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Neuronal voltage-activated Ca2+ channels have received consider- able attention from scientists because of the central role played by Ca2+ in the transduction of electrical activity to chemical signals. The importance of neuronal electrical activity resulting in a rise in intra- cellular Ca2+ is highlighted at the presynaptic nerve terminal. Depo- larization of the presynaptic nerve terminal results in influx of Ca2+ through voltage-activated Ca2+ channels. This Ca2+ influx contributes to the depolarization, but more importantly, the increase in intra- cellular Ca2+ activates biochemical events culminating in vesicular release of neurotransmitter. Electrophysiological studies on presyn- aptic nerve terminals have proved difficult because of access problems and the small size of most terminals. A number of preparations, how- ever, have been amenable to studying presynaptic Ca2+ channel activ- ity. These include: 1.The squid giant presynaptic terminal (1);2.Peptidergic nerve terminals of the rat neurohypophysis (2); and3.Frog motor endings (3). [ABSTRACT FROM AUTHOR]

Ion channels are membrane-spanning protein molecules. They form pores or channels, through which ions flow down their electro- chemical gradients. Channels are characterized by two properties, selectivity and gating. Thus, the pore selects for one or a few ion species, allowing only these to permeate, and the pore can open and close in response to changes in the membrane voltage field, or to the binding of chemical transmitters. Because movement of ions through the pore can be measured by the electrical current they carry, it has been possible to study the functioning of these proteins with high resolution. Channel openings and closings, reflecting changes in protein con- formation, can be detected on the time scale of a few tens of microseconds in some cases, and over the past 10 years, methods allowing study of events in a singlechannel protein molecule have become widely used. [ABSTRACT FROM AUTHOR]

It is generally agreed that ion channels are operationally composed of two functional domains: The selectivity filter comprises the part of the protein that determines which ion may pass and which is retained; the gate determines under which conditions the selected ions may pass. Named according to the mechanisms that regulate the gate, the two groups of ion channels are today known as the ligandgated and the voltagegated channels. [ABSTRACT FROM AUTHOR]

Muscle contraction is affected by the rapid release of Ca2+ ions through Ca2+- conducting channels localized in an intracellular mem- brane compartment, the sarcoplasmic reticulum (SR). The rabbit skel- etal muscle SR Ca2+ release channel has been identified as a high-affinity receptor for the plant alkaloid ryanodine, and purified to apparent homogeneity as a 30s protein complex comprising four identical high- mol-wt polypeptide subunits of relative mol mass (Mr) ~560,000 (it1-it4). Morphological and immunolocalization studies of the ryanodine receptor complex have indicated that the subunits of the tetramer are arranged in the form of a four-leaf clover (1,5,6), and are identical to the previously described bridging structures ("feet") (7) that span the transverse tubular-SR junctional gap (8). [ABSTRACT FROM AUTHOR]

Calcium channels are an essential part of the cellular signal trans duction system, since they produce changes in cytosolic calcium. Three types of voltage-dependent calcium channels (T, L, and N channels) have been identified by electrophysiological and pharmacological tech- niques. L and N-type channels have attracted intensive interest since their opening and closing is modulated by hormones, G proteins, pro- tein kinases, toxins, and drugs. The permeation of calcium through L-type channels is blocked by several organic drugs, the calcium chan- nel blockers (CaCB), including the dihydropyridines (DHP), phenylalkylamines, and benzothiazepines. Each class of CaCB binds to a specific site that is present on the α1-subunit of the Gtype calcium channel. The binding to these CaCB-specific sites is modulated allos terically by the membrane potential, calcium, and the occupancy of the other binding sites (1-3). [ABSTRACT FROM AUTHOR]

Purification of receptor proteins has always been an important and challenging task in advancing the structural characterization and also in providing sequence data for the molecular cloning of the recep- tors. Some of the difficulties in the purification of receptors are: 1.Low concentrations of receptors in the tissue;2.Solubilization of intact receptors from cell membranes; and3.Development of an efficient affinity chromatography system. [ABSTRACT FROM AUTHOR]

