Functional pharmacology in human brain

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Most neurological and psychiatric disorders involve selective or preferential impairments of neurotransmitter systems. Therefore, studies of functional transmitter pathophysiology in human brain are of unique importance in view of the development of effective, mechanism-based, therapeutic modalities. It is well known that central nervous system functional proteins, including receptors, transporters, ion channels, and enzymes, can exhibit high heterogeneity in terms of structure, function, and pharmacological profile. If the existence of types and subtypes of functional proteins amplifies the possibility of developing selective drugs, such heterogeneity certainly increases the likelihood of interspecies differences. It is therefore essential, before choosing animal models to be used in preclinical pharmacology experimentation, to establish whether functionally corresponding proteins in men and animals also display identical pharmacological profiles. Because of evidence that scaffolding proteins, trafficking between plasma membrane and intracellular pools, phosphorylation and allosteric modulators can affect the function of receptors and transporters, experiments with human clones expressed in host cells where the environment of native receptors is rarely reproduced should be interpreted with caution. Thus, the use of neurosurgically removed fresh human brain tissue samples in which receptors, transporters, ion channels, and enzymes essentially retain their natural environment represents a unique experimental approach to enlarge our understanding of human brain processes and to help in the choice of appropriate animal models. Using this experimental approach, many human brain functional proteins, in particular transmitter receptors, have been characterized in terms of localization, function, and pharmacological properties.


Raiteri M. Functional pharmacology in human brain. Pharmacol Rev 2006; 58:162-193.


I. INTRODUCTION

Receptors, transporters, ion channels, and enzymes in the central nervous system (CNS1) exhibit high heterogeneity in terms of structure, function, and pharmacology. These protein isoforms, particularly neurotransmitter receptors, have traditionally been classified by pharmacological differences in the affinities of antagonists/inhibitors. The use of techniques of functional pharmacology, together with the availability of selective ligands, has led to the identification of multiple receptive sites in which the same neurotransmitter binds to elicit various responses. Studies of molecular biology have confirmed the existence of receptor types and subtypes within a single species by showing that pharmacological differences reflect differences in primary structure. It has subsequently become apparent that functionally equivalent receptors between species can display distinct pharmacological profiles and molecular biologists have identified key amino acids responsible for imparting ligand specificity. Clearly, this high receptor heterogeneity increases the likelihood of interspecies differences, thus making of unique importance studies on human brain functional proteins.

Mechanisms by which synaptic strength is modified play critical roles in neurotransmission. In the CNS, modification of the strength of synapses can be produced through neurotransmitters/neuromodulators released by neurons and glia. A classic mechanism by which the strength of synapses can be modulated involves the activation of receptors localized on neuronal axon terminals and termed presynaptic receptors (Starke et al., 1989; Bonanno and Raiteri, 1993; Langer, 1993; Miller, 1998; Raiteri, 2001). Presynaptic receptors can be activated by transmitters/modulators released by the terminals on which the receptors are localized (presynaptic autoreceptors) or by transmitters/modulators originating from neighboring structures (presynaptic heteroreceptors). Although the classic presynaptic modulatory systems involve metabotropic G protein-coupled receptors (GPCRs), presynaptic ionotropic receptors also can modify transmitter release [see Engelman and MacDermott (2004) for a review].

As to neurotransmitter transporters, their roles in brain physiology and pathology are only understood in part. These proteins represent highly sophisticated systems able to play more refined roles than simple transmitter (re)uptake. All known transmitter transporters can, under some conditions, mediate transmitter release directly from the cytosol (Attwell et al., 1993; Levi and Raiteri, 1993). Transporters may regulate the time course of synaptic events by modifying the extent of activation of receptors and the level of their desensitization (Seal and Amara, 1999; Sims and Robinson, 1999). Transporters for different transmitters can coexist and interact with each other on the same neuron [see Raiteri et al. (2002a) for a review]. Interactions between transporters and presynaptic receptors, as well as trafficking of transporters between the plasma membrane and intracellular pools, have been described previously (Reith, 2002).

It is now well accepted that native neurotransmitter receptors and transporters rarely exist as isolated entities; instead, they more frequently consist of "complexes," sometimes composed of several proteins (see, for instance, Husi et al., 2000; Couve et al., 2004). There is convincing evidence that GPCRs can exist as homo- or heterooligomers. Oligomerizations seem to occur physiologically or pathologically and may determine the pharmacology of GPCRs (Bouvier, 2001; Agnati et al., 2003; Milligan, 2004). Functional changes in receptor properties can also originate from the trafficking of GPCRs as well as of ionotropic receptors which takes place under a variety of conditions [see Collingridge and Isaac (2003) and Tan et al. (2004) for reviews]. Another important factor that can elicit changes in the function and pharmacology of native receptors is represented by the interactions between two different receptors or between a receptor and a transporter localized on the same plasma membrane. All these events might provide molecular explanations for native receptor pharmacology that is not easily explained by heterologous expression of human receptors, which are unlikely to find, in a foreign environment, the ingredients present in their natural milieu. Native functional proteins (receptors, transporters, ion channels, and enzymes) represent therefore the best systems when investigating synaptic receptor function, secretory and transport processes and their modulations. Unfortunately, due to serious degradative postmortem changes, functional studies in human brain generally require the availability of fresh brain tissue samples, which represents a major problem in following this approach.

The present article is essentially focused on functional studies performed with fresh human brain tissue not considered in my previous review (Raiteri, 1994). Data from laboratory animal experiments are here discussed only in comparative terms to help in the choice of the animal species possibly representing appropriate models to be used in preclinical studies.


II. CHOLINERGIC RECEPTORS

Muscarinic receptors and their subtypes were the first cholinergic receptors to be functionally identified and characterized in human brain. In particular, a number of studies were published in the early 1990s dealing with muscarinic autoreceptors mediating inhibition of acetylcholine (ACh) release from human neocortex tissues (Feuerstein et al., 1990, 1992; Marchi et al., 1990; Beani et al., 1992). These works have already been considered in my previous review (Raiteri, 1994). Curiously, to my knowledge, no new reports of functional studies on human brain muscarinic receptors have appeared in the last decade. In contrast, there has been increasing interest in the identification and pharmacological characterization of native nicotinic ACh receptors (nAChRs) through functional studies with samples of human brain obtained in neurosurgery.

Nicotine produces several central effects, some of which seem to be beneficial, e.g., increased alertness, reduced anxiety, analgesia, and facilitation of cognitive processes. Subtypes of the nAChR have been proposed as potential targets for drugs to be used in Alzheimer's disease and in other pathological conditions (Lloyd and Williams, 2000; Paterson and Nordberg, 2000); the knowledge of their function, structural subunit composition, and pharmacology in the human CNS is therefore of critical importance for the development of mechanism-based therapeutic approaches.


II.A. Norepinephrine Release Regulation by Nicotinic Receptors

Nicotinic receptors are largely localized on presynaptic axon terminals where they mediate regulation of neurotransmitter release (Wonnacott, 1997). Nicotine was found to cause norepinephrine (NE) release from human neocortex slices through activation of mecamylamine-sensitive mechanisms (Pittaluga et al., 1999). Although, based on experiments with rat brain synaptosomes, nAChRs mediating an increase in NE release can be thought to exist on noradrenergic nerve terminals (Clarke and Reuben, 1996; Luo et al., 1998; Vizi, 1998; Risso et al., 2004), to what extent nicotinic agonists directly act at noradrenergic terminals in human cortex to evoke NE release remains to be established; in fact, the release of NE elicited by nicotine in human cortical slices was in part prevented by glutamate N-methyl-D-aspartate (NMDA) receptor antagonists, suggesting an indirect mechanism whereby nicotine can provoke glutamate release onto NMDA receptors mediating release of NE (Pittaluga et al., 1999). Nicotine has indeed been reported to increase glutamate release in various animal preparations (Gray et al., 1996; Fedele et al., 1998) as well as in human cerebrocortex (Marchi et al., 2002b; see below).

