_INPUT XML FILE_ 12 Other Interneuronal Signals The previous chapters were devoted to what we might now consider the “classical” or perhaps “conventional” neurotransmitters. By this, we mean those transmitters that are synthesized within the neuron (cell body or synaptic terminal) that releases them by activity-dependent, Ca2+-dependent mechanisms to act on discrete receptors largely, but not exclusively, on the neuron, smooth muscle, or gland cell opposite the nerve terminal. However, as the wheels of progress have turned, it seems that once again the more we learn about inter-cellular communication in the nervous system, the more complicated the situation becomes. The purine signals discussed in Chapter 11 provided an appetizer by acting both pre- and postsynaptically, as do many of the other classical transmitters. Nevertheless, through improved methods of substance identification and detection of signal responsiveness, several potent interneuronal signals have been recognized that are not stored in vesicles, or even stored at all, but rather seem to be synthesized and released on demand to act more broadly than the immediate-releasing neuron terminals and to modify the effectiveness of the conventional interneuronal signals. Prostaglandins The prostaglandins are a group of biologically active derivatives of arachidonic acid often referred to as eicosanoids for their basic 20-carbon atom structure. The two major pathways of eicosanoid metabolism are the cyclooxygenase pathway, which yields the prostaglandins and thromboxanes, and the lipoxygenase pathway, which yields the leukotrienes. A minor pathway termed epoxy-genase yields epoxides, which have received scant attention in the central nervous system (CNS). Arachidonic acid is synthesized on demand from dietary linoleic acid by either a G protein-regulated phospholipase A2 or diglyceride lipase activation (Fig. 12–1), and it yields a very broad array of bioactive metabolites, as shown in Figure 12–2. The three major groups of arachidonic acid-derived metabolites are the prostaglandins, thromboxanes, and leukotrienes. Historically, the earliest effect of prostaglandins arose with the recognition in the 1930s that fresh semen could induce contraction of myometrial muscle, and the name arose from the factor's anticipated origin. As chemical detection methods improved in the 1950s, two classes of prostaglandins were recognized—a PGE class that was soluble in ether and a PGF class soluble in phosphate buffer (fosfat in Swedish)—and one of their sites of synthesis localized to seminal vesicles. Subsequent work indicated that virtually every organ could manufacture prostaglandins, and several distinct synthetic pathways were recognized. A major advancement occurred when John Vane proposed that aspirin and several other nonsteroidal anti-inflammatory drugs (NSAIDs) worked by inhibiting the prostaglandin-synthesizing enzyme cyclooxygenase. Work in the 1990s revealed that a second cyclooxygenase (COX-2) (and perhaps a third) was also present in the CNS. Since this enzyme was shown to be induced by inflammatory cytokines, it immediately suggested a separate target for relief of inflammatory pain, with the potential for reduced symptoms from the gastrointestinal tract irritation typically evoked by NSAIDs. COX-2 is also induced in vitro by the neurotransmitter GLU and inhibited by glucocorticoids. Unfortunately, prolonged use of COX-2 inhibitors has untoward cardiovascular side effects, leading to multiple litigious claims and diverting attention from this once-promising therapeutic modification. It is well known that the eicosanoids, particularly the prostaglandin series, play an important modulatory role in nervous tissue, but it has been difficult to write a lucid account of specifically how and where they act. This is primarily due to the fact that they are not stored in tissues, nervous or other, but synthesized on demand, particularly in pathophysiological conditions. They act briefly (some with a half-life of seconds) and at extremely low concentrations (10−10 M). Although indomethacin is a good inhibitor of cyclooxygenase-1, blocking the conversion of arachidonic acid to prostaglandins, there are few specific inhibitors available to block lipoxygenase and epoxygenase. Thus, although it had been postulated that the E series of prostaglandins modulates noradrenergic release, blocks the convulsant activity of pentylenetetrazol, strychnine, and picrotoxin (possibly by increasing the level of γ-aminobutyric acid [GABA] in the brain), and increases the level of cAMP in cortical and hypothalamic slices, these effects were noted in vitro with the addition of substantial amounts of the prostaglandins. There was very little evidence in intact animals to support these neuronal findings. Since we skeptics all hold “in vivo veritas” in higher regard, the physiological relevance of the effect was in doubt. Subsequently, however, direct evidence has established arachidonic acid and lipoxygenases as second messengers. The cascade begins with the binding of a neuroactive agent to its receptor. Then, according to findings from the Axelrod laboratory, the receptor is coupled to G proteins, which may either activate or inhibit phospholipase A2, although this has not been conclusively established for all neural tissues. The activated enzyme promotes the release of arachidonic acid, which will then act intracellularly as a second messenger. Arachidonic acid and its metabolites can also leave the cell to act extracellularly as first messengers on neighboring