672342

research-article2016
JOP0010.1177/0269881116672342Journal of PsychopharmacologyMillan et al.

Commentary

The frontal cortex as a network

hub controlling mood and cognition:
Probing its neurochemical substrates Journal of Psychopharmacology

for improved therapy of psychiatric and

1­–30
© The Author(s) 2016
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DOI: 10.1177/0269881116672342
jop.sagepub.com

Mark J Millan, Jean-Michel Rivet and Alain Gobert

Abstract
The highly-interconnected and neurochemically-rich frontal cortex plays a crucial role in the regulation of mood and cognition, domains disrupted
in depression and other central nervous system disorders, and it is an important site of action for their therapeutic control. For improving our
understanding of the function and dysfunction of the frontal cortex, and for identifying improved treatments, quantification of extracellular pools of
neuromodulators by microdialysis in freely-moving rodents has proven indispensable. This approach has revealed a complex mesh of autoreceptor and
heteroceptor interactions amongst monoaminergic pathways, and led from selective 5-HT reuptake inhibitors to novel classes of multi-target drugs
for treating depression like the mixed α2-adrenoceptor/5-HT reuptake inhibitor, S35966, and the clinically-launched vortioxetine and vilazodone.
Moreover, integration of non-monoaminergic actions resulted in the discovery and development of the innovative melatonin receptor agonist/5-
HT2C receptor antagonist, Agomelatine. Melatonin levels, like those of corticosterone and the “social hormone”, oxytocin, can now be quantified by
microdialysis over the full 24 h daily cycle. Further, the introduction of procedures for measuring extracellular histamine and acetylcholine has provided
insights into strategies for improving cognition by, for example, blockade of 5-HT6 and/or dopamine D3 receptors. The challenge of concurrently
determining extracellular levels of GABA, glutamate, d-serine, glycine, kynurenate and other amino acids, and of clarifying their interactions with
monoamines, has also been resolved. This has proven important for characterizing the actions of glycine reuptake inhibitors that indirectly augment
transmission at N-methyl-d-aspartate receptors, and of “glutamatergic antidepressants” like ketamine, mGluR5 antagonists and positive modulators
of AMPA receptors (including S47445). Most recently, quantification of the neurotoxic proteins Aβ42 and Tau has extended microdialysis studies to
the pathogenesis of neurodegenerative disorders, and another frontier currently being broached is microRNAs. The present article discusses the above
themes, focusses on recent advances, highlights opportunities for clinical “translation”, and suggests avenues for further progress.

Keywords
Microdialysis, frontal cortex, depression, Alzheimer’s, schizophrenia, antidepressant, antipsychotic, Tau, β-amyloid, monoamine, glutamate, GABA,
mGluR, AMPA, NMDA, ketamine

The frontal cortex as a

therapeutically-important centre
controlling mood and cognition
The frontal cortex (FCX) is heavily innervated by ascending mono-
aminergic, cholinergic and histaminergic pathways, contains a rich
population of modulatory GABAergic interneurons, and sends
major projections (mainly glutamatergic) to other cortical regions
and sub-cortical centres (Adolphs, 2009; Alexander et al., 1986;
Lewis, 2012; Millan et al., 2012a; Robbins and Arnsten, 2009; Zanto
et al., 2011). It is a crucial network hub in the control of core func-
tions like mood, cognition and social behaviour. Accordingly, dis-
ruption of the FCX is implicated in the pathogenesis of depression,
schizophrenia and many other central nervous system (CNS) disor-
ders, and the FCX is a pivotal region underlying the therapeutic
actions of pharmacotherapeutic, psychosocial-cognitive-behavioural
and stimulation-based treatments for their control (Anticevic et al.,
2015; Barr et al., 2009; Cerqueira et al., 2007; Holmes and Wellman,
2009; Millan et al., 2012a, 2015, 2016; Navailles et al., 2010).
To improve our understanding of the role of the FCX, and to
acquire insights into potentially improved therapies, it is critical
to characterize interactions amongst the many neuromodulators
underpinning its complex operations. This enterprise has most
effectively been led by preclinical work in rodents. One fun-
damental approach consists in the sampling of extracellular
levels of specific neuromodulators by microdialysis coupled to
various methods of detection and quantification, where possible
Pole for Therapeutic Innovation in CNS disorders, IDR Servier,
Croissy-sur-Seine, France
Corresponding author:
Mark J Millan, Pole for Therapeutic Innovation in CNS disorders, IDR
Servier, 125 chemin de ronde, 78290 Croissy-sur-Seine, France.
Email: mark.millan@fr.netgrs.com

