Psychopharmacology: Treatment of Psychiatric Disorders Jambur Ananth
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1Principles of Psychopharmacology2

Introduction to Neurotransmitters, Receptors, Signal Transduction and Second MessengersChapter 1

Jambur Ananth
 
INTRODUCTION
The nervous system is highly organized to produce an effective communication. The brain receives, processes, and interprets multiple types of sensory stimuli, controls motor stimuli, and records experiences in the form of memory. Thus, it is responsible for cognition, mood, and behavior. The brain is composed of over 100 billion neurons consisting of several distinct cell types, each with a different structure and function. The neurons are the basic structures of the communication system.
 
NEURONS
Neurons are irregularly shaped cells that possess three identifiable parts—the cell body, dendrites, and axons (Fig. 1.1). Most neurons have multiple dendrites which are short and highly branched.
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Fig. 1.1: The description of a neuron
Dendrites receive incoming impulses into the neuron from other neurons. There are specialized spines on the dendrites known as the primary receptive zones which are the sites for receiving impulses from other neurons. The neurons generally have only one axon. The axon is long and divides to form numerous projections. The projections end in highly specialized structures called axon terminals. Axons and their terminals relay neuronal output to other neurons. The impulses come to the cell through the dendrons and go out of the neuron from the axons. The axons do not directly connect with other neurons.
 
Electrical Transmission
Neurons are electrically excitable cells. This property allows them to process information and to communicate with each other. The electrical activity is controlled by selective flow of ions across the neuronal membrane. The membrane itself is impermeable but contains within itself several transport proteins or ion pumps. Neuronal ion channels are selective and tightly gated (controlled). The interior of the cell is negatively charged compared to that of the outside. The degree of polarization at rest is called the resting potential. The excitatory neurotransmitter receptors allow positively charged ions or cations into the cell on stimulation. They make the interior of the cell less negative with respect to the outside, depolarize the membrane, and bring the neuron toward the threshold of firing an action potential. Inhibitory neurotransmitters cause hyperpolarization of the membrane.
 
Neurotransmitters
Neurotransmitters transfer neuronal impulses to other information pathways which lead to later activity including information flow and behavior. They are divided into different classes based on their mode of synthesis (Table 1.1). For example, synthesis of neuropeptides occurs through a messenger dependent process within the cell body.1 The nonpeptide neurohormones are synthesized by enzymes contained in the terminals.
A presynaptic release of a neurotransmitter activates postsynaptic receptor. Thus, the presynaptic electrical impulse goes out of the cell into the synaptic cleft as a chemical impulse. From the cleft, the information travels into the postsynaptic cell through specific sites on the cell called receptors. Understanding the receptor is key in comprehending how the neurons work.
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Table 1.1   Different classes of neurotransmitters
Neurohormones
Releasing hormones
Amino acids
Neuropeptides
Dopamine
ACTH
Glutamate
Endorphins
Norepinephrine
Oxytocin
GABA
Enkephalins
Epinephrine
Vasopressin
Glycine
Somatostatin
Acetyl choline
Melanocyte stimulating hormone
Cholecystokinin
Serotonin
Growth hormone releasing factor
Gastrin
Corticotropin releasing factor
Vasoactive intestinal peptide
Hypothalamic releasing factors
Neuropeptide Y Angiotensin Neurotensin Melatonin
 