cells. Eicosanoids have been shown to mediate the somatostatin-induced opening of an M channel in hippocampal pyramidal cells and the release of VIP in mouse cerebral cortical slices. At the supracellular level, prostaglandins of the E series have been held to be a mediator of fever and prostaglandins of the D series as regulators of sleep. It is thus becoming clear, despite enormous technological difficulties in assaying eicosanoids, that these agents are major messengers. Another exciting chapter of the arachidonic acid story has been told separately, namely the endocannabinoids, which are described later in this chapter. Neurosteroids According to their discoverer, Etienne Baulieu, neurosteroids are those steroids that are synthesized in the nervous system either de novo from cholesterol or by in situ metabolism of blood-borne precursors but are found at levels in the nervous system that are independent of steroid synthesis by adrenals or gonads. Such steroids include at least two previously known steroid precursors, pregnenalone (PREG) and dehydroepiandrosterone (DHEA), that in the nervous system have effects alone or as sulfated esters. In the central and peripheral nervous systems, neurosteroid synthesis has been attributed to oligodendrocytes, astrocytes, and neurons. The peripheral benzodiazepine receptor found on the mitochondrial outer membrane has been suggested to allow cholesterol access to the P450 cleavage enzyme complex on the inner mitochondrial membrane, leading to PREG formation and subsequently to other neurosteroids (Fig. 12–3). In contrast to the endocrine actions of adrenal steroids on the brain, acting at a distance and at very low concentrations, neurosteroids are thought to act locally as either autocrine (acting on the cells that synthesizes them) or paracrine (acting on cells close to the site of synthesis) signals. In this manner, the ability to activate myelin synthesis in oligodendrocytes may provide a reparative effect in multiple sclerosis. In their sulfated ester forms, both PREG and DHEA have been reported to be potent regulators of GABAA and NMDA receptor functions, with PREG-S and DHEA-S inhibiting the effects of GABA; however, these effects on the GABA receptor are reduced if the complex contains a δ subunit. In addition, further characterization of the neurosteroids suggests that a small group of pregnenolone-, progesterone-, or tetrahydrodeoxycorticosterone-derived catabolites are the active moieties in the CNS, namely 3α,5α- and 3α,5β-androsterone, 3α,5β-THP pregnenolone, 3α,5α-THP allopregnenolone, 3α,5α-THDOC allotetrahydroDOC, 3α,5β-THDOC tetrahydroDOC, and 3α,5α- and 3α,5β-androstanol (Morrow, 2007). These are the neurosteroids that are believed to be responsible for the largely GABA-enhancing actions to induce anxiolytic, sedative, and anticonvulsant activity through allosteric modifications at discrete sites on the receptor. Of interest is that systemic doses of ethanol at low to moderate pharmacological levels induce elevation of neurosteroids to levels capable of modifying GABA receptors and contributing to the cellular and behavioral effects of ethanol (see Chapter 18). _CONFIGURATION FILE_ [chapter=section]||[para=p]||[/para]||[subchap1=subsec1]||[head1=h1]||[/head1]||[para=p]||[c=cha]||[/c]||[/para]||[/subchap1]||[/chapter]||[chapnum=secnum]||[title=head]||[/title]||[id=cid]||[/id] ||[/chapnum] _CODE_ #!/usr/bin/perl use warnings; use strict; use Data::Dumper; use XML::Simple; my %xhash=(); my $file= $ARGV[0]; my $simple = XML::Simple->new(ForceArray => 1,KeepRoot => 1); my $xhash = $simple->XMLin($file); print Dumper ($xhash); open FH,"sampleconfg.ini" or die $!; my $line; my %c_hash=(); my $c_hashRef = ''; my @tmpA = (); #sub recur($); foreach $line (){ $line=~s/^(\s+)(.*)/$2/i; $line=~s/(.*?)(\s+)$/$2/i; chomp($line); if ($line ne ''){ $_=$line; $c_hashRef = ''; my @array = split(m/\|\|/, $line); # print @array; # exit; ($c_hashRef,@tmpA) = recur(\@array); # print Dumper($c_hashRef); } } print Dumper($c_hashRef); my %repl; # lookup table: a => 1, etc. traverse($c_hashRef, sub { my ($key, $val) = @_; $repl{$key} = $val; }, "collect" ); # print Dumper \%repl; # debug traverse($xhash, sub { my ($key, $val, $href) = @_; if (exists $repl{$key}) { my $newkey = $repl{$key}; $href->{$newkey} = $val; delete $href->{$key}; } }, "replace" ); print Dumper ($xhash); $simple->XMLout($xhash, KeepRoot => 1, OutputFile => 'pets.fixed.xml', XMLDecl => "", ); print "process over"; sub traverse { my ($hash, $callback, $mode) = @_; traverse($hash->[0], $callback, $mode) if ref($hash) eq "ARRAY"; return unless ref($hash) eq "HASH"; for my $key (keys %$hash) { my $val = $hash->{$key}; if (ref($val)) { traverse($val, $callback, $mode); if ($mode eq "collect") { if (exists $val->{repval}) { $callback->($key, $val->{repval}); } } } if ($mode eq "replace") { $callback->($key, $val, $hash); } } } sub recur($) { my $arrRef = $_[0]; my @procArr = @$arrRef; my $assign = ''; my %c_hash = (); my @tmpArr = @procArr; shift(@tmpArr); my $arr = $procArr[0]; if ($arr=~m/\[(.*?)\=(.*)\]/ig) { $assign = $1; my $tmpVal = ''; $tmpVal = $2; $tmpVal =~ s/\=(.*?)/$1/ig; $c_hash{$assign}{'repval'} = $tmpVal; ($c_hash{$assign}{'addval'},@tmpArr) = recur(\@tmpArr); $arr = shift(@tmpArr); if ($arr) { if ($arr=~m/\[(.*?)\=(.*)\]/ig) { my $key = $1; my $tmpVal = ''; $tmpVal = $2; $tmpVal =~ s/\=(.*?)/$1/ig; $c_hash{$key}{'repval'} = $tmpVal; ($c_hash{$key}{'addval'},@tmpArr) = recur(\@tmpArr); $arr = shift(@tmpArr); } } } return (\%c_hash, @tmpArr); } _OUTPUT XML FILE_
12 Other Interneuronal Signals

