Introduction

For neurons, altering gene expression in response to extracellular signals is a fundamental process; thus the biochemical and physiological changes that occur in neurons during the variety of activities experienced in a lifetime are largely a result of gene activation and suppression by signals received via receptor systems from the environment. The receptors are specialised structures present on the surface of the bilaminar neuron plasma membrane that respond in a specific manner when a structurally specific compound or ligand binds to them.

Activation of these receptor systems occurs via a variety of signal-specific chemical transmitters such as hormones, cytokines, neuropeptides, and neurotransmitters. These eventually modulate the activation (increase in transcriptional activity) or the inhibition (decreased transcriptional activity) of specific genes in the neuron through various types of synaptic transmission.

Synaptic transmission has been conceptualised as a set of processes by which neurotransmitters, acting through their receptors, cause changes in the conductance of specific ion into and out of the neuron to produce excitatory or inhibitory postsynaptic potentials. However, it has become evident that neurotransmitters elicit diverse and complicated effects in target neurons. This has led to the development of a much more complex view of synaptic transmission (Huganir & Greengard 1990).

Activation of receptors can also modulate other activities of the neuron such as glucose uptake and consumption rates, oxygen utilisation, neurotransmitter production, and enzyme concentrations.

Phenotypic and functional development of neurons is accomplished through genetic lineage and environmentally induced gene activation

The initial modulation of genetic events by the environment takes place during the differentiation of stem cells into neurons. Neurons are neurons because they produce the proteins and enzymes necessary to carry out the functions of neurons. They can manufacture axons and dendrites because they are rich in tubulin and microtubule-associated proteins (Black & Baas 1989). They can maintain a membrane potential that varies within a specific range of values depending on the environmental circumstances because they manufacture gated plasma ion channels (Hess 1990). They can communicate with other neurons because they have neurotransmitter-specific enzymes to produce neurotransmitters (Snyder 1992). In other words, all or most of the necessary functions of a neuron are made possible by the activation of the genes that code for the proteins necessary to subserve those functions and the suppression of those genes that do not.

The gene repertoire available for transcription in a neuron is determined by the lineage of the neuron and the stage of commitment and differentiation that the neuron has achieved (Van den Berg C 1986; Pleasure 1992). There are usually temporally and environmentally dependent branch points in the development of a neuron lineage that can determine a particular course of differentiation or development that the neuron will take (Lillien & Raff 1990). For instance, during one critical phase or branch point in the development of a neuron the type of neurotransmitters that the neuron will be producing is determined by the environment in which the axons have come into contact. The determinant factors encountered by the axons are transported via retrograde axonal flow to the nucleus where the signals are interpreted and the appropriate genes activated to manufacture the enzymes necessary to produce the specific neurotransmitters signalled for.

Environmental stimulus is conveyed to the nucleus of the neuron via specialised receptor systems on the membrane

The plasma membrane of a neuron is essential for the survival of the neuron. It encloses the cell and maintains essential differences in composition and ion concentrations between the cytosol of the neuron and the external environment. The plasma membrane is essentially composed of proteins floating in a thin bilayered lipid structure held together by non-covalent interactions. This unique structure forms a relatively impermeable barrier to the passage of most water-soluble compounds. Some of the proteins in the lipid bilayered structure act as structural support, while others act as receptors and transducers that relay information across the membrane about the neuron’s environment (Fig. 3.1). Proteins that span the membrane usually assume an α-helical structure as they pass through the lipid portions of the membrane. This configuration is the most thermodynamically stable, due to interactions with the polar peptide bonds of the polypeptide and the hydrophobic nature of the lipid environment. The transmembrane portion of the protein can pass through the membrane only once, resulting in a single-pass transmembrane protein, or multiple times, resulting in the formation of a multipass transmembrane protein (Fig. 3.2). Multipass transmembrane proteins can form channels in the membrane that can be regulated by a variety of mechanisms (Alberts et al. 1994). Some channels are intimately associated with specialised proteins that act as receptors. The neuron has specific receptor proteins for a variety of chemical compounds known as transmitters.