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2 Journal of Psychopharmacology
accompanied by electrophysiological monitoring of the activity
of the neurons in question (Di Chiara, 1990; Di Chiara et al.,
1996; Flik et al., 2015; Lejeune et al., 1994, 1997; Millan et al.,
2000a,2001b; 2007a: Sharp et al., 1986; Ungerstedt, 1991).
Extracellular levels of neurotransmitters and other neuromodula-
tors generally reflect a balance of tightly-regulated processes of
synthesis and release versus clearance by reuptake and enzymatic
degradation: microdialysis offers a means for probing all these
mechanisms, together with their modulation by potential thera-
peutic agents.
In this regard, an early focus of microdialysis studies was
monoamines, in particular the modulation of their extracellular
levels in the FCX by drugs for treating anxiety, depression and
psychosis – including tricyclic-structured reuptake blockers like
clomipramine, monoamine oxidase inhibitors and selective 5-HT
reuptake inhibitors (SSRIs). Indeed, towards the end of the
1990s, microdialysis was being intensively exploited to answer
two core and interrelated questions: (1) what is the influence of
drugs for treating depression upon extracellular levels of 5-HT,
noradrenaline (NA) and dopamine (DA) in the FCX and other
key regions like the hippocampus and (2) what is the nature of
interactions amongst monoaminergic projections in the FCX. It
was thought – correctly, as it turned out – that clarifying these
issues would help in the conception and evaluation of novel
classes of antidepressant acting via multiple and complementary
mechanisms of action.
A comprehensive review article dedicated to the above themes
was published in this journal by the present authors at the turn of
the Millennium, together with a survey of perspectives for future
progress (Millan et al., 2000a).
Aims and organization of the review
The purpose of the present review is to pick up on those perspec-
tives and to examine the progress that has been made over the past
15 years or so as regards our knowledge of, ability to probe, and
therapeutic exploitation of the FCX in the control of depression
and other CNS disorders. In addition, we consider what might be
achieved over the next decade of progress, and it is emphasized
that the pursuit of microdialysis studies of the FCX and other CNS
regions remains fundamental, not least since such work can
increasingly be “translated” into clinical investigations. Ipso facto,
it would be unwise indeed to discontinue this line of neurochemi-
cal research. Rather than an exhaustive review, the paper high-
lights and visually illustrates some major advances and themes
of the last decade and a half, including a palette of hitherto-
unpublished yet noteworthy observations from the author’s lab.
The paper focusses upon the following (“Top Ten”) facets of
research which have rapidly progressed over the past 15 years or
so and – in most cases – will continue to do so.
•• First, methodological progress that now permit quantifi-
cation of a vast range of neuromodulators in dialysates
from the FCX in comparison to earlier studies that
focussed primarily on monoamines.
•• Second, recent developments in our understanding of
how monoaminergic pathways interact in the FCX, and
incorporating some newer mechanisms – like roles for
5-HT6 receptors and trace amines – that were poorly char-
acterized at the time of the original review.
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•• Third, recently-launched multi-target drugs for treating
depression, like vortioxetine, that were developed around
the notion of more effectively exploiting monoaminergic
interactions in the FCX and other structures controlling
mood and cognition.
•• Fourth, Agomelatine (clinically-available) and other anti-
depressants that target not only monoamines for mood-
relief but also other, complementary, non-monoaminergic
mechanisms.
•• Fifth, modulation of the release of acetylcholine (ACh)
and histamine in the FCX in relation to their control of
cognitive processes – bearing in mind that cognitive dys-
function in depression has been, in comparison with
schizophrenia, neglected.
•• Sixth, measurements of extracellular levels of hormones
(melatonin, corticosterone and oxytocin) that play impor-
tant roles in the pathogenesis and potentially control of
psychiatric disorders.
•• Seventh, the complex challenge of quantifying glutamate,
GABA and other amino acids in FCX dialysates, and
linking their release, reuptake and actions both to mono-
aminergic transmission and to novel strategies for treat-
ing depression and schizophrenia.
•• Eighth, the equally formidable hurdle of quantifying
extracellular levels of proteins incriminated in neurologi-
cal disorders, such as β-amyloid (Aβ) and Tau.
•• Ninth, the translational bridging of preclinical studies of
FCX neurochemistry to work that can be undertaken in
human subjects, notably in the framework of clinical
trials.
•• Finally, some new domains of “R and D” which are likely
to evolve over the coming years.
While this Review covers, then, a substantial amount of terri-
tory, in view of its “Anniversary” nature, the citation list is illus-
trative rather than comprehensive. Further, it has somewhat a
historical flavour in focussing on key articles that have appeared
over the last 15–20 years, and in incorporating a representative
selection of our own work in this field.
Technological progress: from
monoamines to neuromodulators to
hormones to proteins
Dialysis of the FCX in freely-moving animals:
the basic paradigm
The core dialysis paradigm permitting quantification of extracel-
lular levels of neuromodulators in rats or mice is depicted in
Figure 1. As indicated in the Figure and legend, the system is
highly flexible, with one to two probes introduced and measure-
ments undertaken in freely-moving animals fully recovered (for
at least a week) following implantation. Obviously, for all neuro-
modulators determined (Table 1), measurements need to be taken
in “normal” subjects. In addition, studies undertaken during
behavioural tasks, in “knockout and overexpressing” mice, in
response to therapeutic agents, and employing genetic, environ-
mental and pharmacological models for depression and other
CNS disorders, are of interest.
Millan et al. 3

Figure 1.  Schema illustrating typical dialysis probe placement in brain, the diversity of structures and neuromodulators that can be dialysed, and
methods used for detection.
Dialysis can be undertaken with one, two or, albeit rarely, more probes and is performed in freely-moving animals, equipped with a swivel, tether and collection tube
connecting the dialysis probe the sample collector. “CMA 11” is a standard microdialysis probe with a membrane cutoff of 6 kiloDaltons (kDa) sufficient to allow for the
passage of neurotransmitters. However, other probes can be used with cutoffs as high as 300 kDa, notably when proteins are to be measured. As a rule, and for studies of
drug actions, following stabilization of baseline values after probe introduction (an hour or so), samples are taken for a few hours. However, they can be taken round the
clock for 24 hours to cover an entire diurnal cycle (Figure 3). See text and Table 1 for additional information on methodology for quantifying specific neuromodulators in
dialysates.

Simultaneous quantification of 5-HT, DA and
NA in single dialysate samples
Reflecting limited technical means and the comparatively low
sensitivity of detection systems, most of the first wave of dial-
ysis studies was oriented around measures of DA and monoam-
inemetabolites in the striatum (Bonhomme et al., 1995; Di
Chiara, 1990; Leviel et al., 1989; Nisell et al., 1994; Rivet
et al., 1994; Sharp et al., 1986; Ungerstedt, 1991; Zetterström
et al., 1983). This work was subsequently extended to 5-HT
and NA as well as the FCX and other cerebral regions. Further,
there was increasing interest in drugs for treating depression
and in dopamine/serotonin antagonists for treating psychosis
(“atypical antipsychotics”). With some exceptions, experiments
were still performed in anaesthetized animals (Celada et al.,
2004; Gardier et al., 1996; Hertel et al., 1999; Hjorth and
Auerbach, 1996; Jordan et al., 1994; Millan et al., 2007b;
Nomikos et al., 1992; Schoemaker et al., 1997; Westerink
et al., 1987; Zhang and Beyer, 2006).
Profiting from the refinement of detection methods, including
high performance liquid chromatography (HPLC) coupled to
coulometric (electrochemical) detection, in the mid-1990s we
elaborated an exceptionally sensitive and reliable method for
simultaneously and rapidly (minutes) quantifying all three mono-
amines in a single and small volume (20 μL) dialysate sample of
the FCX (Figure 2). Moreover, these studies could be undertaken
in freely-moving rats (Gobert et al., 1998) – and, subsequently,
mice (Di Cara et al., 2011). This proved a decisive advance allow-
ing for the refined characterization of: (1) the control of monoam-
ine release, breakdown and reuptake in the FCX; (2) the influence
of drugs on these parameters and (3) reciprocal interactions
amongst dopaminergic, serotonergic and adrenergic pathways to
the FCX. This work was summarized in a tome which appeared in
this journal (Millan et al., 2000a) and has since been further devel-
oped both by us and by others employing similar techniques (e.g.
Flik et al., 2015; Kehr et al., 2001; Yoshitake et al., 2004).
More recently, mass spectrometry (MS) systems have been
introduced with a similar ability to detect multiple monoamines

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4 Journal of Psychopharmacology

Table 1.  Summary of neuromodulators quantified in microdialysate samples of the frontal cortex, and of the technologies used for their
determination.