A variety of biochemical and physiological studies in recent years have made it clear that the brain possesses specific receptors and puta- tive signal transduction pathways for insulin and the insulin-like growth factors (IGF-I and IGF-II). These observations have challenged tradi- tional ideas concerning the physiological role of these peptides in the CNS, and have compelled us and others to consider that they are important regulators of metabolism and growth and development in the CNS (reviewed in 1). A combination of the techniques used for culturing neuralderived cells and those involved in studying insulin and IGF-I receptors on peripheral nonneural tissue has provided a major stimulus for these studies, the results of which define insulin and the IGFs as neuropeptides. The purpose of this chapter is to present these techniques together in a comprehensive manner so as to pro- vide the reader with an overview of how it is possible to study insulin and the IGF-I receptors in nervous tissue. [ABSTRACT FROM AUTHOR]

This chapter is designed to serve as a guide for neurobiologists interested in studying neurotransmitter receptors and ion channels expressed in the oocytes of the African clawed frog Xenopls laevis. The emphasis is on routinely employed methods, based mostly on the experience gained in our laboratories, but also taking into account the practice of many others. A short introduction provides an over- view of advantages and disadvantages of the system, and of what can be done using it. [ABSTRACT FROM AUTHOR]

The typical mammalian genome contains 3 × lo9bp of DNA. This amount of DNA is believed to include 3-4 × lo4 unique protein- encoding genes. Even complex, highly specialized cells, such as neurons, express a small fraction, perhaps 20%, of all the possible genes, how- ever. A major problem in neurobiology, therefore, is to characterize the mechanisms involved in neural-specific gene expression. Significant progress has been made in our understanding of the factors that restrict the expression of genes to particular cell lineages. A particularly good example is the growth hormone (GH) gene. Growth hormone belongs to a gene family that includes prolactin and chorionic somatomammotropin. Despite a high degree of homology between these genes derived from a single common ancestral gene, GH is exclusively expressed in one cell type in the mammal, the pitu- itary somatotroph. The first step in defining the molecular basis of somatotroph-specific gene expression was the identification of the minimal nucleotide sequences within the GH gene that confer strict pituitary-specific expression. Utilizing pituitaryderived cell lines trans- fected with reporter genes that contained putative cell-specific human or rat GH gene regulatory sequences, several groups of investigators delineated two binding sites in the promoter region that are essential [ABSTRACT FROM AUTHOR]

Recent progress in the field of molecular biology has provided a new method to localize an mRNA signal within a cell or tissue section. This technique, called "in situ hybridization histochemistry," gives us dynamic information about gene expression at individual cellular lev- els. For the detection of a particular target mRNA, a specific probe that has complementary sequence to the target mRNA is used. This probe has to include a reporter molecules in order to visualize the position of the probe-mRNA hybrid on a tissue section (1). Conven- tional in situ hybridization procedures use radioisotopes (e.g., 3H, 35S, 32P) as reporter molecules that can be visualized by autoradiography after hybridization (2-4). The use of autoradiography, both film auto- radiography and emulsion autoradiography, restricts the wide appli- cation of this method because of the following reasons. [ABSTRACT FROM AUTHOR]

Because of the vast organizational complexity of the brain, it is crucial to study gene regulation at the level of individual cells. Most brain regions or individual neurons lying adjacent to each other may contain different neurotransmitters or different receptors, which may differentially regulate the gene of interest. Moreover, the brain expresses a much higher proportion of genes than other tissues, such that there are >50,000 distinct mRNA, whose cellular distribution is unknown. This extreme complexity in biochemical composition means that any study of changes in neuronal gene expression in response to pharmacological manipulations (1) or in response to lesions (2) should be analyzed at the singlecell level for maximal sensitivity to detect changes in heterogeneous cell populations. [ABSTRACT FROM AUTHOR]

The complexity of neural gene expression results from an inter- play of a multitude of signal transduction pathways and trans-acting transcription factors interacting at cis-acting DNA elements (1). Clearly, a major challenge is to define the pathways and molecular mechanism(s) interposed between protein kinase activation (action from the cell surface) and the cis-acting DNA elements (controlling target gene expression, in the nucleus), via intermediary protein factors. In general, investigators performing similar studies on various genes use large amounts of nuclear extracts made from cultured established cell lines. Simplicity of preparation is the main reason for this, as opposed to the difficulty of handling small quantities of primary neural tissues. Unfortunately, if only cell line extracts are used, not only is the infor- mation passed in vivo between cells in their natural context lost, but also information gained from these related established lines may have little physiological relevance to normal cells growing in their normal tissues. The methods presented in this chapter have been used (2,3) and may further be used to permit resolution of complex patterns of DNA-protein interactions generated from behavioral changes occur- ring at the level of the whole animal in healthy or diseased states. [ABSTRACT FROM AUTHOR]