Synthesis and Storage of Neurotransmitters
The neurohormones are synthesized in the neurons and their terminals from essential amino acids. For example, dopamine (DA) is synthesized from the amino acid, tyrosine and serotonin (5HT) from tryptophan. The neuronal cells have the necessary enzymes to convert the amino acids to neurotransmitters by a number of metabolic steps. Tyrosine hydroxylase, an enzyme that participates in the synthesis of catecholamines, is generally saturated with tyrosine. This indicates that changes in the plasma levels of tyrosine do not affect catecholamine synthesis. Tryptophan hydroxylase, on the other hand, is not generally saturated and therefore its plasma level affects 5HT synthesis. Many of the enzymes that participate in neurohormone synthesis require coenzymes such as pyridoxol phosphate.
The neurotransmitters are stored in membrane bound vesicles which are assembled in the cell body and transported down the axon to the nerve terminal. The properties of vesicles have been extensively studied. For example, the NE vesicles are either large or small. The large vesicles contain proteins called chromogranins, and the small ones contain ATP. The large ones discharge neurotransmitters to the cleft when stimulated by electrical impulse. The small vesicles possess a specific APT dependent uptake mechanism in their membrane which enables them to take up and concentrate catecholamine from cytosol. Dense cored vesicles of different sizes have been described in DA neurons. Drugs can act either on the storage or release. Reserpine prevents storage and amphetamine causes release of catecholamines from the vesicles.2
The synthetic route of neuropeptides is different. For example, the pentapeptide leuenkephalin is derived by proteolytic cleavage of proenkephalin A, which contains six metenkephalin and one leuenkephalin sequences. Protein synthesizing machinery of the cell is used for the synthesis of neuropeptides in the ribosomes. The precursor neuropeptide is then packaged to vesicles via the Golgi complex and transported through axonal transport. The vesicles containing neuropeptides may contain a coexisting neurotransmitter.
 
Neurotransmitter Release
The arrival of an action potential at the nerve terminal triggers opening of calcium channels which then leads to neurotransmitter release. This occurs by exostosis of the vesicles. The specialized sites on the presynaptic membrane where the release takes place are called active zones. These regions have dense projections forming a synaptic grid with the exoskeletal filaments. Synaptic vesicles are located close to the active zones and are frequently seen in the areas where synapses are dependent on fast neurotransmitters. The vesicles move toward the active zones, fuse with the presynaptic membrane, and discharge the neurotransmitter to the synaptic cleft close to the postsynaptic receptors. These empty small vesicles are ready to take up the neurotransmitter synthesized in the cytosol. Postsynaptic receptors are located exactly opposite the active zones. The neurohormone reaches the specific target on the postsynaptic fibers. Whether each neurohormone works independently or works with other neurohormones is an important issue. The current consensus is that many neurohormones may work together thereby assisting in cotransmission and neuromodulation.
 
Presynpatic Receptors and Control of Neurotransmitter Release
Many neurotransmitters including DA are known to exert effects at presynaptic receptors located on the nerve terminal. The principle of this interaction is to modulate release of the neurotransmitter frequently in an inhibitory manner. This mechanism can be seen as a means whereby neurotransmitter release is regulated by a form of feedback inhibition by the released neurotransmitter itself. Therefore, there is self-regulation of the release of neurotransmitters. Those receptors which allow self-regulation are called autoreceptors.
Another mechanism of neurotransmitter release occurs when a neurotransmitter from one nerve ending is modulated by another regulatory receptor neurotransmitter. The autoreceptors which modulate another neurotransmitter are called heteroreceptors.
 
Termination of Action of Released Neurotransmitter
In order to provide a satisfactory, rapid responding signaling system at synapses, it is necessary for the neurotransmitter to be released, perform its function, 5and then cease its action. This is achieved by the removal of the neurotransmitter from the synapses by destruction or by reuptake back into the presynaptic vesicles. For DA, 5HT, and others, there are active transport processes. These active transport processes are sodium dependent and are present in the presynaptic membrane. In addition, specific transport proteins have been identified for the transfer of DA and 5HT. The extraneuronal metabolism of catecholamines in the synaptic area depends on the enzyme, catechol-O-methyltransferase.
 
RECEPTORS
The receptor is a protein with a binding site for the neurotransmitter. It is capable of transmitting the signals across the membrane to elicit a change within the cell concerned. There are receptors for growth factors as well as for hormones in the brain. The receptors are a specific part of the cell membrane. Ideally suited to carry on neurotransmission, they consist of extracellular, transmembrane, and intracellular segments (Fig. 1.2). The extracellular segment located outside the cell is the primary binding site for the neurohormone. However, the binding sites for some neurotransmitters are in the transmembrane segment. Psychopharmacological agents work by competing for the binding site or blocking the neurotransmitter.
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Fig. 1.2: Different parts of a receptor
The transmembrane segment is located within the membrane and may serve to hold the receptor firmly in its place. The transmembrane segment for one neurotransmitter may be similar to those of other neurotransmitters thus forming superfamilies. For example, all receptors which use a second messenger system consist of seven transmembrane regions. All the serotonergic and norepinephrinergic receptors, except 5HT3 receptors, belong to this superfamily. Ion gated channel receptors consist of five transmembrane regions which interact with ion channels. The examples are benzodiazepines as well as 5HT3 receptors.
The remaining segment of the receptor is intracellular. These consist of cytoplasmic loops connected with the transmembrane part of the receptors. The intracellular segment interacts with transmembrane part of the receptors and with the intracellular proteins. Their interaction with the intracellular proteins is necessary to trigger the second messenger system.
 