Extracellular neuromodulator Technique employed Limit of detection Basal level in dialysates Key citation

5-HT, dopamine, noradrenaline HPLC-ECD 30–60 pM 400 pM Gobert et al., 1998

5-HT, dopamine, noradrenaline HPLC-MS 70–700 pM 400 pM Gobert, unpub. obs.
Acetylcholine HPLC-ECD 30 pM 1.3 nM Gobert et al., 2003
Acetylcholine HPLC-MS 100 pM 1.3 nM Gobert, unpub. obs.
Glutamate, aspartate, d-serine, PEA HPLC-fluorimetry 2–4 nM 0.33–0.5 µM Gobert et al., 2011
Glycine, l-serine, taurine HPLC-fluorimetry 2–4 nM 2.6–4.5 µM Gobert et al., 2011
Glutamine HPLC-fluorimetry 2 nM 2.3 µM Gobert et al., 2011
GABA HPLC-fluorimetry 2 nM 10 nM Gobert et al., 2011
Histamine HPLC-fluorimetry 0.5 nM 320 nM Rivet, unpub. obs.
Kynurenate HPLC-fluorimetry 0.2 nM 0.8 nM Gobert et al., 2011
Melatonin HPLC-fluorimetry 1 nM 9 nM Rivet, unpub. obs.
Corticosterone Enzymatic immunoAssay 30 pM 700 pM Bouchez et al., 2012
Aβ1-42 & Aβ1-40 ELISA 0.1 & 0.6 pM 0.5 & 2.5 pM Gobert et al., 2014
Tau protein ELISA 1.5 pM 250 pM Gobert et al., 2014
Tau protein HPLC-MS 20 pM 250 pM Gobert, unpub. obs.

The neuromodulators depicted, and the methods indicated, are those which we have studied in our lab. As outlined in the text, other biologically-active molecules can be
determined in extracellular samples, and additional technologies used for detection and quantification. Note the marked differences in basal levels of the neuromodula-
tors in microdialysates and the need to attain appropriate sensitivity for reliable detection. All basal values are from frontal cortex microdialysates except melatonin
(pineal gland) and corticosterone (basolateral amygdala). See also Figures 2 and 3 for visual illustration. Abbreviations: ECD, electrochemical detection; ELISA, enzyme-
linked immunosorbent assay; HPLC, high performance liquid chromatography; MS, mass spectrometry; and PEA, phosphoethanolamine.

in parallel (Bergh et al., 2016; Hows et al., 2004; Kim et al.,
2016). They have the advantage of improved resolution for dis-
tinguishing the individual monoamines, but at the price of lesser
sensitivity (Table1), a potential problem in structures where lev-
els are low – for example, NA in the striatum (Gobert et al.,
2004). In any event, as outlined below, the exploitation of a vari-
ety of procedures has led to further insights into interactions
amongst monoamines and other transmitters in the FCX and sub-
cortical territories, and has facilitated the discovery and charac-
terization of novel classes of psychotropic agent.
Quantifying acetylcholine without the need
for cholinesterase inhibitors
The remarkable efficacy of the ACh-cleaving enzyme, cholinest-
erase, means that extracellular levels of its metabolite, choline,
are vastly (thousand-fold) higher than those of ACh itself. Hence,
a major challenge during the early 2000s remained the need to
reproducibly and sensitively quantify basal levels of ACh in
dialysates without resorting to the addition of cholinesterase
inhibitors to the perfusion medium – or their administration to
animals – in order to artificially boost basal levels of ACh vis-a-
vis choline. Use of these inhibitors has the major disadvantages
of: modifying the sensitivity of muscarinic (M2/M4) autorecep-
tors; perturbing the physiological status of cholinergic neurones;
and interfering with their control by pharmacological agents (see
Gobert et al., 2003; Ichikawa et al., 2000; Kehr et al., 2001).
Initially pioneered by Ichikawa et al. (2000), we further
developed a novel technique, depicted in Figure 2, that oxida-
tively eliminated choline in a pre-column, thereby increasing
assay sensitivity by 10-fold and allowing reliable quantification
of baseline and stimulated levels of ACh in the FCX and other
brain regions (Gobert et al., 2003; Millan et al., 2004). This was
especially opportune in view of: (1) increasing recognition that
the cognitive deficits of schizophrenia are poorly relieved and
even exacerbated by existing drugs for treating psychosis – in
certain cases, like clozapine, reflecting blockade of muscarinic
receptors; (2) the ongoing design of novel classes of agent for
relieving cognitive dysfunction in schizophrenia and other disor-
ders by boosting ACh release in FCX and hippocampus and (3)
the functional relevance of ACh to cognitive and motor dysfunc-
tion in neurological disorders (Graef et al., 2011; Kendall et al.,
2011; Millan et al., 2012a). More recently, we and others have set
up a rather more robust mass spectrometry (MS) technique to
detect and measure ACh (Figure 2) (Hows et al., 2002).
Some examples of work along these lines with potential new
drugs for treating psychosis are given later.
Quantification of histamine compared to the
metabolite 1-methyl-histamine
Another frontocortical neurotransmitter involved in cognitive
processes, in particular attention and vigilance, is histamine
(Raddatz et al., 2010), but it has also proven something of a head-
ache to reliably quantify. Indeed, the vast majority of studies of
histaminergic transmission have indirectly estimated activity on
the basis of concentrations of its principle metabolite, 1-methyl-
histamine – usually quantified in tissue. This strategy has obvious
disadvantages. Not least, for pharmacological agents, it is difficult
to know if any elevation in 1-methyl-histamine levels genuinely
reflects a shift in histamine release as opposed to, say, an altera-
tion in its catabolism and elimination. (Incidentally, it should be
noted that specific reuptake sites for histamine have not been
found, but organic cation transporters appear to be harnessed for
its clearance (Yoshikawa et al., 2013)). Lack of a means to rapidly,
reliably and directly quantify extracellular histamine had been a

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Millan et al. 5

Figure 2.  Evolution of the use of dialysis for the quantification of extracellular levels of neuromodulators: from monoamines to amino acids and
acetylcholine (ACh).
Studies of the response of extracellular levels of monoamines in the FCX to antidepressants, and analysis of interactions amongst frontocortical monoaminergic pathways,
necessitated the setting up of a highly-sensitive method for quantifying 5-HT, NA and DA in single dialysate samples of freely-moving rats. With increasing interest in the
role of the FCX in the control of cognition, a technique for determining levels of ACh in the absence of cholinesterase inhibitors (w/o AChE) became important, initially
by electrochemistry, later by use of mass spectrometry. Another challenge was to establish a procedure for concurrently quantifying a whole range of amino acids and
their “false” relatives, taurine and phosphoethanolamine (PEA), in dialysates, together with both d- and l- forms of serine of which the d isomer gates NMDA receptors by
acting at glycine B sites. Differentiation of d- from l- forms of serine required a separate approach, as outlined in the text. Together these strategies facilitate evaluation
of the interrelationship amongst frontocortical populations of glutamatergic and GABAergic neurons, and between neurons and astrocytes, a key issue for many classes of
psychotropic agent (Figure 4). Note that the amino acid chromotagram shown does not include all standards. Amino acids in order of elution: Asp, apartate; Glu, gluta-
mate; Asn, aparagine; His, histidine; Gln, glutamine; Ser, serine (dl); PEA, phosphoethanolamine; Cit, citrulline; Arg, arginine; Gly, glycine; Thr, threonine; Ala, alanine;
Tau, taurine; Tyr, tyrosine and GABA, γ-aminobutyric acid. AAA is aminoadipic acid that was used as an internal standard (8 ng).

