Michael E. Lewis

Anatomy of CNS opioid receptors

Abstract There is a wide body of evidence to suggest the existence of at least three distinct opioid receptor types in the CNS, referred to as μ, δ, and κ. This paper reviews some of the findings that have led to this conclusion and the anatomical distributions of these sites in the rat brain. Their relation to the opioid peptides and some of the proposed functions mediated by these receptor systems are also discussed.

Insulin-like Growth Factor-I: Potential for Treatment of Motor Neuronal Disorders

Motor neuronal disorders, such as the loss of spinal cord motor neurons in amyotrophic lateral sclerosis or the degeneration of spinal cord motor neuron axons in certain peripheral neuropathies, present a unique opportunity for therapeutic intervention with neurotrophic proteins. Normally, such proteins do not cross the blood-brain barrier, but spinal cord motor neuron axons and nerve terminals lie outside the barrier and thus may be targeted by systemic administration of protein growth factors. Insulin-like growth factor-I (IGF-I) receptors are present in the spinal cord, and, like members of the neurotrophin receptor family, IGF-I receptors mediate signal transduction via a tyrosine kinase domain. IGF-I was found to prevent the loss of choline acetyltransferase activity in embryonic spinal cord cultures, as well as to reduce the programmed cell death of motor neurons in vivo during normal development or following axotomy or spinal transection. Consistent with earlier reports that IGF-I enhances motor neuronal sprouting in vivo, subcutaneous administration of IGF-I increases muscle endplate size in rats. Subcutaneous injections of IGF-I also accelerate functional recovery following sciatic nerve crush in mice, as well as attenuate the peripheral motor neuropathy induced by chronic administration of the cancer chemotherapeutic agent vincristine in mice. Doses of IGF-I that accelerate recovery from sciatic nerve crush in mice result in elevated serum levels of IGF-I which are similar to those obtained following subcutaneous injections of formulated recombinant human IGF-I (Myotrophin) in normal human subjects. Based on these findings, together with evidence of safety in animals and man, clinical trials of recombinant human IGF-I have been initiated in patients with amyotrophic lateral sclerosis and are planned to begin soon in patients with chemotherapy-induced peripheral neuropathies.

Aurintricarboxylic acid protects hippocampal neurons from NMDA- and ischemia-induced toxicity in vivo

Abstract: The polymeric dye aurintricarboxylic acid (ATA) has been shown to protect various cell types from apoptotic cell death, reportedly through inhibition of a calcium‐dependent endonuclease activity. Recent studies have indicated that there may be some commonalities among apoptosis, programmed cell death, and certain other forms of neuronal death. To begin to explore the possibility of common biochemical mechanisms underlying ischemia‐or excitotoxin‐induced neuronal death and apoptosis in vivo, gerbils or rats subjected to transient global ischemia or NMDA microinjection, respectively, received a simultaneous intracerebral infusion of ATA or vehicle. As a biochemical marker of neuronal death, spectrin proteolysis, which is mediated by activation of calpain I, was measured in hippocampus after 24 h. ATA treatment resulted in a profound reduction of both NMDA‐and ischemia‐induced spectrin proteolysis, consistent with the possibility of some common mechanism in apoptosis and other forms of neuronal death in vivo.

Pharmacological Induction of Nerve Growth Factor mRNA in Adult Rat Brain

Three structurally unrelated compounds, all of which induce nerve growth factor (NGF) in cell culture systems, were assessed for their ability to induce NGF mRNA in adult rat brain using a highly sensitive RNAse protection assay. Interleukin-1 beta (0.5-1 pmol) and 1,25-dihydroxyvitamin D3 (25-25,000 pmol) were extremely potent inducers of NGF mRNA, being respectively at least 50,000 and 4000 times more potent than 4-methylcatechol. These compounds elicited an approximate twofold increase in NGF mRNA in both the hippocampus and cortex, without altering beta-actin mRNA levels after a single intracerebroventricular injection. The duration of NGF induction was dependent on the compound administered. For example, the elevation of NGF mRNA elicited by interleukin-1 beta peaked at 8 h and lasted for at least 24 h. In contrast, the induction of NGF after 1,25-dihydroxyvitamin D3 and 4-methylcatechol administration peaked between 4 and 8 h and was not apparent 24 h after injection. These results demonstrate induction of NGF mRNA in vivo by administration of physiological or pharmacological agents and differentiate these agents by potency and duration of action. Further, these findings indicate that pharmacological induction of NGF may be a viable strategy for the treatment of neurodegenerative disorders such as Alzheimers disease.