“ ” “ ” The previous chapters were devoted to what we might now consider the classical or perhaps conventional neurotransmitters. By this, we mean those transmitters that are synthesized within the neuron (cell body or synaptic terminal) that releases them by activity-dependent, Ca -dependent mechanisms to act on discrete receptors largely, but not exclusively, on the neuron, smooth muscle, or gland cell opposite the nerve terminal. However, as the wheels of progress have turned, it seems that once again the more we learn about inter-cellular communication in the nervous system, the more complicated the situation becomes. The purine signals discussed in provided an appetizer by acting both pre- and postsynaptically, as do many of the other classical transmitters. Nevertheless, through improved methods of substance identification and detection of signal responsiveness, several potent interneuronal signals have been recognized that are not stored in vesicles, or even stored at all, but rather seem to be synthesized and released on demand to act more broadly than the immediate-releasing neuron terminals and to modify the effectiveness of the conventional interneuronal signals. 2+ Chapter 11

Prostaglandins

The prostaglandins are a group of biologically active derivatives of arachidonic acid often referred to as eicosanoids for their basic 20-carbon atom structure. The two major pathways of eicosanoid metabolism are the cyclooxygenase pathway, which yields the prostaglandins and thromboxanes, and the lipoxygenase pathway, which yields the leukotrienes. A minor pathway termed epoxy-genase yields epoxides, which have received scant attention in the central nervous system (CNS). Arachidonic acid is synthesized on demand from dietary linoleic acid by either a G protein-regulated phospholipase A or diglyceride lipase activation ( ), and it yields a very broad array of bioactive metabolites, as shown in . The three major groups of arachidonic acid-derived metabolites are the prostaglandins, thromboxanes, and leukotrienes. Fig. 12–1 Figure 12–2 2

' — — Historically, the earliest effect of prostaglandins arose with the recognition in the 1930s that fresh semen could induce contraction of myometrial muscle, and the name arose from the factor s anticipated origin. As chemical detection methods improved in the 1950s, two classes of prostaglandins were recognized a PGE class that was soluble in ether and a PGF class soluble in phosphate buffer ( in Swedish) and one of their sites of synthesis localized to seminal vesicles. Subsequent work indicated that virtually every organ could manufacture prostaglandins, and several distinct synthetic pathways were recognized. A major advancement occurred when John Vane proposed that aspirin and several other nonsteroidal anti-inflammatory drugs (NSAIDs) worked by inhibiting the prostaglandin-synthesizing enzyme cyclooxygenase. Work in the 1990s revealed that a second cyclooxygenase (COX-2) (and perhaps a third) was also present in the CNS. Since this enzyme was shown to be induced by inflammatory cytokines, it immediately suggested a separate target for relief of inflammatory pain, with the potential for reduced symptoms from the gastrointestinal tract irritation typically evoked by NSAIDs. COX-2 is also induced by the neurotransmitter GLU and inhibited by glucocorticoids. Unfortunately, prolonged use of COX-2 inhibitors has untoward cardiovascular side effects, leading to multiple litigious claims and diverting attention from this once-promising therapeutic modification. fosfat in vitro