serious and persistent hassle in the screening for H3 autoreceptor
antagonists – which disinhibit histamine release – for the treat-
ment of Alzheimer’s disease and schizophrenia (Millan et al.,
2012a; Raddatz et al., 2010). In view of this issue, as illustrated in
Figure 3, we set up a dialysis procedure for sensitive detection of
histamine and its separation from 1-methyl-histamine. Other
experts have also elaborated approaches for studying the impact
of histamine H3 antagonists and additional drug classes upon
extracellular levels of histamine in the brain, and for deciphering
the interrelationships between histaminergic and monoaminergic
transmission (Flik et al., 2011, 2015).
Adrenergic and serotonergic input to the FCX modulates the
release of histamine, so elevations in its levels may contribute to
the influence of drugs for treating depression upon cognition (see
below) (Flik et al., 2015; Sanchez et al., 2015). Further, while H3
antagonists per se are unlikely to be effective drugs for treating
depression, association of H3 antagonist properties with inhibi-
tion of 5-HT reuptake has attracted interest (Barbier et al., 2007):
such drugs are best characterized neurochemically using micro-
dialysis measures of extracellular levels of histamine.
Differentiating and quantifying a broad
palette of d- and l-amino acids
Glutamatergic pyramidal neurons interact locally with monoam-
inergic, cholinergic and other classes of frontocortical neurons,
they project to other cortical lobes, and they also connect the
FCX with subcortical structures like the basal ganglia, hippocam-
pus and thalamus (Alexander et al., 1986; Millan et al., 2012a;

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6 Journal of Psychopharmacology

Figure 3.  Evolution of the use of dialysis for the quantification of extracellular levels of neuromodulators: from histamine and kynurenate to all-day
sampling of hormones.
Rather than estimating the activity of histaminergic pathways by dosing 1-methyl-histamine in tissue, it was desired to establish a procedure for differentiation of this
metabolite from histamine in dialysates by fluorimetry. Kynurenate is an endogenous antagonist at NMDA, AMPA and α7-nicotonic) receptors but it does not need to
be derivatized like amino acids, so it was necessary to devise a separate method for its independent quantification, as shown in Figure 2. For both corticosterone and
melatonin, continuous 24 h dialysis permitted pursuit of their circadian rhythms in the brain throughout the entire day: corticosterone peaks just around lights off, while
melatonin peaks in the middle of the dark cycle. Note the parallel shifts in its precursor, N-acetyl-melatonin, and the anti-correlated levels of 5-HT from which melatonin
is generated in the pineal gland. Exceptionally, these assays were initially established in the basolateral amygdala (high density of receptors for corticosterone) and
pineal gland (site of melatonin synthesis), respectively.

Musazzi et al., 2015; Sanacora et al., 2008). Equally pivotal to
the operation of the FCX, GABAergic interneurons exert a pro-
nounced (usually inhibitory) tone on monoamines and other fron-
tocortical neurotransmitters (Lewis, 2012; Millan et al., 2012a,
2016). Moreover, they play an important role in the synchroniza-
tion of glutamatergic networks to orchestrate the top-down con-
trol of many functions like mood, cognition, social behaviour,
reward, motor performance – as well as impulsive and compul-
sive behaviours (Lewis, 2012; Millan et al., 2012a, 2016; Park
et al., 2015). A disruption of glutamatergic and GABAergic neu-
rones in the FCX is strongly implicated in the pathophysiology of
disorders like depression and schizophrenia. For example, a
hypoactivity of FCX-localized NMDA receptors is implicated in
the induction of psychosis, so drugs that increase activity at the
co-agonist glycine B site – of which the endogenous ligands are
glial-derived glycine and d-serine – have been sought as potential
new drugs for treating psychosis (Danysz and Parsons, 1998;
Fossat et al., 2012; Heresco-Levy and Javitt, 2004; Kantrowitz
and Javitt, 2010; Millan, 2005a; Park et al., 2015; Wolosker,
2011; Yang and Svensson, 2008). Further, there is considerable
interest in glutamatergic agents as potential new drugs for treat-
ing depression (see below).
These observations emphasize the crucial importance of
understanding the patterns and regulation of glutamate and
GABA release in the FCX, of the respective significance of neu-
rons and astrocytes in this regard, and or integrating measures of
other – usually ignored – amino acids like aspartate (Figure 4)
(Errico et al., 2015; Eulenburg and Gomeza, 2010; Fossat et al.,
2012; Gobert et al., 2011; Millan, 2005a; Pehrson and Sanchez,
2014; Tsai and Lin, 2010; Wolosker, 2011).
One approach to address all this is microdialysis. Though it
lacks the rapid time resolution of voltammetry and electrophysi-
ology, and questions persist concerning the significance of gluta-
mate levels in dialysates, it is indispensable for evaluating the
actions of pharmacological agents which should inherently have
substantial residence times and sustained functional effects

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Millan et al. 7

Figure 4.  Schematic depiction of the interrelationships amongst glutamatergic and GABAergic neurons, and amongst neurons and astrocytes, in the
frontal cortex.
The figure incorporates amino acids, “false” amino acids and kynurenate as quantified in dialysates of FCX (Figures 2 and 3). Note the complex patterns of interplay
between astrocytes and neurons as regards the generation, reuptake and release of neuromodulators: microdialysis has proven important in their characterization.
In addition, there are many interactions mediated via distinct classes of receptor, not all of which can be shown: for example, there are inhibitory GABAB receptors
on glutamatergic terminals. The postsynaptic neurone could be another glutamatergic (pyramidal) cell, a GABAergic interneurone or a monoaminergic terminal etc.
For example, GABAergic neurons are under the excitatatory control of NMDA receptors (see text). Similarly complex configurations occur in other structures, like the
ventrotegmental area where clusters of dopaminergic cell bodies projecting to the FCX are located. Kynurenate also behaves as an antagonist at AMPA receptors and
α7-nicotinic receptors. Abbreviations as follows: Asc-T: alanine, serine, cysteine transporter; Asp, aspartate; D-AAO, d-amino acid oxidase; EAAT, excitatory amino acid
transporter; IDO, indolamine 2,3-dioxygenase; GABAT, GABA transaminase; GAD, 1-glutamic acid decarboxylase; GAT, GABA transporter; Glu, glutamate; Gln, glutamine;
Gln-ase, glutaminase; Gly; glycine; GlyT1, glycine transporter; K-3-OH, kynurenine 3-hydroxylase; KAT, kynurenine aminotransferase; Kyn, kynurenine; NR, NMDA receptor;
Ser-HMT, serine hydroxymethyltransferase; SNTA, specific neutral amino acid transporter; TCA, tricarboxylic acid cycle; TDO, tryptophan 2,3-dioxygenase; Xc-, cysteine/
glutamate antiporter; and Trp, trytophan.