Structural and Functional Analysis of Receptors
Receptors are classified according to their mode of action as well as their location. Based on their action, two kinds of receptors including the ion gated channel and G protein linked receptors have been identified (Table 1.2). There are differences in the structure and function of these two groups of receptors. In addition, according to their location, they are classified as presynaptic, postsynaptic, autoreceptor, terminal, and intracellular receptors. Each neuron upon stimulation releases a neurohormone.
Table 1.2   Two classes of receptors
Receptor type
Ion gated channel
Second messenger linked
Response time
Within milliseconds
100s of milliseconds to minutes
Structure
5 subunits and an ion channel
7 subunits with a receptor binding site
Second messenger
No second messengers
Adenylate cyclase Phospholipase C
Other names
Class I
Inotropic
Class II
Metabotropic
G protein
No
Involved
 
Fast Ion Channel Lined Receptors
Biochemical analysis has shown that ion gated channel receptors have five subunits which are glycosylated integral membrane proteins. These combine to form an oligomeric protein array containing the receptor binding site, a central ion channel and various additional binding sites for other molecules. For example, the GABAa receptor is modulated by compounds such as benzodiazepines, steroids, alcohol, barbiturates, and picrotoxin. It is assumed that modulatory site exists in the receptor for each of these compounds. The NMDA class of glutamate receptor is modulated by glycine and phencyclidine related compounds. The predicted three dimensional structures of each subunit based on hydropathy analyses show a common structural motif of four-membrane spanning alpha which are then packed together. The ligand binding site is thought to be formed from the extracellular amino terminal segment. The ion channel is thought to be formed from the second membrane 6spanning segment of each subunit. Phosphorylation sites have been identified on the large intracellular loop and this phosphorylation is involved in regulating the rate of sensitization. Examples of this group are GABAa, acetyl choline, glutamate, 5HT, 5HT3, and glycine receptors.
 
Second Messenger System Linked Receptors
Second messenger linked receptors do not contain intrinsic ionic channels. Rather, they transduce signals to the interior of the cell by activating G proteins. G proteins can produce complex but subtle changes in neurons. They can act on the ion channels specially the K and Ca channels to alter the intrinsic excitability of the cell. They can activate or suppress a number of enzyme systems. All DA, 5HT, and NE receptors except one (5HT3) are G protein linked. They are also an integral part of the membrane glycoproteins, containing the receptor binding site. G proteins link these receptors to their effectors, adenylate cyclase, and phospholipase C. The gene cloning techniques have revealed that all G protein linked receptors contain a seven transmembrane alpha helices (Fig. 1.3). The sites for glycosylation are near the extracellular amino acid terminus of the proteins while the sites for phosphorylation are in the third intracellular loop and in the cytoplasmic carboxy terminal region. The phosphorylation sites may be important in short term sensitization of the receptor.3 The helices are bound together by disulfide bonds and thus provide a cavity within the membrane into which the ligand may bind. The action of G proteins seems to occur on parts of the second and third cytoplasmic loops and the carboy-terminal tail.4 There are a limited number of G proteins which interact with a number of different receptors.
 
Receptor Assay
There are two important types of in vitro receptor assays. These two types of assays are based on the ability of a substance to bind to a receptor or the ability of a neurotransmitter to stimulate a response following receptor binding. In the first type, the radioligand binding assay, the binding of radioactive ligand to the receptors is measured. For example, D2 dopamine receptors may be assayed by the binding of the radiolabelled drug 3H spiperone.
In the second method, the immediate postreceptor event can be used to assay the receptors. Such assays are termed as functional assays. For many receptors, the receptor occupancy by specific ligands leads to changes in the activities of one of the enzymes such as adenylate cyclase or phospholipase C. Therefore, these receptors can be assayed by measuring the changes in the activities of these enzymes.
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Fig. 1.3: G protein linked receptor
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Fig. 1.4: G proteins: Structure and function
 