All receptors for chemical transmitters have three things in common:

Receptors can be either directly or indirectly linked to ion channels

These two different types of linkage are determined by two different genetic programming families of receptors.

Receptors that gate ion channels directly are called inotropic receptors. Upon binding of a transmitter, the receptor undergoes a conformational change that allows the ion channel to open. The receptor is part of the same molecular structure that composes the channel. The activation of inotropic receptors produces fast synaptic actions (milliseconds in duration), e.g. ACh receptor at the neuromuscular junction (Fig. 3.3).

Receptors that indirectly gate ion channels are called metabotropic receptors. These types of receptors act through a special series of interlinked proteins called G-proteins. G-proteins are so named because of their ability to bind the guanine nucleotides, guanosine triphosphate (GTP), and guanosine diphosphate (GDP). Four major types of G-proteins are involved in transduction of signals produced by neurotransmitter binding, Gs, Gi/o, Gq, and G12, and multiple subtypes exist for each.

Activation of these types of receptors often activates a second messenger such as cyclic AMP (cAMP) or diacylglycerol in the cytoplasm of the neuron. Other prominent second messengers in brain include cyclic GMP (cGMP), calcium, phosphatidylinositol (PI), inositol triphosphate (IP3), arachidonic acid, and nitric oxide (NO) (Duman & Nestler 2000) (Fig. 3.3).

Many second messengers then act on a variety of intracellular kinases or enzymes to promote or inhibit cellular functions. Such intracellular processes can produce rapid changes in neuronal function such as changes in ionic conductance across the membrane. These second messenger processes can also produce short- to medium-term modulatory effects on neuronal function, such as regulation of the responsiveness of the neuron to the same or different neurotransmitters (transmitter modulation) via changes in receptor sensitivity. Relatively long-term modulatory effects on neuronal function, including changes achieved through the regulation of gene expression, can also be regulated by the actions of second messengers on other intracellular components called third messengers. Such changes can last seconds to minutes and include altered synthesis of receptors, ion channels, and other cellular proteins, or for much longer periods ultimately resulting in forms of learning and memory (Fig. 3.4).

Free levels of intracellular Ca⁺⁺ can act as a potent second messenger in a number of different pathways

Under normal conditions the intercellular concentration of free Ca⁺⁺ ions is under strict regulatory control. Receptor activation can result in increases in free intercellular Ca⁺⁺ ion levels in two different ways involving two types of mechanisms that operate to different extents in different cell types. Neurotransmitter receptor activation can alter the flux of extracellular Ca⁺⁺ ions into neurons or can regulate release of Ca⁺⁺ ions from intracellular stores. Once released, Ca⁺⁺ ions can exert multiple actions on neuronal function via intracellular regulatory proteins. Receptors can directly regulate the conductance of specific voltage-gated Ca⁺⁺ channels via coupling with G-proteins. In addition, activation of other second messenger systems can alter Ca⁺⁺ channel conductance. Depolarisation of a neuron by any means will activate voltage-gated Ca⁺⁺ channels, which will lead to the flux of Ca⁺⁺ into the cells. Finally, extracellular Ca⁺⁺ can pass through some ligand-gated channels, such as the nicotinic cholinergic and glutamate N-methyl-D-aspartate (NMDA) receptors (Duman & Nestler 2000).

The structure of chromatin can regulate gene transcription induced by receptor stimulation

In human neurons, DNA is contained in the nucleus of the cell. The nucleus is also the site of DNA replication and transcription. Chromatin is formed from subunits of nucleosomes, which are chromosomes intimately associated with histone proteins. A chromosome is composed of extremely long molecules of DNA. The DNA not actively involved in transcription processes is stored in supercoiled structures that drastically reduce the space requirements in the nucleus for the storage of DNA. Chromatin is not only structurally important in this storage role but also acts in the regulation of gene expression by inhibiting transcription factors access to binding sites on DNA. Activation of a gene requires that the chromatin or nucleosomal structure be modified to allow the binding of regulatory proteins to the appropriate subset of genes. This is accomplished by a specialised group of proteins referred to as activator proteins that remodel the chromatin and expose core promoter sites on the appropriate genes. This permits the binding of yet another complex of proteins called general transcription factors to the core promoter site on the DNA. This complex of general transcription factors can then recruit and bind with RNA polymerase to enter the transcription initiation phase of the replication process (Workman & Kingston 1998).