(Gobert et al., 2011; Van der Zeyden et al., 2008). To this end,
then, we developed a robust and sensitive system capable of con-
currently quantifying more than a dozen amino acids in dialysates
of FCX (Figure 2). This was based on naphthalene-2,3-dicarboal-
dehyde/cyanide derivitization coupled to HPLC binary gradient
reverse phase separation and fluorescence detection. In addition,
we introduced a technical innovation (derivatization with ortho-
phthaldehyde and Boc-l-cysteine for chiral separation) to allow
for complementary differentiation of “d” (NMDA co-agonist)
versus “l” (precursor) forms of serine (Gobert et al., 2011). As
can be seen from Figure 2, it was also possible to determine lev-
els of taurine in parallel. This osmoregulatory and neuroprotec-
tive compound behaves as a weak GABAergic agonist and is
under the excitatory control of NMDA receptors (Gobert et al.,
2011; Wu et al., 2005). While the underlying mechanisms are not
entirely clear, taurine appears to positively affect cognition,
mood and motivation, especially when combined with other pro-
cognitive and energy-promoting agents (Curry and Stasio, 2009).
The simultaneous quantification of phosphoethanolamine (PEA)
(a catabolic product of membrane-enriched phosphotidylethan-
olamine) provides a readout for a loss of cellular integrity due to
excitotoxicity (Vance, 2008) – as provoked by, for example, a
high dose of NMDA (Table 2) (Gobert et al., 2011). Finally, in a
separate run using contrasting conditions (mobile phase, sodium
acetate (20 mM), zinc acetate (200 mM) and methanol (10%)),
fluorimetric measurement of the endogenous NMDA, AMPA and
α7-nicotonic receptor antagonist, kynurenate (kynurenic acid),
was achieved (Erhardt et al., 2009; Gobert et al., 2011; Millan,
2005a).
As described elsewhere, all these procedures and measures
were subjected to rigorous pharmacological characterization
(Table 2) (Gobert et al., 2011). The following key examples may
be briefly mentioned (see Table 2). First, the GABAB autorecep-
tor and heteroceptor agonist, Baclofen, reduced levels of GABA
itself and also those of the excitatory and co-localized transmit-
ters, glutamate and asparate. Second, NMDA elevated levels of
both taurine and PEA, and reduced levels of d-serine, actions
abolished by the competitive NMDA receptor antagonist, CPP.

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8 Journal of Psychopharmacology

Third, levels of kynurenate were increased by administration of

50.1 ± 3.9a
15.8 ± 0.4

17.5 ± 0.1
16.7 ± 0.4
14.3 ± 0.9

15.6 ± 1.0
13.6 ± 2.0

17.2 ± 1.1
14.5 ± 1.1

vehicle. As previously (Gobert et al., 2011), values shown represent “Area under the curve” analysis: that is, the global drug influence expressed across the 3 h dialysis period as % × minutes/1000. Doses are in mg/kg, but note that
15.6 ±0.9

the dose of ketamine was 80.0 for its influence on amino acids. Data are means ± SEMs with N values between 5 and 8. aP < 0.05 to corresponding vehicles in Newman–Keuls test following ANOVA. Abbreviations: ago, agonist at the
its precursor, kynurenine, an effect blocked by inhibition of the

The findings shown, which incorporate some documented in Gobert et al. (2011), are based upon dialysis studies performed over a period of 3 h following injection of the agent shown, with effects determined in comparison to
converting enzyme, amino-oxyacetate (Gobert et al., 2011).
PEA

ND
This microdialysis procedure is highly instructive for exam-
25.6 ± 2.2a ining the actions of drugs for treating depression and psychosis

18.4 ± 0.9a

19.8 ± 1.0a

21.2 ± 1.4a
15.8 ± 0.6

17.6 ± 0.5

15.1 ± 1.1
15.3 ± 1.3
17.7 ± 1.6

16.9 ± 1.2
as well as pro-cognitive agents (see below). Nonetheless, a word

Glycine B site on NDMA receptors; ant, antagonist; Ch Bl, Channel Blocker; co-ago, co-agonist; DA, dopamine; Gly-RI, glycine reuptake inhibitor; NA, noradrenaline; ND, not determined; PEA, phosphoethanolamine.
15.1 ±0.5
Taurine

of caution, especially for glutamate. With the exception of

blockers of glutamate reuptake, its extracellular levels are quite
resistant to modification, even for mechanisms that are known to
influence glutamatergic transmission (Gobert et al., 2011; Van
12.0 ± 0.9a
14.1 ± 0.9a
13.8 ± 1.1a
17.0 ± 1.0
17.1 ± 1.0
15.1 ± 2.6

19.7 ± 1.3
18.6 ± 1.0

18.5 ± 1.3
16.8 ± 1.4
15.7 ±0.4
der Zeyden et al., 2008). Thus, the sensitivity of this and other
GABA

dialysis systems to small and rapid alterations in glutamate

release “felt” by postsynaptic glutamatergic receptors may be
limited and insufficient for monitoring dynamic shifts in signal-
162.5 ± 16.7a

ling. Also, the architecture of various classes of glutamatergic

14.6 ± 1.1a

28.9 ± 2.3a
24.4 ± 0.4a
17.1 ± 0.5

15.5 ± 0.4
16.8 ± 1.1
19.8 ± 1.1

16.5 ± 1.2

15.9 ± 0.8

17.1 ± 0.6

receptor at synapses is complex with both intra- and extra-

Glycine

synaptic distributions: localized differences in concentration

differentially seen by these populations cannot be registered by
microdialysis (Danysz and Parsons, 1998; Papouin and Oliet,
Table 2.  Influence of glutamatergic agents upon dialysate levels of monoamines and amino acids in the frontal cortex of freely-moving rats.

2014). Thus, for glutamate in particular, the importance of other

571.1 ± 194.6a

measures of its activity, especially those with rapid time resolu-

14.9 ± 0.8a

13.3 ± 1.0a
17.5 ± 0.4

15.4 ± 0.5
17.7 ± 0.6
18.4 ± 0.7
16.0 ± 0.8

17.9 ± 1.3
18.6 ± 0.5
18.7 ± 0.9

tion should be underlined (Alvarsson et al., 2015).

d-Serine

Twenty four hour monitoring of centrally-

active hormones implicated in psychiatric
13.3 ± 1.3a
19.0 ± 0.7a

12.7 ± 1.5a

11.6 ± 1.0a
16.7 ± 0.7

16.1 ± 1.5

15.8 ± 0.7
15.4 ± 0.5
16.3 ± 1.3

13.7 ± 0.7
15.0 ± 1.2
Aspartate

disorders: melatonin, corticosterone and

oxytocin
Kynurenate provides a convenient link to a further set of proce-
14.6 ± 1.4a
20.6 ± 0.2a

14.4 ± 1.0a
17.6 ± 0.5

17.9 ± 1.7
16.1 ± 0.4

16.5 ± 0.9
15.9 ± 0.7
22.4 ± 4.1

16.0 ± 1.0
Glutamate

dures that were established for dosing central levels of hormones

16.7 ±1.0

implicated in the pathogenesis and management of depression

and other psychiatric disorders. Like kynurenate, melatonin is
ultimately derived from the precursor, tryptophan, albeit follow-
ing an alternative biosynthetic route in which 5-HT is trans-
27.5 ± 2.2a
25.5 ± 2.8a