The release of NE evoked by nicotine from human cerebrocortical slices prepared from specimens removed during neurosurgery for intractable severe epilepsy was external Ca2+-dependent and attenuated by the N-type voltage-sensitive calcium channel (VSCC) blocker {omega}-conotoxin GVIA (Woo et al., 2002), suggesting activation of VSCCs following depolarization provoked by nicotine. As the nAChRs involved are insensitive to {alpha}-bungarotoxin, the authors tend to exclude the theory that {alpha}7 subunit-containing nAChRs play a relevant role, although the use of some other selective antagonist would be advisable. Interestingly, Woo et al. (2002) found that the nicotine-evoked release of NE was prevented by nitric-oxide synthase inhibitors, indicating the involvement of nitric oxide (NO). Moreover, the nicotine effect was attenuated by guanylyl cyclase inhibitors and potentiated by the phosphodiesterase inhibitor zaprinast, consistent with the involvement of cyclic GMP. Although the authors, based on a few results with glutamate receptor antagonists, proposed that endogenous glutamate does not play a role in the nicotine effect, the stimulation by nicotine of the NO/cyclic GMP pathway would seem compatible with the idea that, in human neocortical slices, nicotine causes release of glutamate onto NMDA receptors mediating both NO production (Maura et al., 2000) and NE release (Fink et al., 1992a).

Nicotinic receptors mediating NE release have recently been characterized by Amtage et al. (2004) using fresh specimens of human neocortex obtained during surgical access to remove epileptic or neoplastic tissue. Slices were prepared, prelabeled with [3H]NE, and exposed in superfusion to nicotinic receptor ligands. The authors compared the release of [3H]NE from human neocortex slices to that from rat neocortex slices. The nicotine-evoked release of [3H]NE from human slices was found to be reduced in part by glutamate receptor antagonists, consistent with the findings and interpretation given by Pittaluga et al. (1999) that only part of the nAChRs involved in NE release are located on noradrenergic neurons, whereas some are on glutamatergic neurons from which glutamate is released onto noradrenergic terminals. Of note, Amtage et al. (2004) report that nicotinic agonists seem unable to evoke release of [3H]NE from rat neocortical slices, which would make it inappropriate to use the rat cortex as a model to screen for NE release-enhancing nicotinic receptor agonists.

In their work Amtage et al. (2004) characterized pharmacologically the subunit composition of the nAChRs-mediating enhancement of NE release in the human neocortex by using a number of selective nAChR antagonists. The nicotine-evoked [3H]NE release was sensitive to {alpha}-conotoxin MII, an antagonist that selectively blocks {alpha}3beta2- and {alpha}6-containing nAChRs. On the other hand, {alpha}-conotoxin AuIB, a selective {alpha}3beta4-subtype antagonist, failed to block the nicotine effect, thus excluding the involvement of {alpha}3beta4 subunit-containing nAChRs, in contrast to some observations on rat brain tissue (Vizi and Lendvai, 1999; Anderson et al., 2000).

Surprisingly, Amtage et al. (2004) found no evidence for the presence of nicotinic autoreceptors on cholinergic terminals in both human and rat neocortex. The authors admit that their "conclusion is in contrast to previous studies with mice and rats which suggest the occurrence of presynaptic nicotinic autoreceptors in the rodent brain." But the negative findings of Amtage et al. (2004) particularly contrast with results by Marchi et al. (2002b) showing the presence of release-enhancing nicotinic autoreceptors on cholinergic axon terminals isolated from human neocortical tissue (see below). The reasons for these discrepancies may be manifold, although it seems that the use of human (or rat) slices prevents in some way the identification of presynaptic receptors that can be clearly observed in superfused isolated axon terminal preparations.


II.B. Nicotinic {alpha}7 Receptors and Glutamate Release

The existence of presynaptic nAChRs able to mediate glutamate release had been suggested by different authors, based on electrophysiological and neurochemical studies in the laboratory animals (McGehee and Role, 1995; Gray et al., 1996; Radcliffe and Dani, 1998). However, direct demonstration that release-regulating nAChRs exist on glutamatergic axon terminals both in rat and human brain was obtained only recently (Marchi et al., 2002b). Experiments performed with synaptosomes prepared from fresh specimens of human cerebral cortex showed that nAChR agonists evoked release of glutamate, an effect prevented by {alpha}-bungarotoxin or by methyllycaconitine at concentrations compatible with their selective blockade of nAChRs containing {alpha}7 subunits. Similar results have been obtained using synaptosomes from the rat corpus striatum (Marchi et al., 2002b) and, more recently, the rat hippocampus and cortex (unpublished results). It may be added that the {alpha}7 nAChRs involved are presynaptic heteroreceptors localized on glutamatergic axon terminals because the superfusion system used only shows release modulations of a given transmitter when they are consequent to direct actions on the synaptosomes releasing that transmitter, whereas indirect effects are prevented (see Raiteri and Raiteri, 2000).

The {alpha}7 nicotinic receptors able to mediate glutamate release in the human cerebral cortex might be involved in the cognition-enhancing activity of nicotine (see Levin and Simon, 1998). The pharmacological similarity between human and rat nicotinic heteroreceptors mediating potentiation of glutamate release suggests that the rodent receptor could represent a useful model in the development of new {alpha}7 nAChR ligands.


II.C. Nicotinic Autoreceptors

When isolated human neocortex nerve endings, prelabeled with [3H]choline, were exposed in superfusion to nicotine or to ACh (in the presence of atropine to block inhibitory muscarinic autoreceptors), an increase in the release of [3H]ACh was observed (Marchi et al., 2002b). The releasing effect of ACh + atropine was insensitive to {alpha}-bungarotoxin or to methyllycaconitine, thus excluding the involvement of {alpha}7 nAChRs. The following considerations support the view that these autoreceptors mediating positive feedback regulation of ACh release are {alpha}4beta2 nAChRs as previously suggested for the rat nicotinic autoreceptor (Wilkie et al., 1996): 1) dihydro-beta-erythroidine, which completely blocked the effect of ACh at the autoreceptor, has been reported to be reasonably selective for the {alpha}4beta2 subtype (Albuquerque et al., 2000); 2) the EC50 values for (-)-nicotine (1 µM) and ACh (5 µM) in activating human autoreceptors are in keeping with those found at {alpha}4beta2 receptors heterologously expressed in different systems (McGehee and Role, 1995; Gopalakrishnan et al., 1997); 3) nicotine was significantly more potent on human {alpha}4beta2 than on human {alpha}4beta4, {alpha}2beta4,{alpha}3beta2, and {alpha}3beta4 receptors (Stauderman et al., 1998); 4) {alpha}4beta2 nAChRs constitute the major subtype lost in the cortex and hippocampus of Alzheimer's patients (Warpman and Nordberg, 1995), compatible with their presence on degenerating cholinergic axon terminals.