The process of transcription can be divided into three steps: initiation, elongation, and termination. Regulation of gene expression can and does occur at each of these steps in the neuron; however, the transcription initiation phase seems to be the most highly regulated step involving extracellular signalling mechanisms.

In humans, three different types of RNA polymerases, I, II, and III, are involved with the transcription of different types of DNA. Polymerase I is involved in the transcription of ribosomal RNAs (rRNA). Polymerase II is involved in the transcription of messenger RNA (mRNA) and another subset of RNAs known as snRNA, which are involved in splicing RNA segments. Polymerase III is involved in the transcription of a number of smaller RNA types including transfer RNA (tRNA) (Struhl 1999) (Fig. 3.5).

In humans, the expression of highly complex genes requires that additional transcriptional activators are necessary for the transcriptional process to function. These additional transcriptional activation complexes are referred to as functional regulatory elements or transcription factors that bind to specialised sites within the structure of the gene. These functional transcription factors determine the unique pattern of expression for each gene, both in the normal course of development and in response to environmental stimuli. Aspects of gene expression under the control of various transcription factors include the cell type in which the gene is expressed, the time during development when the gene is expressed, and the level to which it will be expressed (Collingwood et al. 1999).

Several families of transcription factors as well as several modes of activation or inhibition of these factors have been identified. For example, the cAMP response element binding protein (CREB) family of transcription factors activate transcription of genes to which they are linked when they are phosphorylated by cAMP-dependent protein kinase (protein kinase A). Protein kinase A is activated in the presence of cAMP (De Cesare & Sassone-Corsi 2000).

The CREB family of transcription factors can also be activated by other second messengers such as Ca⁺⁺ bound by calmodulin that can activate a variety of protein kinases upon entering the nucleus of the neuron. These kinases can in turn phosphorylate CREB, resulting in the activation of transcription of the specific CREB-linked gene (Nestler & Hyman 2002).

Dissemination of the receptor stimulus throughout the neuron

The environmental stimulus – whether it be a growth hormone, a neurotransmitter, or a hormone – must get its signal from the receptors on the neuron cell membrane to the transcriptional controlling factors in the nucleus in order for production of the necessary proteins that it calls for.

Some signalling molecules such as hydrophobic hormones (glucocorticoids, oestrogen, and testosterone) can gain direct access to the nuclear apparatus by their lipid-soluble chemical structure that allows them the ability to transverse the highly hydrophobic bilayered lipid plasma membrane dependent mainly on their concentration gradients. These hormones then bind with intracellular hormone receptors that carry them through the cytoplasm and across the nuclear membrane where they bind and alter the conformation of transcriptional factors (Evans & Arriza 1989; Lin et al. 1998).

Other signalling molecules such as Ca⁺⁺ ions gain access through specific ion channels present in the neuron plasma membrane (Hess 1990).

Protein hormones, growth factors, peptide neuromodulators, and neurotransmitters must act on their transcription protein targets indirectly by either inducing a change in a transmembrane protein channel related to their receptor proteins (Lester & Jahr 1990) or by inducing a change in linked intramembrane proteins. Changes in these linked intramembranous proteins called G-proteins eventually result in the release of intracellular ions or the generation of intracellular second messenger such as cAMP, diacylglycerol, and inositol triphosphate (Berridge & Irvine 1989; Huang 1989), which then activate directly or through other intermediates the transcription factors in the nucleus such as CREB (Fig. 3.6).

The number of known second messengers is still relatively small. Response specificity is achieved through one of the following methods:

Prolonged activity of second messengers can lead to a variety of damaging effects on a neuron, ranging from inappropriate activity to transformation and cell death. This has resulted in the formation of a variety of regulatory mechanisms in the neuron to control the concentration and temporal activity of second messengers. Most commonly, the second messengers are sequestered by intracellular proteins, or degraded by intracellular enzymes within milliseconds of release in the neuron (Boekhoff et al. 1990). Because of their size and short span of activity, second messengers are limited in their ability to act over the long term and limited in their specificity for precise and effective modulation of protein transcription. These shortcomings have led to the search for a third messenger system within the neuron that can function very specifically and over long periods to modulate gene expression.