26.3 ± 2.8a

22.2 ± 1.7a
26.1 ± 3.6a
31.0 ± 5.2a
18.0 ± 0.6

19.5 ± 1.9

16.4 ± 0.6
20.5 ± 1.1

20.3 ± 0.5

formed (by 5-HT-N-acetyltransferase) into its acetylated

derivative, N-acetyl-5-HT (nor-melatonin), which is then con-
DA

verted into melatonin by hydroxy-indole-O-methyl transferase

(Catena-Dell’Osso et al., 2012). The synthesis of melatonin
25.8 ± 1.8a
29.4 ± 4.4a

24.7 ± 2.6a

26.0 ± 3.3a
20.6 ± 1.4

20.5 ± 1.1

18.8 ± 1.5
20.8 ± 1.2
21.8 ± 0.9
20.4 ± 1.1

21.1 ± 1.2

occurs in the pineal gland which was the logical place, rather than
the FCX, to validate a fluorometric method that we devised for its
measurement. Moreover, in view of the pronounced diurnal
NA

rhythm in secretion of melatonin, and its crucial role in the circa-

dian scheduling of other rhythms and behaviours (Catena-
16.7 ± 0.5

16.7 ± 0.9
18.0 ± 1.1
17.1 ± 1.2
15.1 ± 0.2
17.5 ± 2.2
14.4 ± 0.9

17.4 ± 2.2
18.3 ± 0.6
18.1 ± 2.3
17.4 ±1.0

Dell’Osso et al., 2012), this was an opportunity to set up a system

allowing for monitoring of the daily fluctuations of extracellular
5-HT

levels of neuromodulators over 24 h. As shown in Figure 3, the

fluorometric strategy chosen permitted not only demonstration of
NMDA co-ago
NMDA co-ago

the night-time peak in melatonin levels but also, and rather ele-
NMDA Ch Bl
mGluR5 ant
GABAB ago

AMPA PAM
NMDA ago
NMDA ant

gantly, a concomitant elevation in N-acetyl-5-HT coupled to an

Activity

Gly-RI
Gly-RI

anti-correlated reduction in their precursor, 5-HT. The sensitivity

—

of this procedure was sufficient to allow deployment in the FCX

and other brain structures, and it is obviously informative as a
direct measure of how pharmacological agents and animal mod-
ORG 24598 (10.0, s.c.)
LY392,098 (10.0, s.c.)
Ketamine (10.0, s.c.)

Sarcosine (800, i.p.)

Baclofen (10.0, s.c.)

els for depression influence the diurnal organization of biological

d-Serine (160, s.c.)
Glycine (800, i.p.)
Vehicle (s.c., i.p.)
NMDA (40.0, s.c.)
Drug (dose, route)

MPEP (10.0, i.p.)

rhythms.
CPP (20.0, s.c.)

Another hormone which likewise displays a striking circadian

rhythm is corticosterone (CS), secreted by the adrenal gland
downstream of the hypothalamo-hypophyseal axis. CS secretion
is a typical response to “stress” and closely linked to multiple – if

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Millan et al. 9
not all – CNS disorders (Cerqueira et al., 2007; Lupien et al.,
2009). Indeed, protracted and uncontrolled increases in CS secre-
tion are incriminated in the etiology of anxio-depressive states,
disruption of mood and cognition, and structural and functional
changes in corticolimbic structures like the FCX, hippocampus
and amygdala (Cerqueira et al., 2007; De Quervain et al., 2009;
Holmes and Wellmann, 2009; Lupien et al., 2009; Millan, 2003,
2006). The central actions of CS reflect its recruitment of nuclear
mineralocorticoid receptors (high affinity) and glucocorticoid
receptors (low affinity, mainly activated during stress and peak
circadian levels) (De Quervain et al., 2009; Lupien et al., 2009).
Antagonists of glucocorticoid receptors have attracted interest
for the control of psychotic depression, but issues of safety ham-
pered their development and they no longer appear to be in active
research (Millan, 2006). Notwithstanding the importance of cen-
tral actions of CS, its levels have almost invariably been moni-
tored in the periphery, so it was useful to set up a sensitive
enzymatic immunoassay for reliably quantifying the far lower
levels of CS in the brain. This was undertaken in the basolateral
amygdala in view of its well-documented role in the cerebral
response to CS under conditions of stress, and to complement
studies performed elsewhere of hippocampal levels of CS in
mice and rats (Béracochéa et al., 2011; Droste et al., 2009).
Pharmacological, psychological and mild physical stressors all
provoked robust and time-constrained elevations in basolateral
amygdala levels of CS which were abolished by cojoint blockade
of central vasopressin1B and corticotrophin releasing factor1
receptors upstream of the hypothalamo-pituitary axis (Bouchez
et al., 2012; Murat et al., 2012). Furthermore, the circadian
rhythm of CS secretion, coordinated by the suprachiasmatic
nucleus (Nader et al., 2010), could be fully reproduced by dialy-
sis quantification in the basolateral amygdala, with the character-
istic increase commencing in anticipation of the wake (dark for
rats) period, peak levels attained and maintained over several
hours during this active phase of the cycle, and a return to base-
line occurring just before the onset of the inactive (lights on)
period (Figure 3) (Mohawk et al., 2007). Quantification of CS
levels in the FCX is also feasible and of especial interest since it
is there that CS exerts several actions, such as modulation of
glutamatergic transmission, related to the pathophysiology of
depression (Dwivedi et al., 2015; Musazzi et al., 2015;
Palamarchouk et al., 2009).
Another hormone to evoke is oxytocin in view of its impor-
tant role, expressed across the FCX and several cerebral struc-
tures, in the promotion of social behaviour and social cognition
(Bukovskaya and Shmukler, 2016; Goodson and Thomson,
2010). Disruption of these social functions is a fundamental
feature not only of autism-spectrum disorders but also of
schizophrenia, depression and even neurological disorders like
Alzheimer’s disease (Adolphs, 2009; Goodson and Thomson,
2010; Henry et al., 2016; Millan et al., 2012a). Thus, modulation
of extracellular levels of oxytocin in discrete brain regions under
conditions of rewarding or conflicting social interaction, and in
response to depression or pharmacological agents, is of consider-
able interest, yet there is surprisingly little information available.
While we have regrettably not had the opportunity to measure
this social hormone ourselves, several procedures have been pro-
posed in order to do so (Mabrouk and Kennedy, 2012). Their util-
ity and promise has been unveiled in behavioural paradigms like
fear-based learning and social interaction (Ross et al., 2009;
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Wotjak et al., 2008; Zoicas et al., 2014). It would be particularly
interesting to evaluate the influence of novel (pharmacotherapeu-
tic and other) strategies for treating autism and schizophrenia
upon the release of oxytocin by dialysis procedures. More gener-
ally, this remains a fruitful line of investigation, and numerous
other hormones – peptidergic, steroid and other – interrelated to
depressive and other psychiatric states, warrant analysis by dialy-
sis procedures (Clough, 2005; Wotjak et al., 2008).
Procedures for detecting and measuring
extracellular levels of neurotoxic proteins in
dialysates
Specialist teams have been studying dialysis levels of the neuro-
toxic proteins, Aβ42 and Tau, for several years in humans, mostly
under conditions of traumatic brain injury (Marklund et al., 2009;
2014; Ungerstedt and Rostami, 2004). Further, CSF levels of
Aβ42, total Tau and phosphorylated Tau are considered reliable
biomarkers of early Alzheimer’s disease (Hulstaert et al., 1999;
Olsson et al., 2016). Ironically, there have been very few reports
on extracellular levels of Aβ42 and Tau in rodents – despite the
frantic levels of research incriminating this infamous double act
in the pathogenesis of Alzheimer’s disease (Iqbal et al., 2016).
One simple reason is that it is very difficult to detect and quantify
Aβ42 and Tau in the extracellular environment – in comparison
to the fairly straightforward task of measuring these neurotoxic
proteins in tissue. Nonetheless, employing a microdialysis tech-
nique coupled to enzyme-linked immunosorbent assay (ELISA)
quantification, Cirrito et al. (2003) showed that soluble Aβ42 is
released into brain interstitial fluid in mice overexpressing amy-
loid precursor protein.
We have recently established our own microdialysis proto-
cols to address this issue (Gobert et al., 2014). Using the P301L
mouse (Lewis et al., 2000), it has proven possible to simultane-
ously quantify levels of Aβ1–40 and Aβ1–42 (ca. 4000 Daltons
each in size) and total Tau protein (ca. 50,000 Daltons) in dialy-
sis samples of the FCX using an ELISA assay coupled to elec-
trochemiluminescence detection (Figure 5) (Gobert et al.,
2014). Inasmuch as the dialysis membrane used for this work
permits passage of molecules of up to about 300,000 Daltons
(Figure 1), all these peptides can easily penetrate into the probe.
While it is likely for Tau that microdialysis detects a mixture of
monomers, dimers and small oligomers (up to hexamers),
Aβ1–40 and Aβ1–42 may be present as even larger conglomerates
– though in both cases proteins must be soluble for passage,
detection and quantification. By use of a single site ELISA, it
may soon be possible to specifically distinguish Tau oligomers
from monomers (Takeda et al., 2016). Another complication is,
however, the vast array of different forms of Tau created by
post-translational modifications like phosphorylation and acet-
ylation (Iqbal et al., 2016). For their detection and differentia-
tion, proteomics and phosphoproteomics plus ELISA may
prove helpful, but this remains difficult in view of the very low
quantities of individual variants of Tau in the extracellular
space (Brinkmalm et al., 2015; Fu et al., 2015; Takeda et al.,
2016). Thus, rigorous microdialysis detection and quantifica-
tion of neurotoxic proteins in rodents, parallelling work under-
taken in human CSF (Brinkmalm et al., 2015; Olsson et al.,
2016; Takeda et al., 2016), remains a work in progress.
10 Journal of Psychopharmacology