It seems important that the nicotinic autoreceptors localized on cholinergic terminals represent distinct nAChRs compared with the nicotinic heteroreceptors present on glutamatergic nerve endings: these two native subtypes of nAChR have different neuronal localization, subunit composition, function, and pharmacology (Marchi et al., 2002b). The pharmacological diversity offers a number of opportunities in terms of therapeutic intervention: autoreceptor ({alpha}4beta2) agonists and heteroreceptor ({alpha}7) agonists are expected to reinforce the acetylcholine-glutamate pathway by increasing, respectively, ACh release and glutamate release. Moreover, the pharmacology of these native human nAChRs seems very similar to that of the corresponding native rat receptors. Thus, monitoring glutamate and ACh release from rat brain synaptosomes may represent a convenient approach to functionally evaluate novel ligands at {alpha}7- or {alpha}4beta2-containing nAChRs.


III. ADRENERGIC RECEPTORS

The existence of several types and subtypes of adrenergic receptors and the consequent possibility of species differences would require extensive characterization of human adrenoceptors before appropriate animal models are chosen. However, functional studies of adrenergic receptors in human brain have focused exclusively on {alpha}2-receptors regulating neurotransmitter release. Of these studies, those describing {alpha}2-adrenergic regulation of ACh and glutamate release (Beani et al., 1992), of GABA release (Ferraro et al., 1993) and of serotonin release (Raiteri et al., 1990; Feuerstein et al., 1993) from human cerebral cortex tissues have already been considered in my previous review (Raiteri, 1994).


III.A. Norepinephrine Release Regulation through Autoreceptors

Similarly to what had been shown in several laboratory animal species [see Starke et al. (1989) for a review], human neocortex autoreceptors exhibit an {alpha}2-adrenoceptor pharmacology (Raiteri et al., 1992). Experiments with human neocortical slices prelabeled with [3H]NE and stimulated electrically showed that the Ca2+-dependent and tetrodotoxin-sensitive release of the catecholamine was inhibited by clonidine (an {alpha}2 agonist) and by oxymetazoline (an {alpha}2 agonist that displays high-affinity for {alpha}2A/{alpha}2D receptors and low affinity for the {alpha}2B/{alpha}2C subtypes). Moreover, the human autoreceptors were insensitive to prazosin and to ARC239 (antagonists that exhibit high affinity for {alpha}2B and {alpha}2C adrenoceptors but low affinity for {alpha}2A/{alpha}2D subtypes). Based on these results, obtained with the few selective ligands available in the early 1990s, it was concluded that the presynaptic {alpha}2-autoreceptors that modulate NE release in the human neocortex are not {alpha}2B/{alpha}2C but are either {alpha}2A or {alpha}2D (Raiteri et al., 1992).

It was subsequently established that {alpha}2A- and {alpha}2D-adrenoceptors are species orthologs, of which only one occurs in a given species and that humans possess the {alpha}2A version, whereas rodents possess the {alpha}2D version (Sastre and García-Sevilla, 1994; Bylund, 1995). Human neocortical {alpha}2-autoreceptors therefore belong to the {alpha}2A subtype of the adrenoceptor.

In 2000, Feuerstein et al. reinvestigated the subtype to which the {alpha}2-autoreceptors belong in the human neocortex by using nine antagonists, including prazosin and ARC239, of which the authors could evaluate the dissociation constants (pKB values) at the autoreceptors. The pKB values of prazosin and ARC239 were the lowest (more than 2 orders of magnitude lower than that of rauwolscine), in keeping with the previous findings of Raiteri et al. (1992) showing insensitivity of the human autoreceptors to the two compounds. Compared with binding or functional results from the literature, the pKB values of the nine antagonists tested by Feuerstein et al. (2000) correlated best with the antagonist affinities at {alpha}2A binding sites, leading the authors to the definitive conclusion that the human neocortex presynaptic autoreceptors are {alpha}2A. In considering that the rodent release-regulating {alpha}2-autoreceptors belong to the {alpha}2D subtype (Limberger et al., 1995), whereas the rabbit and pig cortical autoreceptors are {alpha}2A (Limberger et al., 1995; Trendelenburg et al., 1997), it seems appropriate to use brain cortical tissues from guinea pigs or pigs as human autoreceptor models.


IV. DOPAMINE RECEPTORS AND TRANSPORTERS

IV.A. Dopamine Autoreceptors

The existence of release-regulating dopamine (DA) autoreceptors in human brain was investigated in slices from fresh specimens of human neocortex that were labeled with [3H]DA and stimulated electrically (Fedele et al., 1993). Whereas the selective DA-1 receptor agonist SKF38393 did not affect the electrically evoked release of DA, quinpirole, an agonist at the receptors of the DA-2 family, with preference for the DA-2/DA-3 subtypes, inhibited the evoked release in a concentration-dependent manner. Quinpirole was antagonized by (-)-sulpiride, a DA-2 receptor antagonist with preference for the DA-2/DA-3 subtypes.

In a subsequent work, Fedele et al. (1999) investigated the pharmacological profile of DA autoreceptors in human neocortex further, to establish the subtype (DA-2, DA-3, or DA-4) to which the receptors belong. The quinpirole inhibition of the electrically evoked DA release was unaffected by the selective DA-4 receptor antagonist L-745,870 and by the selective DA-3 receptor antagonist S14297, leading to the conclusion that, in human neocortex, the release of DA in the terminal region of midbrain dopaminergic neurons is regulated through autoreceptors of the DA-2 subtype.

To identify a readily accessible model to be used in the development of selective DA-2 receptor ligands, Fedele et al. (1999) compared the results from human neocortical slices with those obtained in three animal systems: rat neocortical slices, rat striatal slices, and rat mesencephalic neuronal cultures, from which DA release was electrically stimulated. Results from rat striatal slices, mesencephalic neurons and human neocortical slices were superimposable, whereas quantitative differences emerged in the case of rat cortical slices.


IV.B. Drugs of Abuse and Dopamine Release

Drug addiction is believed to result from the reinforcing properties of drugs of abuse on central reward systems, in particular on mesolimbic DA pathways (Di Chiara and Imperato, 1988; Koob et al., 1998). Although microdialysis experiments in rodents indicate that ethanol can evoke DA release in the nucleus accumbens (Di Chiara and Imperato, 1988; Rossetti et al., 1992; Weiss et al., 1993), studies on the effect of alcohol consumption by humans on dopaminergic transmission are very rare. Boileau et al. (2003) measured DA release in the ventral striatum/nucleus accumbens of six healthy subjects in response to alcohol oral ingestion using positron emission tomography and the DA receptor ligand [11C]raclopride. Previous experiments in primates had shown that the binding of [11C]raclopride is inversely proportional to the extracellular DA concentration in the striatum [see Laruelle (2000) for a review]. Boileau et al. (2003) observed a significant reduction in [11C]raclopride binding potential bilaterally in the ventral striatum/nucleus accumbens, indicative of increased extracellular DA. The above technique had previously been used in humans to evaluate extracellular DA augmentation in response to various psychostimulants (Carson et al., 1997; Schlaepfer et al., 1997; Drevets et al., 2001; Volkow et al., 2001; Leyton et al., 2002; Martinez et al., 2003). Altogether, these findings support the idea that mesolimbic dopaminergic activation is a common property of abused drugs in humans.


