). Thus, growth factor signals can converge on the same transcriptional regulatory element as calcium and cAMP dependent signals. The importance of interdependencies between multiple transcription factors in regulating c-fos transcription is becoming increasingly evident. Binding of the coactivator CREB binding protein (CBP) to CREB phosphorylated at serine 133 stimulates transcription under some circumstances by coupling CREB and SRF, bound to the promoter region of the c-fos gene, together with general transcriptional factors. However, transcriptional activity can also be repressed by binding of pp90RSKt o the CBP in response to activation of the Ras pathway by growth factors (124).