The appearance and maintenance of differentiated neural cell types in eukaryotic organisms can be largely attributed to characteristic spa- tial and temporal regulatory cascades evoking cell-specific gene expres- sion. Since gene expression causes information passage from DNA to RNA to proteins and since most mammalian brain protein coding DNA is expressed as messenger RNA (mRNA) (1,2), it appears that the con- trol of mRNA levels is a critical intermediate step in producing brain- specific patterns of gene expression. Of the many transcriptional mechanisms known, RNA initiation is recognized as an important modulator of expression (3-5). [ABSTRACT FROM AUTHOR]

Many proteins that are expressed in the brain, in particular mem- brane-associated proteins, are the products of multigene families. The HT (serotonin) receptor (1), GABA receptor (2), glutamate receptor (3), Na+ channel (4), muscarinic acetylcholine receptor (5), and gly- tine receptor (6) are examples of neural proteins that are encoded by multiple genes. A central issue in cell and molecular biology involves characterization of the functional differences between closely related members of a family of proteins. One approach to this problem involves biochemical analysis of individual protein isoforms, either in situ, or after transfection of cognate cDNAs into suitable recipient cell lines. A second, intersecting line of approach is localization of indi- vidual members of a multigene family within specific anatomical com- partments or cell types. Structure-function analysis, in conjunction with subcellular localization studies, represents a powerful approach for understanding the physiological significance of isoform diversity [ABSTRACT FROM AUTHOR]

All cells have a large number of proteins; for instance, a single hepatocyte has about lo4 different proteins, and a number of them are involved in tissue-specific functions. In neuronal cells, there are additional 104 protein species, which are predicted, from kinetic of DNA-RNA hybridization studies, to be involved in specialized neural functions and in intricate systems of communication (1,2). At present, it is possible to separate and analyze numerous proteins using two-dimensional gel electrophoresis; however, only around 103 protein species in a given tissue can be detected by this method even when combined with the silver-staining technique. In contrast, if a cDNA library from mRNAs is screened, using molecular biological techniques, about 105-106 independent clones, each of which is expected to encode different proteins, can be easily isolated and screened. Recent advances in the techniques of genetic engineering encourage the neuroscientist to search for molecules underlying neuronal functions. [ABSTRACT FROM AUTHOR]

The polymerase chain reaction (PCR; 1) employs a pair of oligonucleotide primers, one sense and one antisense, with respect to the template DNA. During repeated cycles of heat denaturation, annealing of the oligonucleotide primers to the template and extension of the primers by the enzyme taq polymerase, the intervening fragment is amplified. The use of this thermostable polymerase has led to the automation and subsequent widespread use of this technique. This technique has a number of different applications (2) and, in particular, provides a powerful alternative approach to conventional library screening. [ABSTRACT FROM AUTHOR]

Chemically synthesized oligonucleotides are powerful tools in the molecular biologist's repertoire. This chapter describes the design of oligonucleotides and their applications, with particular reference to their use in the isolation and characterization of recombinant DNA clones. Examples of their use are taken either from the literature or from work carried out in our laboratory. Two specific areas are covered: the design of oligonucleotides, based on peptide sequence derived from a purified protein, for use in the isolation of clones from recombinant DNA libraries, and the design of consensus oligonucleotide probes, corresponding to conserved sequences in multigene families, for use in the isolation and characterization of such related genes. We do not cover the use of oligonucleotides for the localization of mRNA transcripts by in situ hybridization or for the analysis of mRNA steady-state levels by Northern blot analysis since this has been described elsewhere (1,2). A detailed discussion of all of the factors involved in the formation and melting of oligonucleotide/target DNA duplexes is beyond the scope of this treatise; for this, readers are referred to the work of Lathe (3). However, formulae for the calculation of melting temperatures and the application of the base analog inosine are described in the Notes. [ABSTRACT FROM AUTHOR]