IV.C. Drugs of Abuse and Dopamine Transporters

Dopamine transporters (DATs) are targets for cocaine and other psychostimulants (Amara and Kuhar, 1993). In particular, cocaine potentiates dopaminergic transmission by blocking the reuptake of DA, leading to elevations in the synaptic level of the neurotransmitter. In vivo imaging studies in humans and in vitro binding studies in postmortem human brain have shown that chronic cocaine abuse results in an increase in DAT binding site density in the nucleus accumbens [see Mash et al. (2002) for references]. These data do not indicate, however, whether the increase in DAT binding sites reflects and increase in the function of DA transporters. Mash et al. (2002) measured uptake of [3H]DA in metabolically active synaptosomes isolated from cryoprotected human brain specimens. The uptake of DA was elevated 2-fold in the ventral striatum from cocaine users compared with age-matched drug-free control subjects. This seems to be the first demonstration of adaptations in DA uptake in postmortem human brain. Based on the results, it is likely that chronic cocaine use, by causing an increase in DAT levels, results in a reduced amount of synaptic DA available to stimulate postsynaptic receptors. According to Mash et al. (2002), "the need to maintain homeostasis within central dopaminergic systems may be one of the factors that drives the compulsive use of cocaine."


V. SEROTONINERGIC RECEPTORS

Pharmacological studies as well as molecular cloning of 5-hydroxytryptamine (5-HT; serotonin) receptors have revealed high receptor heterogeneity. Seven major types of the 5-HT receptor have been identified and termed 5-HT1 to 5-HT7. Most of these receptor types are heterogeneous and occur as subtypes. Species homologs of the same receptor subtype may exist which, despite high structural homology, may display pronounced pharmacological differences (Hoyer and Middlemiss, 1989; Hoyer et al., 1994; Martin and Humphrey, 1994; Hartig et al., 1996).

Among the functions that have been attributed to 5-HT receptors, animal studies have shown that 5-HT receptor activation can mediate modulation of the release of various neurotransmitters. A number of functional studies with native human brain 5-HT receptors have been performed and deal with both autoreceptors and heteroreceptors.


V.A. Release of Serotonin and Control by Human 5-Hydroxytryptamine 1B Autoreceptors

Human neocortical slices, prelabeled with [3H]5-HT, were found to release the [3H]indoleamine upon electrical stimulation. This release was Ca2+-dependent and could be inhibited by exogenous 5-HT. The inhibition was prevented by methiothepin, a broad spectrum 5-HT receptor antagonist (Schlicker et al., 1985). These results show that release-inhibiting presynaptic 5-HT autoreceptors, previously found to be present in the rat brain (Cerrito and Raiteri, 1979; Göthert and Weinheimer, 1979), also exist in the human brain.

Based on pharmacological studies, rat brain 5-HT autoreceptors had originally been classified as 5-HT1B subtype (Engel et al., 1986; Maura et al., 1986). However, 5-HT1B binding sites could not be detected in the human brain (Hoyer et al., 1986); therefore, it was expected that the human autoreceptors would have been pharmacologically different from those in the rat brain.

A subtype of the 5-HT binding site, termed 5-HT1D, was then identified in human brain (Herrick-Davis et al., 1988; Hoyer et al., 1988) as well as in the brain of other species lacking the 5-HT1B site (Bruinvels et al., 1992) and found to display a regional distribution similar to that of the 5-HT1B site in rodents (Waeber et al., 1989). On the basis of this new information, receptors regulating the release of [3H]5-HT from human neocortex slices (Galzin et al., 1992) and human neocortex synaptosomes (Maura et al., 1993) were proposed to belong to the 5-HT1D subtype.

Subsequent cloning studies revealed that 5-HT1D receptors are heterogeneous: two members of the subfamily, which were called 5-HT1D{alpha} and 5-HT1Dbeta, were identified in the human brain (Hartig et al., 1992). Interestingly, replacement by site-directed mutagenesis of the threonine residue present at position 355 in the human 5-HT1Dbeta receptor with the corresponding asparagine found in rodent 5-HT1B receptors was found to render the pharmacology of the receptors, originally quite different, essentially identical (Oksenberg et al., 1992; Parker et al., 1993). The human 5-HT1Dbeta and the rodent 5-HT1B receptors are encoded by a corresponding gene and may have the same biological functions in the two species. Having assumed this, it was proposed that the human autoreceptor be classified as 5-HT1Dbeta (now termed h5-HT1B) (Maura et al., 1993; Fink et al., 1995).

A clear confirmation that, in human cerebral cortex, the terminal 5-HT autoreceptor is of the h5-HT1B subtype came from experiments with an h5-HT1B selective ligand, SB-236057, which was shown to block the human terminal autoreceptor in a release study with human neocortex slices stimulated electrically (Middlemiss et al., 1999).

The available literature shows that h5-HT1B receptors differ pharmacologically from the rodent r5-HT1B receptors (Oksenberg et al., 1992; Parker et al., 1993). Therefore, the rodent autoreceptor cannot be useful as a model for the human counterpart. Other animal species, including guinea pig, pig, and rabbit, possess 5-HT autoreceptors that pharmacologically resemble the h5-HT1B subtype and may represent appropriate models for the human 5-HT autoreceptor.


V.B. Glutamate Release and Modulation by Human 5-Hydroxytryptamine 1D Receptors

Animal studies suggest that 5-HT can interact with glutamate through multiple receptors to inhibit excitatory transmission in the CNS [see Maura et al. (1998) and references therein]. Because excessive glutamate release has been implicated in a number of pathophysiological conditions, understanding how the release of the excitatory transmitter can be regulated in the human CNS may lead to novel therapeutic avenues.

Several years ago we found that the release of glutamate from rat cerebellar synaptosomes could be potently inhibited through the activation of an unknown subtype of the 5-HT1 receptor that we suggested be named 5-HT1D (Raiteri et al., 1986). When the rat 5-HT1D (now termed r5-HT1D) was cloned, it was proposed to represent the species homolog of the human 5-HT1D (now h5-HT1D) receptor (Hamblin and Metcalf, 1991; Hartig et al., 1996).

To investigate whether 5-HT could directly modulate the release of glutamate in human brain, the efflux of glutamic acid and its modulation by 5-HT were studied in synaptosomal preparations from fresh neocortical samples obtained from patients undergoing neurosurgery to reach deeply sited tumors. Depolarization with high-K+ elicited Ca2+-dependent release of endogenous glutamate, which was inhibited by exogenous 5-HT and by the 5-HT1B/1D selective agonist sumatriptan. The agonist effects were blocked by concentrations of ketanserin known to antagonize preferentially the h5-HT1D versus the h5-HT1B receptors (Maura et al., 1998).

These results represent the first functional characterization of a 5-HT1D receptor in the adult human brain. In considering that the human terminal autoreceptor is h5-HT1B, the emerging pharmacological differences between h5-HT1B auto- and h5-HT1D heteroreceptors (see below) may lead to the development of novel therapeutic agents. Inhibition by 5-HT of glutamatergic transmission may be useful in a variety of pathological conditions including epilepsy [see Clough et al. (1996) and references therein], ischemia (Marcoli et al., 2004) (see also section XIII.C.) and depression [see Bonanno et al. (2005) and references therein].