K-252a and Staurosporine Promote Choline Acetyltransferase Activity in Rat Spinal Cord Cultures

Abstract: The protein kinase inhibitor K‐252a increased choline acetyltransferase (ChAT) activity in rat embryonic spinal cord cultures in a dose‐dependent manner (EC50 of ∼100 nM) with maximal stimulatory activity at 300 nM resulting in as much as a fourfold increase. A single application of K‐252a completely prevented the marked decline in ChAT activity occurring over a 5‐day period following culture initiation. Of 11 kinase inhibitors, only the structurally related inhibitor Staurosporine also increased ChAT activity (EC50 of ∼0.5 nM). Effective concentrations of K‐252a were not cytotoxic or mitogenic and did not alter the total protein content of treated cultures. Insulin‐like growth factor I, basic fibroblast growth factor, ciliary neurotrophic factor, and leukemia inhibitory factor yielded dose‐dependent increases in ChAT activity in spinal cord cultures. The combination of K‐252a with insulin‐like growth factor‐l or basic fibroblast growth factor increased ChAT activity up to eightfold over that of untreated controls, which was greater than that observed with each compound alone. K‐252a combined with ciliary neurotrophic factor or leukemia inhibitory factor demonstrated no additive or synergistic effects on ChAT activity. These results suggest that there are multiple mechanisms for the regulation of ChAT activity in spinal cord cultures. The enhancement of spinal cord ChAT activity by K‐252a and Staurosporine defines a new neurotrophic activity for these small organic molecules and raises the possibility that they may activate some regulatory elements in common with the ciliary neurotrophic factor and leukemia inhibitory factor family of neurotrophic proteins.

Induction of NGF by isoproterenol, 4-methylcatechol and serum occurs by three distinct mechanisms

Evidence is provided that isoproterenol, 4-methylcatechol and serum induce NGF by three separate mechanisms. Isoproterenol and 4-methylcatechol induced NGF and NGF mRNA in mouse fibroblast L929 cells in either the presence or absence of serum. Propranolol prevented NGF induction by isoproterenol, but not by 4-methylcatechol or serum. All possible combinations of these inducers resulted in additive increases in the levels of NGF and NGF mRNA.

Autoradiographic study of irreversible binding of [3H]β-funaltrexamine to opioid receptors in the rat forebrain: comparison with μ and δ receptor distribution

Abstract β-Funaltrexamine (β-FNA) is an irreversible μ antagonist and a reversible κ agonist in in vivo and in vitro tests. However, whether it pr produces irreversible δ antagonism is controversial. In binding studies, it is clear that β-FNA does not bind irreversibly (it does reversibility) to κ receptors. Yet there is no consensus as to whether β-FNA binds irreversibly to μ and/or δ receptors. In this study, irreversible binding of [ 3 H]β-FNA to opioid receptors was examined in rat forebrain sections in the presence of 200 mM NaCl and its distribution compared with those of μ and δ opioid receptors, labeled by [ 3 H][ d -Ala 2 , MePhe 4 , Gly-ol 5 ]enkephalin ([ 3 H]DAMGO) and [ 3 H][ d -Pen 2 , d -Pen 5 ]enkephalin ([ 3 H]DPDPE), respectively. Irreversible binding of [ 3 H]β-FNA was determined as thebinding that remained following 5 washes at room temp. for 1,5,20, 20, and 20 min each. Non-specific binding was defined by including 10 μM naloxone, β-chlornaltrexamine (β-CNA), or β-FNA in the incubation mixture. At 37δC, specific irreversible binding of [ 3 H]β-FNA to opioid receptors reached a plateau at 10nM in 60 min, and constituted 50–70% of total irreversible binding. Series of 4 sections of similar anatomical levels were labeled with [ 3 H]DAMGO, [ 3 ]β-FNA, [ 3 H]β-FNA + 10 μM naloxone, β-CNA, or β-FNA, and [ 3 H]DPDPE, resp., and exposed to [ 3 H]-Ultrofilm. The distribution of [ 3 H]β-FNA (5 nM) irreversible labeling is very similar to that of [ 3 H]DAMGO, i.e. patches and subcallosal streaks in caudate-putamen, patches in nucleus accumbens, dense labeling in thalamus, and more binding in the rostral than caudal striatum. As expected, the labeling pattern is quite different from that of [ 3 H]DPDPE. When 10 nM [ 3 H]β-FNA was used, the pattern of irreversible binding was similar to that seen with 5 nM, except that the background was higher. [ 3 H]β-FNA (5 or 10 nM) labeling was reduced by 10 μM naloxone and completely abolished by 10 μM β-FNA or β-CNA. However, when 25 or 50 nM [ 3 H]β-FNA was used, the specific binding was obscured by extremely high nonspecific binding. Thus, at low concentrations, [ 3 H]β-FNA irreversibly labels μ opioid receptors, whereas at high concentrations, it loses specificity for μ receptors.