Molecular Genetics and Receptors
Genetic cloning of receptors has opened new doors in the study of receptors. Isolation of the DNA sequence coding for receptors has allowed the amino acid sequences to be determined, and hydropathy analysis has helped in understanding receptor models. Hydropathy analysis entails running the sequential analysis of hydrophobic amino acids to form membrane spanning alpha helices. Genetic cloning has shown considerable diversity in those receptors with several isoforms. The ability to express cloned receptor genes at high levels facilitates the study of receptors at the protein level using biophysical techniques.
 
Down Regulation
Whereas short term stimulation can lead to desensitization, longer stimulation can lead to alterations in the number of receptors at the cell surface. When there is a reduction in the number of receptors, it is called down regulation. A critical event is the clustering of receptor regions on the cell surface called coated pits where coated vesicles bud off into the cell interior. Subsequently, they loose their coat to become smooth vesicles. The smooth vesicles then fuse with endosomes and receptors. Later, they can either cycle back into the plasma membrane or become degraded.
 
INTRACELLULAR CHANGES
 
G Proteins
G proteins [Rodbell 1992] are so named because of their ability to bind to the guanine nucleotides, guanine triphosphates (GTP), and guanine diphosphate (GDP). They are heterotrimeric proteins consisting of α, β and γ subunits (Fig. 1.4). Distinct α units confer specific functional activity on the different types of G proteins but all of them share similar β and γ subunits. The β and γ subunits act as a membrane anchor for the G protein.
G proteins couple receptors to specific intracellular effector systems. Under resting conditions, they are bound to GDP and are unattached to the receptors or intracellular effector proteins (Fig. 1.4). When the receptor is acti-vated by binding to the specific ligand, the receptor ligand complex attaches to the α subunit of the G proteins. This leads to three important changes: (1) The α unit exchanges its GDP to GTP. (2) The β and γ units get dissociated. (3) The receptor is released from the G protein. These changes lead to the release of free α units bound to GTP. The system returns to its normal state as soon as the ligand is released from the receptor and the GTPase activity that resides in the α unit hydrolyzes the GTP to GDP.
G proteins bring out changes in the second messenger systems such as cyclic AMP, cyclic GMP, and major 8metabolites of phosphatidyl inositol including inositol triphosphate (IP3) and diacylglycerol. The alpha unit of the G proteins along with the ATP activates adenylate cyclase to release cyclic AMP. Nitrous oxide appears to act as an intracellular messenger in mediating the ability of certain neurotransmitters to activate guanylate cyclase. Most of the effects of second messengers are produced by addition or removal of phosphate groups from specific amino acid residues.
Lithium's action is a typical example of the interaction of G protein and the phosphoinositide second messenger system. Lithium in therapeutic concentration inhibits the intracellular enzyme, inositol monophosphatase, leading to depletion of free inositol.57 This results in accumulation of inositol 1 monophosphate and reduction of free inositol. The diacylglycerol, phosphatidic acid, and cystidine monophosphate phosphatidase are elevated.
 