V.C. Pharmacological Diversity between Human 5-Hydroxytryptamine 1B and Human 5-Hydroxytryptamine 1D Receptors

h5-HT1B and h5-HT1D receptors have been reported to have relatively low (63%) overall amino acid homology; in contrast, for a series of 19 structurally diverse compounds, the two cloned human receptors were originally found to be nearly indistinguishable in their binding affinities (Hartig et al., 1992; Weinshank et al., 1992). This view, based on radioligand binding experiments with cell lines expressing the cloned h5-HT1B and h5-HT1D receptors, is no longer tenable in the light of the functional studies of native receptors carried out with human neocortex synaptosomes (Marcoli et al., 1999). This study analyzed the pharmacological profiles of the presynaptic h5-HT1B autoreceptors regulating the depolarization-evoked release of [3H]5-HT and of the presynaptic h5-HT1D heteroreceptors regulating the depolarization-evoked release of endogenous glutamate from human cerebral cortex synaptosomes. The nerve terminals were exposed in superfusion to eight serotonergic ligands during depolarization. Whereas two compounds, sumatriptan and methiothepin, behaved similarly at the auto- and at the heteroreceptors, the behavior of the other six compounds at the h5-HT1B autoreceptor differed strikingly from that at the h5-HT1D heteroreceptor (Table 1). Of note, some drugs known as 5-HT receptor antagonists, in particular metergoline and GR127935, behaved as blockers at the autoreceptors but exhibited potent intrinsic activity at the heteroreceptors, thus strongly inhibiting glutamate release.


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TABLE 1 Pharmacological diversity between h5-HT1B autoreceptors regulating 5-HT release and h5-HT1D heteroreceptors regulating glutamate release in human neocortex Data taken from Marcoli et al. (1999).

Sumatriptan
- h5-HT1B: Agonist
- h5-HT1D: Agonist

Methiothepin
- h5-HT1B: Antagonist
- h5-HT1D: Antagonist

Ketanserin
- h5-HT1B: Inactive
- h5-HT1D: Antagonist

GR127935
- h5-HT1B: Antagonist
- h5-HT1D: Agonist

Metergoline
- h5-HT1B: Antagonist
- h5-HT1D: Agonist

SB-224289
- h5-HT1B: Antagonist
- h5-HT1D: Inactive

BRL-15572
- h5-HT1B: Inactive
- h5-HT1D: Antagonist

(+)-WAY 100135
- h5-HT1B: Inactive
- h5-HT1D: Agonist
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SB-224289, 2,3,6,7-tetrahydro-1'-methyl-5-[2'-methyl-4'(5-methyl-1,2,4-oxad iazol-3-yl)biphenyl-4-carbonyl]furo[2,3f]indole-3-spiro-4'-pipe ridine hydrochloride; BRL-15572, 3-[4-(4-chlorophenyl)piperazin-1-yl]-1,1-diphenyl-2-propanol HCl; (+)-WAY 100135, N-tert-butyl-3-4-(2-methoxyphenyl) piperazin-1-yl-2-phenylpropanamide
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To conclude, our functional studies of native h5-HT1B and h5-HT1D receptors in the cerebral cortex show that the two receptors, originally reported to be pharmacologically identical (Weinshank et al., 1992), exhibit in fact a strikingly different pharmacology. The finding that compounds known to behave as pure 5-HT receptor antagonists in experiments with laboratory animals become full agonists at native 5-HT1D receptors in human brain may be due to several reasons, including the exsistence of a high reserve of h5-HT1D receptors. Whatever the explanation, considering the variability of the results obtained with cell systems expressing recombinant receptors, as well as the species variations in ligand affinities previously observed (see Zgombick et al., 1997), it is evident that functional studies with native human receptors remain of unique importance.

The pharmacological diversity of human 5-HT1B and 5-HT1D receptors augurs well for the potential development of receptor-selective drugs. In particular, the clear differences existing between release-regulating h5-HT1B terminal autoreceptors and h5-HT1D terminal heteroreceptors in human brain open the possibility for development of drugs that are selective autoreceptor antagonists or heteroreceptor agonists or both and that may represent novel mechanism-based therapeutic approaches potentially useful in conditions characterized by defective serotonergic transmission or/and excessive glutamatergic transmission including depression, epilepsy, and neurodegenerative diseases.


V.D. GABA Release and Modulation by 5-Hydroxytryptamine

Feuerstein et al. (1996a) investigated the presence of 5-HT receptors regulating the release of GABA provoked by electrical stimulation of human and rabbit neocortical slices. The slices were prelabeled with [3H]glutamine to study the release of endogenously formed [3H]GABA. The release of [3H]GABA elicited by electrical stimulation was external Ca2+-dependent and tetrodotoxin-sensitive. The addition of sumatriptan, a 5-HT1B/1D receptor agonist, decreased the release of [3H]GABA, whereas methiothepin, a broad spectrum 5-HT receptor antagonist, prevented the sumatriptan effect. The use of slices does not permit a precise localization of the receptor, whether on the soma/dendrites or the terminals of GABA neurons or on both. As to the subtype of the 5-receptor involved, the authors proposed the 5-HT1D as the most likely. As discussed above, in 1996, the human 5-HT1D nomenclature comprised two receptors termed 5-HT1D{alpha} and 5-HT1Dbeta (now h5-HT1D and h5-HT1B); accordingly Feuerstein et al. (1996a) proposed that the 5-HT1D receptor regulating GABA release in human neocortex was the 5-HT1D{alpha} or 5-HT1Dbeta subtype. The pharmacology of this receptor could be characterized more precisely by using the tools now available (see Marcoli et al., 1999).

Interestingly, in rabbit neocortex slices, the release of GABA could not be inhibited by sumatriptan. In addition, 5-carboxamidotryptamine, a broad spectrum 5-HT1 agonist, was unable to affect the release of GABA evoked by electrical stimulation of rabbit neocortex, suggesting that receptors of the 5-HT1 type regulating GABA release may not exist on GABAergic neurons of the rabbit neocortex.


V.E. Serotonin Inhibition of the N-Methyl-D-aspartate Receptor/Nitric Oxide/Cyclic GMP Pathway

The NMDA receptor/NO/cyclic GMP pathway and its possible modulation by 5-HT were studied in slices of human neocortex (Maura et al., 2000). Exposure of slices to NMDA caused cyclic GMP elevation, which was blocked by NO synthase and guanylate cyclase inhibitors. The NMDA effect was potently prevented by 5-HT or by the 5-HT2 receptor agonist (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane. Further pharmacological analysis with 5-HT2A, 5-HT2B, and 5-HT2C receptor antagonists and the novel selective 5-HT2C agonist Ro60-0175 showed that the 5-HT2 receptor involved belongs to the 5-HT2C subtype.

The 5-HT2C receptor is not the only serotonergic receptor involved, however. In fact, the 5-HT1A agonist 8-OH-DPAT inhibited the NMDA-evoked cyclic GMP response, an effect blocked by selective 5-HT1A antagonists.

The work by Maura et al. (2000) also described the ability of the antidepressant trazodone to inhibit the NMDA receptor/NO/cyclic GMP pathway through the activation of 5-HT2C receptors. The mechanism of action of trazodone as an antidepressant is not well understood, although a number of authors have proposed interactions with the serotonergic system [see Maura et al. (2000) for references]. The finding that trazodone, at concentrations compatible with those reached during antidepressant treatment, inhibited the NMDA receptor-mediated production of NO and cyclic GMP in human neocortical slices through receptors of the 5-HT2C subtype suggests that activation of 5-HT2C receptors could be relevant to the antidepressant activity of trazodone and, possibly, of selective serotonin reuptake inhibitors that also indirectly activate 5-HT2C receptors. It should be noted that antidepressants were reported to produce adaptive changes, generally inhibitory, of NMDA receptor functions (Leonard, 1997; Nowak et al., 1998).