Human mitochondrial DNA (mtDNA) is a closed circular genome of 16,569 bp (1), encoding 13 subunits of four enzyme complexes (Complexes I, III, IV, and V) in the oxidative phosphorylation system (2). Mutations of mtDNA have been demonstrated to be associated with various neuromuscular diseases. Point mutations of mtDNA are reported in MELAS syndrome (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes) (3,4), MERRF syndrome (myoclonus epilepsy associated with ragged-red fibers) (5,6), Leber's disease (hereditary optic neuropathy) (7), a type of encephalomyopathy (8), and fatal infantile cardiomyopathy (9). Deletions of mtDNA are observed in Kearns-Sayre syndrome and chronic progres sive external ophthalmoplegia (CPEO) (10). It is also proposed that accumulation of mtDNA mutations is an important contributor to several degenerative diseases and the aging process (11). Evidence supporting this hypothesis has been presented in Parkinson's disease (12,13), cardiomyopathy (14,15), and presbycardia (16). Therefore, the analysis of mtDNA mutations seem to be increasing their importance both in clinical neurology and in the basic neurological science. [ABSTRACT FROM AUTHOR]

Generally, genomic DNA is used either for the construction of genomic libraries or for Southern blot analysis. For several reasons, it is frequently preferable in the field of invertebrate neurobiology to screen, at least initially, genomic rather than complementary DNA (cDNA) libraries for sequences of interest (for example, see refs. 1-3). First, low-abundance mRNAs (e.g., those encoding neuroreceptors; see refs. 2-4) have only a very small probability of being present in cDNA libraries. Second, the expression of genes that encode neuronal proteins may be subject to developmental regulation, and the corresponding mRNAs may be present in the organism only at certain stages (4). Third, it is well established, at least in vertebrates, that certain neuron-specific proteins (e.g., ligandgated ion channels, voltagegated ion channels, and guanine nucleotide-binding protein-coupled receptors) are encoded by gene families (see refs. 5′7). For the complete characterization of such families, therefore, the screening of genomic libraries maybe preferable, since each gene should ideally be present in the library at an equivalent level. In contrast, sequences encoding individual members of gene families are normally present in cDNA libraries at frequencies that are dependent on the abundance of their corresponding mRNAs. Fourth, the genome sizes of some well studied invertebrate species are quite small when compared with those of vertebrates: For example, the haploid genome sizes of the nematode Caenorhabditis elegans and of the fruit fly Drosophila melunogaster are approx 8.0 × 107 and 1.4 × lo8 bp, respectively (8). In contrast, the haploid genome size of humans is 2.8 × 109 bp (8). Therefore, when using representative genomic libraries from such invertebrates, it is possible to know whether the entire genome has, indeed, been screened. After DNA sequence analysis has confirmed the identity of a particular genomic clone, subfragments can be used to determine the abundance of the corresponding neuronal mRNA by Northern blot analysis and to investigate the genomic complexity by Southern blot analysis. [ABSTRACT FROM AUTHOR]

Conventional agarose gel electrophoresis is a widely used technique for the analysis of many kinds of biological molecules, including fragments of DNA. It has one major limitation, namely its inability to resolve DNA fragments of greater than approx 30 kb in length. In human genetics, the enormous length of the genome (3 million kb) makes it necessary to have techniques capable of analyzing much larger DNA molecules, and it was in response to this that the methods of pulsed field-gel electrophoresis (PFGE) were developed. The procedure was first described by Schwartz and Cantor (1), and since then numerous variations and modifications have been published (2-4). These methods all have in common the use of two alternately switched (pulsed) electric fields, arranged at an angle of between 90 and 180 degrees. Conventional gel electrophoresis uses a single, continuous electric field. In PFGE, the molecules are forced to change direction each time the field is switched, and the time taken for a large DNA molecule to reorient itself in response to the change in field is a direct function of its size. Hence, at each pulse, longer molecules become retarded relative to shorter ones because of their longer reorientation time, and over the course of the electrophoretic run, a separation is achieved. The theory of the method was described in detail by Southern et al. (5). [ABSTRACT FROM AUTHOR]