Figure 5.  Evolution of the use of dialysis for the quantification of extracellular levels of neuromodulators: quantification of Tau protein and
ß-amyloid in dialysates of the FCX.
The proteins were all determined with a highly sensitive and selective ELISA (enzyme-linked immunosorbent assay) technique, as outlined in the text and Table 1. Their
levels were determined in “P301L” mice bearing a Tau mutation associated with Tau over-production as compared to their wild-type counterparts with the same genetic
background. In comparison to wild-type animals, Tau levels were significantly higher in P301L mice at 16 months when behavioural symptoms, cognitive deficits and
neuropathological changes appear. Conversely, there was no difference to wild-type mice for levels of ß-amyloid 1–40 or 1–42. Data are means ± SEMs. *P < 0.01 to wild-
type animals in Newman–Keuls test following ANOVA with time as a dependent variable. N was 6–8 per value.

Extracellular levels of other biologically-
important molecules
The above discussion evoked a quite wide spectrum of biologi-
cally-active substances found in the extracellular space and rele-
vant to the genesis and control of depression and other CNS
disorders, but the list is not exhaustive. Second messengers
(cAMP and cGMP) and growth factors like brain derived neuro-
trophic factor (BDNF), for example, merit a brief comment, as
well as other neuropeptides like substance P, somatostatin and
neurotensin (Cunha et al., 2010; Pepicelli et al., 2004; Vaka et al.,
2011; Wojtak et al., 2008; Yoshitake et al., 2013).
Dialysis quantification of pharmacological
agents
Finally, at least in the academic environment, the major focus of
dialysis is on quantitation of extracellular levels of endogenous
neuromodulators, yet monitoring of pharmacological agents
themselves is likewise feasible, providing an indication of entry
into the brain, distribution and (with appropriate calculations)
concentrations attained cerebrally (Cremers et al., 2012, 2016;
Deguchi et al., 2002; De Lange et al., 2005). This strategy is
complementary to (and may confirm or refute) observations
made to forecast passage in the brain using: (1) in silico pre-
diction; (2) cellular models of blood–brain-barrier passage and
(3) measures of interactions with endothelial cell-enriched
P-glycoproteins – that eject many compounds from the brain
(Cremers et al., 2012, 2016; Ejsing et al., 2005; Josserand et al.,
2006).
Ideally, one can simultaneously dose the drug under investi-
gation in the FCX as well as the levels of the neurotransmitter
which it is modifying: for example, extracellular levels of DA
together with its precursor, l-DOPA, following administration
of the latter to rats (De Deurwaerdère et al., 2016). This
approach has been less enacted than one might imagine, yet it
remains a translationally-relevant option for providing direct
answers to the question of just how well an agent actually gets
into the brain, and whether it reaches concentrations sufficient
to exert actions of interest – beneficial or undesirable (Cremers
et al., 2012, 2016; Weber et al., 2013). Another use is for moni-
toring long-term studies where it is important to be sure of the
cerebral concentration of a drug throughout treatment. For
example, chronic administration of fluoxetine over three weeks
leads to a large (more than two-fold) elevation in basal extracel-
lular levels of 5-HT in FCX not owing to fluoxetine-induced
5-HT1A autoreceptor desensitization per se, but mainly due to
the long half-life of its bio-active metabolite, nor-fluoxetine,
which was found at very high levels in microdialysates. In con-
trast, this was not the case for the SNRI, S33005, which has a
shorter half-life and no equivalent bioactive metabolite (Gobert
et al., unpublished observations; Millan et al., 2001a).