Altogether, the results available from experiments with fresh human brain tissues indicate that 5-HT is a major inhibitory agent of glutamatergic transmission in the human cerebral cortex able to act at multiple sites (Fig. 1). Not only can serotonin inhibit the evoked release of glutamate from nerve terminals by acting at presynaptic h5-HT1D receptors (Maura et al., 1998); it also can inhibit events triggered by glutamate release, i.e. the NMDA receptor/NO/cyclic GMP pathway, by acting at postsynaptic receptors of the 5-HT2C and 5-HT1A subtype. Agonists at human 5-HT1D, 5-HT2C, and 5-HT1A receptors or antagonists at 5-HT1B autoreceptors, deserve attention as potentially useful drugs in neuropathologies with underlying defective serotonergic or excessive glutamatergic transmission.


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FIG. 1. Multiple 5-HT receptors regulate glutamatergic transmission in the human neocortex. Serotonin released from 5-HT terminals can activate 5-HT1B autoreceptors mediating negative feedback regulation of 5-HT exocytosis and 5-HT1D heteroreceptors mediating inhibition of release from glutamate (GLU) terminals. Serotonin, acting at postsynaptic 5-HT1A and 5-HT2C receptors, can also depress the NO/cyclic GMP production elicited by activation of postsynaptic ionotropic glutamate receptors.
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VI. GABA RECEPTORS

Ionotropic GABAA receptors have been the object of a number of functional studies with human brain tissue removed to treat drug-resistant epilepsies (see section XII.A.1.). As to receptors of the GABAB type, several reports deal with these metabotropic GABA receptors in human brain and are mainly focused on the existence of GABAB receptor subtypes [see Bonanno and Raiteri (1993) and Bowery et al. (2002) for reviews].


VI.A. GABAB Receptor Subtypes: Pharmacological Evidence

1. GABAB Autoreceptors.
The release of GABA from human cerebral cortex synaptosomes depolarized in superfusion with high-K+ was inhibited by the GABAB receptor agonist (-)-baclofen, but not by the GABAA agonist muscimol, suggesting the presence of release-regulating GABAB autoreceptors in human neocortex GABAergic terminals (Bonanno et al., 1989). The existence of GABAB autoreceptors in human CNS was confirmed when the introduction of potent, selective GABAB receptor antagonists (Froestl et al., 1995) facilitated studies of GABAB receptors. In particular, the human GABAB autoreceptor was characterized using three antagonists: phaclofen, CGP35348 and CGP52432 (Fassio et al., 1994). The effect of (-)-baclofen on the evoked release of GABA from human neocortex synaptosomes was differentially reduced by the three antagonists: CGP52432 was by far the most potent (IC50 = 0.09 µM); phaclofen was much less potent (IC50 = 70 µM); CGP35348 was ineffective up to 100 µM. These results parallel those previously obtained with the neocortex of the rat (Bonanno and Raiteri, 1993; Lanza et al., 1993), indicating that human and rat cortical autoreceptors are pharmacologically very similar (Table 2).


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TABLE 2 GABAB receptor subtypes regulating transmitter release in human and rat neocortex Reported are IC50 values of GABAB receptor antagonists tested against (-)-baclofen. Reproduced from Bowery et al. (2002).
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2. GABAB Heteroreceptors Regulating Neuropeptide Release.
Human neocortex synaptosomes released cholecystokinin-like immunoreactivity (CCK-LI) and somatostatin-like immunoreactivity (SRIF-LI) in a Ca2+-dependent manner when depolarized in superfusion with high-K+. The GABAB receptor agonist (-)-baclofen inhibited both the evoked release of CCK-LI and SRIF-LI. The effect of (-)-baclofen was mimicked by CGP47656 and blocked by CGP35348 or CGP52432. Interestingly, in experiments of GABA release, CGP47656 behaved as an antagonist at the GABAB autoreceptors (Bonanno et al., 1996; Raiteri et al., 1996). Together, these results indicate that 1) CCK- and SRIF-releasing terminals in human neocortex possess release-inhibiting GABAB heteroreceptors; 2) these receptors differ pharmacologically from the human GABAB autoreceptors since the latter are CGP35348-insensitive (Fassio et al., 1994) and can be blocked by CGP47656; 3) human heteroreceptors regulating CCK and SRIF release seem to be pharmacologically similar to the corresponding receptors found in rats (Gemignani et al., 1994; Table 2). Cholecystokinin has been implicated in anxiety (Woodruff and Hughes, 1991; Harro et al., 1993); thus, the finding that inhibitory GABAB heteroreceptors present on CCK-releasing terminals differ pharmacologically from the GABAB autoreceptors mediating inhibition of GABA release suggests that GABAB heteroreceptor-selective agonists may have therapeutic potential as novel anxiolytics. As to the somatostatinergic system, reduced release of SRIF seems to play a role in cognitive impairments typical of Alzheimer's disease (Gabriel et al., 1993) or accompanying other pathologies; selective GABAB heteroreceptor antagonists could lead to increased SRIF release and consequently to cognitive improvement (see below).

A GABAB receptor antagonist, CGP36742, had been reported to improve the performances of mice, rats, and monkeys in tests covering diverse manifestations of learning and memory (Mondadori et al., 1993; Froestl et al., 1995). We examined the ability of CGP36742 to block release-regulating GABAB receptors. In particular, CGP36742 was tested against the inhibition of the depolarization-evoked release of GABA, glutamate, CCK, and SRIF produced by (-)-baclofen in rat and human neocortex axon terminals (Bonanno et al., 1999). It was found (Table 2) that CGP36742 potently antagonized the inhibition by (-)-baclofen of the release of SRIF from both rat (IC50 = 0.14 µM) and human (IC50 ~ 5 µM) neocortex synaptosomes. In contrast, the effects of (-)-baclofen on GABA, glutamate, and cholecystokinin release were insensitive to CGP36742 at concentrations up to 100 µM. CGP36742 is the first GABAB receptor antagonist displaying great selectivity for the GABAB presynaptic receptors regulating SRIF release. Considering the implication of the neuropeptide in cognitive processes, disinhibition of SRIF release with activation of SRIF receptors may represent one of the mechanisms involved in the behavioral activity of CGP36742. In the rat brain, the CGP36742-induced somatostatin (sst5) receptor activation was found to facilitate the function of NMDA receptors (Pittaluga et al., 2000, 2001a), which are known to play a primary role in cognitive processes. If humans respond as rats, CGP36742 (now termed SGS742) could act according to the scheme shown in Fig. 2 Interestingly, CGP36742/SGS742 is the first GABAB receptor antagonist entered into clinical trials. In a phase II double-blind, placebo-controlled study in 110 patients with mild cognitive impairment, oral administration of the drug significantly improved attention as well as working memory. A second phase II clinical trial in 280 patients with Alzheimer's disease is underway (Froestl et al., 2004).