Second Messengers
The second messengers are cyclic AMP, GMP, inositol triphosphate (IP3), and diacylglycerol. On one side, they are linked to G proteins which can either activate or inhibit adenylate cyclase. The active form of adenylate cyclase is the complex with an alpha unit of G protein bearing GTP and magnesium. The α unit of the G protein dissociates from βγ upon binding with GTP. The activated adenylate cyclase converts ATP into cyclic AMP. Cyclic AMP signals within the cell activation of a cyclic AMP dependent protein kinase A. This in turn leads to phosphorylation of proteins in the cell.
Inactivation of adenylate cyclase is driven by hydrolysis of GTP to GDP by the GTPase activity. What happens to the α units within the cell, is not clear. A theory proposed by Rodbell8 indicates that these units are still bound to the membrane. Another enzyme, phosphodiesterase hydrolyzes cyclic AMP into AMP and reduces cyclic AMP levels within a cell, once a stimulatory signal comes to an end.
Phospholipase C hydrolyzes the minor membrane phospholipid phosphotidyl inositol bis phosphate (PIP2), to inositol triphosphate IP3, and diacylglycerol (DAG). The enzyme can be activated by a group of G protein linked receptors leading to increased IP3 and DAG both of which are second messengers.9 These two second messengers are associated with different cellular effects. IP3 releases CA from endoplasmic reticulum and DAG activates a separate CA dependent protein kinase.
Protein kinases activated by second messengers are named after the second messengers that activate them. The cyclic AMP activated protein kinase is named cyclic AMP protein kinase. In addition, the brain contains many other protein kinases such as protein tyrosine kinase. Through second messenger mediated activation of enzymes called protein kinases, neurotransmitters regulate protein phosphorylation. Protein kinases transfer phosphate groups from ATP to serine, threonine, or tyrosine residues in specific substrate proteins. By transferring a phosphate group from ATP to substrate protein, protein kinases alter the conformation and thus function of the target protein. All receptor desensitization investigated has been shown to be due to phosphorylation of receptors. Phosphorylation alters the responsiveness of the ion gated channels, regulates the enzymes that synthesize neurotransmitters, and controls diverse biological functions within neurons.
Table 1.3   Neuronal proteins regulated by phosphorylation
• Enzymes involved in neurotransmitter synthesis
• Enzymes involved in the regulation of second messengers
• Cytoskeletal proteins
• Ion channels
• Neurotransmitter receptors
• Protein kinases
• Synaptic vesicle proteins
• Transcription factors
• Protein phosphatase inhibitors.
 
Third Messengers
Phosphates by virtue of their charge and size, change the conformation of substrate proteins and thereby change their function. Neurotransmitter receptor stimulation would increase levels of cyclic AMP and of activated cyclic AMP dependent protein kinase. Activated protein kinase phosphorylates many proteins. Many classes of neuronal proteins are regulated by phosphorylation (Table 1.3). These phosphoproteins are called the third messengers.
By transferring a phosphate group from ATP to substrate protein, protein kinases alter the conformation and thus function of the target protein. All receptor desensitization investigated has been the result of phosphorylation of receptors. Phosphorylation alters the responsiveness of the ion gated channels, regulates the enzymes that synthesize neurotransmitters, and controls diverse biological functions within neurons. Protein phosphorylation plays a fundamental role in the regulation of neuronal gene expression. Such regulation appears to be achieved through a subset of the proteins that regulate transcription including transcription factors, RNA polymerase, and a variety of other nonhistone and histone nuclear proteins.
 
CREB Proteins
CREB proteins play a major role in mediating the effects of cyclic AMP on gene expression (Fig. 1.5). They appear to be constitutively synthesized so that they exist in neurons roughly at constant concentrations.
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Fig. 1.5: Cascade of neurotransmission
The primary mechanism by which the CREB proteins are regulated is through phosphorylation by cyclic AMP dependent protein kinase. Under resting conditions, they are localized to the nucleus where they are bound to a particular DNA sequence named cyclic AMP response element (CRE), without significant transcriptional activity. Activated protein kinase would then be translocated into the nucleus where it would phosphorylate and activate cyclic AMP response binding protein (CREB). The activated CREB occupies the CRE receptor on the genes which leads to the transcription of messenger RNA. The messenger RNA translocates back to the cytoplasm and produces a protein. Phosphorylation of CREB proteins activates their transcriptional activity. The CREB protein changes ultimately produce a number of effects in the cell activity including reduction in the number of receptors, and down regulation. Many drugs have an effect on CREB proteins and modify their action on the central nervous system.
 
Fourth Messenger System
Protooncogenes are normal cellular genes that cause cellular transformation. There are a number of protooncogenes each serving a specific function. They are also called “Immediate Early Genes” because their transcription is transiently activated in neurons within minutes without the requirement for new protein synthesis in response to neurotransmitters and drugs. Such cellular IEGs include members of the related fos and jun families and Zif 268. The fos family of genes has a prefix c in animals and v in virus. The protein produced by the gene, c-fos, is called Fos. It is present in very small quantity in many neuronal cells under resting conditions. Diverse stimuli including drugs can activate the IEGs by stimulating the production of a calmodulin dependent protein kinase. c-fos mRNA produced in response to a stimulus is then translocated into the cytoplasm where it is translated into new Fos protein. Fos protein is then translocated back to the nucleus where in conjunction with a member of the Jun family, it binds to the AP1 site on the promoter regions of the numerous target genes and regulates their expression. The importance of the fos proteins in psychiatry has been described in Chapter 7 on “New Antipsychotic Drugs.”
This protein kinase in turn phosphorylates CREB protein that is already present in the cell and bound to a CRE in the fos gene promoter. Phosphorylation of the CREB protein activates its transcriptions activity and leads to increased fos mRNA expression. The finding that depolarizing stimuli can increase Fos lead to the suggestion that Fos can be used to map neuronal activity in the brain.10
 