Localization of Nerve Growth Factor Receptor mRNA in the Rat Basal Forebrain with in Situ Hybridization Histochemistry

Summary1.In situ hybridization histochemistry was used to localize nerve growth factor receptor (NGFR) mRNA in the adult rat basal forebrain.2.In emulsion-dipped sections35S-labeled RNA antisense probes produced a high density of silver grains over cells located in the medial septum, vertical and horizontal limbs of the diagonal band of Broca, and nucleus basalis.3.This distribution of NGFR mRNA overlaps with the distribution of NGFR protein localized using immunocytochemical techniques.4.No hybridization signal was detected when sections were hybridized with a35S-labeled RNA sense (control) probe.5.We suggest that NGFRs are synthesized in these basal forebrain nuclei and transported to terminal areas where NGF is thought to be bound and internalized, an initial step in the many actions of this neurotrophic factor.

Nonradioactive detection of vasopressin and somatostatin mRNA with digoxigenin-labeled oligonucleotide probes.

In situ hybridization histochemistry has become a powerful tool to study the regulation of selected mRNA species in various regions of the central nervous system (CNS). Several laboratories have successfully used this technology to localize relatively rare mRNAs within the cytoplasm of individual neurons. In the main, hybridization studies are performed with radiolabeled oligonucleotide, cDNA or cRNA probes. Although high resolution autoradiographic images have been successfully produced with these radiolabeled probes, the level of background, and the prolonged autoradiographic exposure time (6-10 weeks) required with %and 3H-labeled probes has often limited their utility. Although autoradiography has permitted the identification of cellular profiles, the degree of resolution achieved with radiolabeled probes has never approached that normally obtained with enzyme-generated signals ( e . g . , immunohistochemistry). In recent years, several nonradioactive markers have been developed to detect specific nucleotide sequences under a variety of hybridization conditions. Several different classes of enzymes or haptens can be directly conjugated to nucleotides using conventional chemical techniques. Histochemical studies in the nervous system have focused on the use of alkaline phosphatase or horseradish peroxidase for the detection of antigen and hybridization signals. A few laboratories have utilized these enzymes conjugated to streptavidin to detect biotinylated probe^.^,^ However, the successful use of biotinylated probes to detect rare mRNAs has been limited. One laboratory has published an alternative method which chemically incorporates modified bases with functionalized “linker arms” into synthetic oligonucleotide^.^ These linker arms can be conjugated to several different enzymes. Although we have successfully used this method for high resolution detection of arginine vasopressin mRNA in rat CNS,8 the sensitivity of these probes is limited by the addition of only a single enzyme ( e .g . , alkaline phosphatase) into the nucleotide sequence. Moreover, it is not a routine procedure for histochemical laboratories to conjugate alkaline phosphatase to nucleotides by this method. Recently, we developed a new method for nonradioactive in situ hybridization histochemistry.’ This method, based on the conjugation of digoxigenin with dUTP” (Boehringer Mannheim Biochemicals) provides a higher degree of resolution than radio-

In Situ Hybridization Combined with Retrograde Fluorescent Tract Tracing

In situ hybridization histochemistry is one of the most recent additions to neurobiological methods. In this method, a labeled DNA or RNA sequence is hybridized to its specific complementary messenger RNA (mRNA) in histological sections and visualized by autoradiography or histochemistry. The method originated in the field of molecular genetics and was originally used for localization of specific DNAs in metaphase chromosomes (Gall and Par-due, 1969; Jones and Robertson, 1970; Jacob et al., 1971). Later, globin mRNA was detected in dispersed mammalian cells (Harrison et al., 1973). The early studies are good sources for information on techniques and their application to the analysis of invertebrate development (Capco and Jeffery, 1978; An-gerer and Angerer, 1981; McAllister et al., 1983; Cox et al., 1984). This chapter focuses on the mammalian CNS. Most commonly, probes are radioactively labeled, but biotinylated probes have also been tried (Singer and Ward, 1982; Varndell et al., 1984; Binder et al., 1986). An advantage of using radioactively labeled probes is that grain counting will give a relative quantification of mRNA levels in specific cells (Szabo et al., 1977; Brahic and Haase, 1978; Griffin et al., 1985; Uhl and Sasek, 1986; Wilcox et al., 1986a,b; Young et al., 1986b; Chronwall et al., 1987). Compared to immunohistochemistry, in situ hybridization offers the advantage of localizing the anatomic site for protein synthesis, not merely detecting the presence of the protein.