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FIG. 2. Schematic representation of the neurotransmitter interactions that may underlie the cognitive enhancing activity of CGP36742 (now termed SGS742). GABA and somatostatin (SRIF) are often colocalized in interneurons, although purely GABAergic terminals (not shown) also exist. Glutamate (GLU; red dots) evokes release of norepinephrine (NE; brown dots), GABA (blue dots), and SRIF (green dots) by activating ionotropic NMDA/AMPA receptors located on the respective axon terminals. GABA can inhibit its own release and SRIF release through different GABAB receptor subtypes. CGP36742 (SGS742) binds with high affinity to the GABAB heteroreceptor (violet) modulating SRIF release but with very low affinity to the GABAB autoreceptor (blue) modulating GABA release (Bonanno et al., 1999). SRIF positively modulates NE release by activating sst5 receptors (green) colocalized with NMDA receptors on noradrenergic terminals. When NMDA is added to slices, GABA is released and inhibits the release of SRIF. CGP36742 (SGS742) prevents this GABAergic inhibition, allowing the neuropeptide to reach the sst5 receptors on NE terminals. These SRIF receptors trigger activation of NMDA receptors through the phospholipase C (PLC)-inositol trisphosphate (IP3)-protein kinase C (PKC) pathway (Pittaluga et al., 2005).
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3. GABAB Heteroreceptors Regulating Glutamate Release.
In a comparative study on the release of endogenous glutamate and endogenous GABA from human neocortex synaptosomes, the effects of three selective GABAB receptor antagonists on the inhibition of the depolarization-evoked release of the two amino acids elicited by (-)-baclofen were investigated (Bonanno et al., 1997). Phaclofen antagonized the effect of (-)-baclofen on GABA release but did not modify that on the release of glutamate. The inhibition by (-)-baclofen of the release of GABA was insensitive to CGP35348 which, in contrast, blocked the heteroreceptors sited on glutamatergic terminals. Finally, CGP52432, added at 1 µM, blocked GABAB autoreceptors, but was ineffective at the heteroreceptors. The results (Table 2) show that the release of GABA and glutamate evoked by depolarization of human neocortex nerve terminals can be affected differentially through pharmacologically distinct GABAB receptors. These human receptors seem to be very similar to those present in the rat neocortex (Bonanno and Raiteri, 1992), suggesting that the rat receptors may be useful models in the development of selective ligands.


4. The Mystery of GABAB Receptor Subtypes.
Receptor heterogeneity is a general phenomenon. Types and subtypes of receptors have often been identified pharmacologically on the basis of their differential blockade by antagonists. Looking at the data reported in Table 2, the existence of subtypes of the GABAB receptor seems therefore undeniable, with strong similarities between human and rat receptors.

The problem is that, as a rule, pharmacological heterogeneity has been found to reflect structural diversity. This seems not to be the case for GABAB receptors, however. The structure of GABAB receptors is rather peculiar, the receptors being heterodimers composed of two subunits, GABAB1 and GABAB2; heterodimer formation is obligate for functional expression of GABAB receptors [see Bowery et al. (2002) for a review]. The mystery lies in the so far unsuccessful attempts to identify other subunits and the increasing belief that GABAB1 and GABAB2 exclusively form all GABAB receptors and that there are no further subunits to be discovered. According to some authors [see, for instance, the review by Couve et al. (2004)], the long-standing controversy on the existence of multiple GABAB receptor subtypes in the CNS may be explained by assuming the existence and the different composition of "GABAB receptor complexes" and signaling machinery in different neurons. In other words, GABAB1 and GABAB2 subunits would associate with several proteins, giving rise to complexes (see Couve et al., 2004) that differ between neurons and may underlie the pharmacological diversity of the same heterodimer. Although the mystery remains unsolved, the differential blockade by GABAB antagonists of receptors (or receptor complexes), sited on different neurons and performing different functions, represents a pharmacological heterogeneity that can be exploited to obtain therapeutically useful GABAB receptor ligands.


VII. GLUTAMATERGIC RECEPTORS

Excitatory amino acids have been the object of an impressive number of studies; however, functional glutamatergic transmission in human brain has been poorly investigated. Electrophysiological studies on glutamate receptors in human brain slices are described in section XII.A.1. in relation to epilepsy. Reports showing that activation of NMDA and non-NMDA ionotropic glutamate receptors can evoke release of NE from human neocortical slices (Fink et al., 1992a), that the NE release evoked by NMDA or kainic acid can be inhibited by ethanol (Fink et al., 1992b), and that high D-glucose, at concentrations compatible with hyperosmolar diabetic coma, differentially modifies the NMDA-evoked release of GABA and NE (Fink et al., 1994) were considered in my previous review (Raiteri, 1994). It should be noted that glutamate ionotropic and metabotropic receptors exist as subtypes having discrete pharmacological profiles, which makes interspecies differences quite likely; more intense investigation of functional glutamatergic transmission in human brain is therefore required.


VII.A. Metabotropic Glutamate Receptors

1. Phosphatidylinositol Turnover.
Glutamate metabotropic receptors linked to phosphatidylinositol hydrolysis have been identified in human cerebellum (Nicoletti et al., 1989) and neocortex (Morari et al., 1991; Dubeau et al., 1992). An interesting receptor-receptor interaction seems to occur in human cerebral cortical slices where the activation of the phosphatidylinositol turnover by quisqualate was found to be prevented by NMDA, suggesting that glutamate metabotropic receptor functions in the human neocortex are negatively modulated by NMDA receptor activation (Morari et al., 1991).

Morari et al. (1995) subsequently reported on the occurrence of another receptor-receptor interaction in the human brain. The authors observed that the stimulation of the phosphatidylinositol turnover brought about by glutamate metabotropic receptor agonists could be regulated through activation of ionotropic AMPA receptors. In particular, a submaximal AMPA and metabotropic receptor activation resulted in positive cooperation. Clearly, these early works were based on determinations of phosphatidylinositol hydrolysis in the presence of non-subtype-selective metabotropic receptor agonists and would therefore deserve further consideration taking advantage of the selective ligands now available.


2. Release of Acetylcholine.
In a recent work (Feligioni et al., 2003) carried out with nerve terminals prepared from human neocortex, it was found that activation of metabotropic glutamate receptors of group I elicited release of ACh through the production of inositol trisphosphate in cholinergic nerve endings. In contrast, exposure of rat cholinergic nerve endings to a metabotropic group I agonist failed to evoke ACh release. Interestingly, the HIV-1 protein Tat provoked ACh release both from human and rat synaptosomes; however, whereas in human neocortex nerve terminals Tat activates inositol trisphosphate-linked metabotropic group I receptors to elicit ACh release, the viral protein acts in rats at an as yet unidentified receptor that mediates ACh release through internal Ca2+ mobilization triggered by cyclic adenosine diphosphoribose (Feligioni et al., 2003) (see section XIV.B. for more details).


VII.B. N-Methyl-D-aspartate Glutamate Receptors

1. Release of Norepinephrine.
The stimulation of NE release by NMDA/glycine observed in human neocortical slices by Fink et al. (1992a) could also be seen in synaptosomes isolated from human neocortical tissue (Pittaluga et al., 1996) indicating the existence of presynaptic release-enhancing NMDA receptors on human noradrenergic axon terminals. In the absence of glycine, the HIV-1 coat protein gp120 allowed NMDA to enhance the release of NE from human noradrenergic nerve endings (Pittaluga et al., 1996) (see section XIV.A.). Experiments with NMDA receptor antagonists led to the conclusion that gp120 mimics glycine at the receptor coagonist site on the NR1 subunit, being ~4 orders of magnitude more potent than the natural coagonist glycine. Of interest, gp120, which also acts at the NMDA receptors present on rat noradrenergic terminals (Pittaluga and Raiteri, 1994), is unable to mimic glycine at the NMDA receptors sited on rat striatal dopaminergic terminals (Pittaluga et al., 2001b). The gp120 potentiation of the NMDA-evoked NE release in human brain was blocked by memantine (Pittaluga et al., 1996), a low-affinity, uncompetitive, open-channel blocker of the NMDA receptor (Lipton and Chen, 2004) and this finding might have therapeutic implication (see section XVI.A.).