GENETIC CHANGES
The interaction of extracellular ligands with their receptors on the cell membrane elicits the flow of information into the cytoplasm via a number of signal transduction pathways. These do not end in the cytoplasm but extend to the nucleus where they are capable of bringing about changes in the gene expression. These protein kinases can actually enter into the nucleus and regulate expression of genes. This is a ubiquitous mechanism of adaptation to the environment. This regulation does not affect the gene DNA sequence. Phosphorylation of nuclear proteins (transcription factors) is the most important mechanism by which neurotransmitters regulate gene expression. Thus, the G protein linked transmission has both short term and long term effects. From a psychopharmacological perspective, two types of studies involving gene expression in the nervous system are useful. These are: (1) Monitoring gene expression will provide information into the anatomical areas responsible for neurobiological responses. (2) The molecules that couple second messengers to gene expression as well as the products of these genes offer new insight into neuropharmacological intervention.10
Gene expression can be activated by normal physiological processes, by drugs and by experience. A class of proteins termed as transcriptional factors play a role in transsynaptic regulation of gene expression. Cellular responses to external stimuli can be transcription dependent or independent. Transcription independent responses are mediated by second messenger system molecules that control the posttranscriptional modifications of the preexisting substrates such as ion channels, membrane receptors, and cytoskeletal proteins. The long term response involves the second messenger system altering genes. This is achieved by activation of a preexisting factor by phosphorylation or by translocation into the nucleus. The other mechanism requires de novo synthesis of a transcription factor in response to stimulation of the cell. The newly synthesized factor can then activate or repress other genes.
REFERENCES
  1. Coyle JT. Introduction to the world of neurotransmitters and neuroreceptors. In: RF Harris and AJ Frances (Eds). Annual Review Vol 4 American Psychiatric Press, Washington Press,  Washington DC,  1985.
  1. Suzler D, Rayport S. Amphetamines and other dopaminergic psychostimulants reduce pH gradients in midbrain dopaminergic neurons and chromaffin granules: A mechanism of action. Neuron 5: 797–808, 1990.
  1. Bouvier M, Collins S, O’Dowd BF et al. Two distinct pathways for cyclic AMP mediated down regulation of the beta 2-adrenergic receptor. Phosphorylation of the receptor and regulation of its mRNA level. J Biol Chem 264: 16786–92, 1989.
  1. Liggett SB, Carson MG, Lefkowitz RJ et al. Coupling of mutated form of the human beta 2-adrenergic receptor to Gi and Gs: Requirement for multiple cytoplasmic domains in the coupling process. J Biol Chem 266: 4816–21, 1991.
  1. Berridge MJ, Downes CP, Hanley MR. Neural and developmental actions of lithium: A unifying hypothesis. Cell 59: 411–19, 1989.
  1. Casebolt T, Jope RS. Long-term lithium treatment selectively reduces receptor coupled inositol phospholipid hydrolysis in rat brain. Biol Psychiatry 25: 329–40, 1989.
  1. Manji HK, Lennox RH. Long term action of lithium: A role for transcriptional and posttranscriptional factors regulated by protein kinase C. Synapse 16: 11–28, 1994.
  1. Rodbell M. The role of hormone receptors and GTP-regulatory proteins in membrane transduction. Nature 284: 17–22, 1985.
  1. Meldrum, E, Parker PJ, Carozzi A. The PidIns-PLC super-family and signal transduction. Biochem Biophysics Acta 1092: 49–71, 1991.
  1. Morgan JI, Curran T. Stimulus-transcription coupling in the nervous system. Annual Rev Neurosci 14: 421–52, 1991.