Extracellular signals result in activation of a third messenger system coded for by immediate early genes

Third messengers are groups of nuclear proteins known as translational factors that are induced by a variety of extracellular signals. These proteins bind to specific nucleotide sequences in the promoter and enhancer regions of genes (Mitchell & Tjian 1989). The third messengers are coded by immediate early genes (IEGs), also referred to as primary response genes or competence genes. The proteins encoded for by immediate early genes in concert with other transcriptional factors exert powerful excitatory or inhibitory effects on the initiation of RNA synthesis (Pleasure 1992). Many of the IEGs were initially recognised because they are the normal nuclear homologues of transforming retroviral oncogenes, which are the class of gene released by viruses immediately upon entering a host cell.

The most fully studied IEG is c-fos. The c-fos gene has three binding sites for CREB and is activated by neurotransmitters or other stimuli that stimulate the production of cAMP in the neuron (Ahn et al. 1998). Changes in the tertiary structure of c-fos gene are detectable within one minute after cell stimulation, first appearing in regulatory regions of the gene and then propagating to decoding regions of the gene. The half-life of the c-fos gene and the protein it codes for are very short, in the range of 20–30 minutes. This time frame of activation is much shorter than other proteins of a structural or enzymatic nature but is many orders of magnitude greater than the half-life of the second messengers.

Fine-tuning of the effects of third messengers is accomplished through a complex network of controls. Since there are now over a hundred IEGs and corresponding proteins composing third messengers, a very complex matrix of interactivity which would allow complicated but minor variances in linear and temporal combinations of third messengers for various functions can be developed (Pettersson & Schaffner 1990; Ptashne & Gann 1990) (Table 3.1). For example, when fos binds to DNA as a heterodimer complex with another third messenger protein called jun the transcription of the target protein – usually tyrosine hydroxylase, neurotensin, neuromedin, or a proenchephalin – is dramatically increased (Gizang-Ginsberg & Ziff 1990; Kislauskis & Dobner 1990). However when fos binds to DNA on its own it inhibits the transcription of c-fos, its coding gene (Gius et al. 1990).

Table 3.1 Diversity of pro-oncogenes thus far discovered

Class	Proto-oncogene nomenclature	Homologue
Receptor ligand	c-sis Int-2 hst

Transmembrane tyrosine kinases Membrane-associated tyrosine kinases   Non-tyrosine kinase receptors Serine/threonine kinases   G-protein-like   Signal transduction enzymes Nuclear proteins Zinc finger proteins Leucine zipper protein Helix-loop-helix  

Reproduced with permission from Discussions in Neuroscience, 7(4) (August 1991), Elsevier Science Publishers BV.

The fos family of genes may act as a molecular switch within the neuron

Under resting conditions the concentration of c-fos mRNA and protein in the neuron are extremely low, but c-fos expression can be dramatically increased by a variety of stimuli (Correa-Lacarcel et al. 2000). For example, experimental induction of a grand mal seizure causes marked increases in c-fos mRNA within the brain within 30 minutes and induces the formation of c-fos protein within 2 hours (Sonnenberg et al. 1989). The fos-like proteins are highly unstable and return to normal values within 4–6 hours. Administration of other substances such as cocaine or amphetamine causes a similar pattern of expression in the striatum (Graybiel et al. 1990; Hope et al. 1994). With repeated activation, the c-fos family of genes become refractory to the stimulus, and other isoforms of the fos proteins which express very long half-lives in brain tissue are expressed and accumulate in specific neurons in response to repeated stimulus (Pennypacker et al. 1995; Chen et al. 1997). The accumulation of these proteins remains in the neurons long after the stimulus has ceased. The prolonged presence of the fos-like proteins may act as a molecular switch inside the cell, shutting off or modulating responses to repeated stimulus. The true functional significance of the sustained presence of these fos