. The "Kynurenate Test."
A biochemical assay for putative cognition enhancers, termed the "kynurenate test," was introduced by Pittaluga et al. (1997). In this test, the NMDA-evoked release of NE in rat hippocampal slices is antagonized by kynurenic acid and putative cognition enhancers are tested for their ability to counteract the kynurenate antagonism. Several behaviorally active compounds were evaluated; based on the results, the kynurenate test seems to represent a useful assay for putative cognition enhancers acting through the glutamate system via NMDA receptors (Pittaluga et al., 1997).

It was important to establish whether compounds displaying activity in the rat brain responded similarly in a kynurenate test performed with human brain tissue. Slices from human neocortex were exposed to NMDA; the evoked release of NE was antagonized by kynurenic acid. This antagonism was potently prevented by behaviorally active compounds (aniracetam, oxiracetam, D-cycloserine, and the glutamate analog CR2249, but not its enantiomer CR2361) previously reported to positively respond in the kynurenate test performed with rat hippocampal slices (Pittaluga et al., 1999). The similarities between the data obtained with human neocortex slices and those obtained in the rat suggest that the kynurenate test performed with rat brain slices represents a useful assay to study cognition-enhancing drugs.


VIII. NEUROPEPTIDE RECEPTORS

Neuropeptides are important modulators of neurotransmitter function, many of them being costored and coreleased with classic transmitters (Hökfelt et al., 2000). Although a number of studies have been performed in which release of neuropeptides from human brain tissue, particularly somatostatin and cholecystokinin, was characterized (see section VI.A.2.), very few reports deal with neuropeptide receptors in human CNS. Results from experiments with laboratory animals show that opioids can regulate neurotransmitter release, although with important species differences [see Feuerstein et al. (1996b) and references therein]. It is therefore important to investigate the modulation of release by opioids in human brain and, in particular, to characterize the subtypes of opioid receptor involved.


VIII.A. Release of Acetylcholine Mediated by Opioid Receptors

Feuerstein et al. (1996b) studied the effects of various opioid receptor ligands on the release of ACh evoked by electrical stimulation or by K+ depolarization in slices of human neocortex. The evoked release of ACh was inhibited by the {delta}-opioid receptor agonist [D-Pen2,D-Pen5]-enkephalin and by the {kappa}-opioid receptor agonist U50488. These effects were prevented, respectively, by the {delta}-opioid antagonist naltrindole and the {kappa}-opioid antagonist norbinaltorphimine. Exposure to the µ-opioid receptor agonist D-Ala2-NMePhe4-Gly-ol5-enkephalin also produced ACh release inhibition; however, this effect 1) was blocked by {delta}- and {kappa}-opioid antagonists and 1) could not be seen with the µ-opioid agonists morphine and (+)-nortilidine. Interestingly, naltrindole augmented, on its own, the evoked release of ACh, suggesting activation of {delta}-opioid receptors by endogenous opioid peptides. Feuerstein et al. (1996b) also suggested a localization of {delta}- and {kappa}-opioid receptors, based on experiments in which the release of ACh was elicited by high-K+ in the presence of tetrodotoxin. Because the K+-evoked ACh release was inhibited by U50488, but not by DPDPE, the authors proposed that {kappa}-opioid receptors are presynaptic on cholinergic terminals, whereas {delta}-receptors are on cortical interneurons. The nature of these interneurons remains to be established, however, because the lack of effect of GABAA and GABAB receptor agonists on ACh release tends to exclude the involvement of GABA interneurons.

In a subsequent work, Feuerstein et al. (1998) attempted to identify the transmitter of the interneuron acting through heteroreceptors on cholinergic terminals of the human neocortex. The authors found that the inhibition of the K+-evoked ACh release by DPDPE disappeared in the presence of octreotide, a somatostatin2 receptor agonist, suggesting that a somatostatinergic interneuron is responsible for the indirect effects of {delta}-opioid receptor activation. The latter receptor was subclassified as the {delta}1 subtype, based on the finding that the {delta}1-opioid receptor antagonist 7-benzylidennaltrexon prevented the DPDPE inhibition of ACh release. The authors concluded that somatodendritic {delta}1-opioid receptors sited on somatostatin-containing interneurons mediate inhibition of somatostatin release onto cholinergic terminals provided with facilitatory somatostatin2 receptors, resulting in {delta}1 receptor-mediated (indirect) depression of ACh release.

The article made no mention of the neuronal distribution of somatostatin which is largely colocalized with GABA in interneurons (Hendry et al., 1984; Somogyi et al., 1984). Because a previous report (Feuerstein et al., 1996b) excludes the involvement of GABA receptors in the {delta}-opioid inhibition of ACh release, the findings raise the possibility that {delta}1 receptor activation selectively affects the release of the cotransmitter somatostatin, although the existence of some "pure" somatostatinergic neurons should not be excluded.


VIII.B. Release of Norepinephrine Mediated by Opioid Receptor-Like 1 Receptors

Nociceptin/orphanin FQ (N/OFQ) is a neuropeptide identified as an endogenous ligand for the opioid receptor-like 1 (ORL1) receptor (Calò et al., 2000; Mogil and Pasternak, 2001). The peptide was reported to inhibit the release of NE from slices of various brain areas of mouse, rat, and guinea pig (Schlicker et al., 1998; Trendelenburg et al., 2000).

The effects of N/OFQ on the release of NE were comparatively studied in electrically stimulated slices of human and rat neocortex (Rominger et al., 2002). N/OFQ decreased the evoked release of [3H]NE in both species; the effects of N/OFQ were prevented by the ORL1 antagonist J-113397 which, on its own, was unable to increase the evoked release of [3H]NE suggesting the absence of a tonic activation by endogenous N/OFQ. Although the efficacy of N/OFQ was significantly lower in human than in rat slices, the affinities of the peptide for ORL1 receptors and the pA2 values of the ORL1 antagonist seem to be very similar in the two species.


IX. CANNABINOIDS AND CANNABINOID RECEPTORS

The CNS effects of {Delta}9-tetrahydrocannabinol, the main active constituent of marijuana and hashish and related compounds are mediated by cannabinoid receptors of the CB1 type. These are Gi protein-coupled receptors, the activation of which leads to inhibition of adenylyl cyclase. CB1 receptors exist on presynaptic axon terminals and their activation was shown in animal studies to cause inhibition of the release of neurotransmitters, possibly consequent to inhibition of VSCCs [see Schlicker and Kathmann (2001) for a review]. Cannabinoids have been reported to modulate neurotransmitter release from fresh human brain tissue, as summarized below.


IX.A. Release of Norepinephrine

The effects of synthetic cannabinoid receptor ligands on the release of [3H]NE were comparatively analyzed in slices of human, guinea pig, rat, and mouse hippocampal tissue (Schlicker et al., 1997). The cannabinoid receptor agonist WIN55,212-2 inhibited NE release in human and guinea pig hippocampal slices, but not in slices of rat and mouse hippocampus. The CB1 receptor antagonist SR141716 markedly attenuated the WIN55,212-2-induced inhibition of NE release, suggesting that activation of CB1 receptors mediate inhibition of NE release in human and guinea pig hippocampus. The modest augmentation of NE release provoked by SR141716, when added alone to human slices, suggests that CB1 receptors may not be tonically activated in the human cerebrocortex.

The hippocampal formation is one of the brain regions with the highest level of CB1 receptor expression (Herkenham et al., 1990). Ca