γ [ ] “ ” It is well known that the eicosanoids, particularly the prostaglandin series, play an important modulatory role in nervous tissue, but it has been difficult to write a lucid account of specifically how and where they act. This is primarily due to the fact that they are not stored in tissues, nervous or other, but synthesized on demand, particularly in pathophysiological conditions. They act briefly (some with a half-life of seconds) and at extremely low concentrations (10− M). Although indomethacin is a good inhibitor of cyclooxygenase-1, blocking the conversion of arachidonic acid to prostaglandins, there are few specific inhibitors available to block lipoxygenase and epoxygenase. Thus, although it had been postulated that the E series of prostaglandins modulates noradrenergic release, blocks the convulsant activity of pentylenetetrazol, strychnine, and picrotoxin (possibly by increasing the level of -aminobutyric acid GABA in the brain), and increases the level of cAMP in cortical and hypothalamic slices, these effects were noted with the addition of substantial amounts of the prostaglandins. There was very little evidence in intact animals to support these neuronal findings. Since we skeptics all hold veritas in higher regard, the physiological relevance of the effect was in doubt. in vitro in vivo 10

Subsequently, however, direct evidence has established arachidonic acid and lipoxygenases as second messengers. The cascade begins with the binding of a neuroactive agent to its receptor. Then, according to findings from the Axelrod laboratory, the receptor is coupled to G proteins, which may either activate or inhibit phospholipase A , although this has not been conclusively established for all neural tissues. The activated enzyme promotes the release of arachidonic acid, which will then act intracellularly as a second messenger. Arachidonic acid and its metabolites can also leave the cell to act extracellularly as first messengers on neighboring cells. Eicosanoids have been shown to mediate the somatostatin-induced opening of an M channel in hippocampal pyramidal cells and the release of VIP in mouse cerebral cortical slices. At the supracellular level, prostaglandins of the E series have been held to be a mediator of fever and prostaglandins of the D series as regulators of sleep. It is thus becoming clear, despite enormous technological difficulties in assaying eicosanoids, that these agents are major messengers. 2

Another exciting chapter of the arachidonic acid story has been told separately, namely the endocannabinoids, which are described later in this chapter.

 Neurosteroids According to their discoverer, Etienne Baulieu, neurosteroids are those steroids that are synthesized in the nervous system either from cholesterol or by metabolism of blood-borne precursors but are found at levels in the nervous system that are independent of steroid synthesis by adrenals or gonads. Such steroids include at least two previously known steroid precursors, pregnenalone (PREG) and dehydroepiandrosterone (DHEA), that in the nervous system have effects alone or as sulfated esters. In the central and peripheral nervous systems, neurosteroid synthesis has been attributed to oligodendrocytes, astrocytes, and neurons. The peripheral benzodiazepine receptor found on the mitochondrial outer membrane has been suggested to allow cholesterol access to the P450 cleavage enzyme complex on the inner mitochondrial membrane, leading to PREG formation and subsequently to other neurosteroids ( ). de novo in situ Fig. 12–3 δ In contrast to the endocrine actions of adrenal steroids on the brain, acting at a distance and at very low concentrations, neurosteroids are thought to act locally as either autocrine (acting on the cells that synthesizes them) or paracrine (acting on cells close to the site of synthesis) signals. In this manner, the ability to activate myelin synthesis in oligodendrocytes may provide a reparative effect in multiple sclerosis. In their sulfated ester forms, both PREG and DHEA have been reported to be potent regulators of GABA and NMDA receptor functions, with PREG-S and DHEA-S inhibiting the effects of GABA; however, these effects on the GABA receptor are reduced if the complex contains a subunit. A α α α β α β α α α α α β α α α β In addition, further characterization of the neurosteroids suggests that a small group of pregnenolone-, progesterone-, or tetrahydrodeoxycorticosterone-derived catabolites are the active moieties in the CNS, namely 3 ,5 - and 3 ,5 -androsterone, 3 ,5 -THP pregnenolone, 3 ,5 -THP allopregnenolone, 3 ,5 -THDOC allotetrahydroDOC, 3 ,5 -THDOC tetrahydroDOC, and 3 ,5 - and 3 ,5 -androstanol (Morrow, 2007). These are the neurosteroids that are believed to be responsible for the largely GABA-enhancing actions to induce anxiolytic, sedative, and anticonvulsant activity through allosteric modifications at discrete sites on the receptor. Of interest is that systemic doses of ethanol at low to moderate pharmacological levels induce elevation of neurosteroids to levels capable of modifying GABA receptors and contributing to the cellular and behavioral effects of ethanol (see ). Chapter 18