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Millan et al. 11
Monoaminergic interplay in the FCX:
overview of core interactions and
recent developments
Regulation of frontocortical monoaminergic
transmission as a basis for therapeutic
progress
Millan et al. (2000a) was principally devoted to the interrelation-
ships amongst monoaminergic pathways innervating the FCX,
how they regulate their own and each other’s activity via reup-
take sites, auto- and heteroceptors, and how improved knowledge
of these interactions might be used as a basis for developing
novel classes of antidepressant. The following paragraphs sum-
marize this core paradigm and some recent – though surprisingly
minor – refinements in our understanding of the interplay
amongst frontocortical monoaminergic projections. It is worth
noting in this context that widely-heralded studies in genetically-
modified mice largely converged with findings acquired using
boring-old – yet selective and well-characterized – pharmaco-
logical agents.
In the subsequent sections, key examples are given of mono-
aminergic antidepressants that were conceived, discovered,
developed and – in two cases – launched based on an improved
understanding of interactions amongst monoaminergic pathways
in the FCX (Millan, 2006, 2009, 2014b; Sahli et al., 2016;
Sanchez et al., 2015).
At a somewhat later date, this purely monoaminergic frame-
work of FCX function was expanded to embrace complementary
non-monoaminergic mechanisms. This led to the elaboration of
further multi-target concepts for improving the treatment of
depression (Millan, 2006), including the therapeutically-
employed agomelatine (De Bodinat et al., 2010; Millan et al.,
2003; 2005), which is likewise evoked below.
How monoaminergic terminals interact in the
FCX: updating the paradigm
Serotonergic, adrenergic and dopaminergic projections to the
FCX are all equipped with reuptake sites for 5-HT (SERT), NA
(NAT) and DA (DAT), respectively, though the latter are scarce
and DA is also cleared by the more numerous NATs on adrener-
gic terminals. SERTs and NATs are, of course, the targets of
SSRIs and NARIs, respectively, and they are both concomitantly
blocked by 5-HT/NA reuptake blockers (SNRIs) like venlafaxine
and S33005 (Barnes and Sharp, 1999; Blier and De Montigny,
1994; Celada et al., 2004; Millan et al., 2000a, 2001a).
Serotonergic, adrenergic and dopaminergic neurons also possess
inhibitory autoreceptors: 5-HT1A and 5-HT1D (cell body)/5-HT1B
(terminal), α2-AR (cell bodies and terminals) and D2/D3(cell bod-
ies and terminals) (Barnes and Sharp, 1999; Gardier et al., 1996;
Hjorth and Auerbach, 1996; Hjorth et al., 2000; Lejeune et al.,
1997; Lejeune and Millan, 1998; McDevitt and Neumaier, 2011;
Millan et al., 1995, 2000a, 2000b, 2000c; Rivet et al., 1994). It is
now clear that the α2A-AR subtype predominates as an autore-
ceptor but α2C-ARs fulfill a complementary and subsidiary role
(Gobert et al., 1998, 1999, 2003; Hertel et al., 1999; Millan
et al., 2000a, 2000b; Milan, 2009). D2 autoreceptors are quanti-
tatively predominant in rodents, but D3 autoreceptors are also
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functionally active and, inasmuch as they are more prevalent in
human dopaminergic cell bodies, they may be of greater clinical
significance than usually thought – also as a mechanism of
neuroprotection (Collo et al., 2012; Joyce and Millan, 2007;
Lejeune et al., 1997; Pich and Collo, 2015).
While dopaminergic neurons do not exert any marked influ-
ence on their serotonergic and adrenergic counterparts, the latter
restrain dopaminergic and serotonergic pathways via  α2-AR
heteroceptors localized on their terminals: mirroring autorecep-
tors, α2A-ARs appear to predominate as heteroceptors over their
α2C counterparts (Gobert et al., 1999, 2003; Hertel et al., 1999;
Millan et al., 2000a, 2000b, 2000c; Milan, 2009). Importantly
α2-AR heteroceptors on dopaminergic terminals are tonically
active, whereas those on serotonergic terminals are not, at least as
judged by the consequences of their blockade. Beta-ARs may
also have some minor excitatory influence on dopaminergic pro-
jections (Celada et al., 2004; Gobert and Millan 1999a; Hjorth
and Sharp, 1993) which are subject to a stabilizing and globally
inhibitory influence of α1A/1B-ARs at the level of their cell bodies
(Lejeune et al., 1994).
As for serotonergic fibres, their influence on neighbouring
dopaminergic and adrenergic projections is pronounced, com-
plex and – even today – not fully understood (Blier and El
Mansari, 2013; Millan et al., 2000a; Milan, 2009; Sanchez et al.,
2015). The best documented control is expressed via 5-HT2C
receptors which exert a robust and inhibitory influence on DA
and NA release in FCX: irrespective of whether these sites show
constitutive activity in native tissue, their inhibitory control is
exerted tonically (Dekeyne et al., 2008; Di Giovanni and De
Deurwaerdère, 2016; Millan et al., 2000a, 2003; Millan, 2005b).
Reconciling the excitatory actions of 5-HT2C receptors with
inhibition of adrenergic and dopaminergic pathways has neces-
sitated the presence of intervening GABAergic interneurons,
located both in the ventrotegmental area and locus coereleus
and at the terminal level in the FCX (Di Giovanni and De
Deurwaerdère, 2016; Millan, 2005b; Santana and Artigas, 2016).
A modest excitatory impact of 5-HT2A receptors has also been
seen on DA and NA liberation, possibly direct and/or heterosyn-
aptic and passing by excitatory glutamatergic input (Celada
et al., 2013; Gobert and Millan, 1999b). Despite some intriguing
(or odd to the skeptic) observations in knockout mice and their
staunch defence by a dwindling handful of devotees, 5-HT2B
receptors are the poor relatives of 5-HT2A and 5-HT2C receptors
and merely lurk in the background. In any event, in addition to
postsynaptic sites, there may be a small population of 5-HT2B
receptors on 5-HT neurons that exert a variable, excitatory influ-
ence on 5-HT release, and 5-HT2B sites were speculated to be
somehow involved in the actions of SSRIs (Diaz et al., 2016;
Launay et al., 2006; McDevitt and Neumaier, 2011). While they
should not be ignored, compared to their big brother, 5-HT2C
receptors, 5-HT2B receptors do not seem to have a prominent
role in the control of monoaminergic transmission in the FCX.
Recently, a role for 5-HT3 receptors in the control of mono-
aminergic transmission was revived, possibly by excitation of
GABAergic neurons, but the robustness and therapeutic rele-
vance of this control is uncertain (Celada et al., 2013; Mørk et al.,
2012; Riga et al., 2016; Sanchez et al 2015). 5-HT4 receptors
have been increasingly implicated in the mediation of antidepres-
sant properties, even leading to the suggestion that they can (indi-
rectly) desensitize 5-HT1A autoreceptors (Samuels et al., 2016;
12 Journal of Psychopharmacology
Vidal et al., 2014). 5-HT5 receptors appear to be present in the
raphe, but there is little or no information on control of seroton-
ergic (or catecholaminergic) transmission, perhaps due to a lack
of pharmacological tools (Millan, 2006; Thomas, 2006). As for
5-HT6 receptors, there is evidence for a facilitatory influence of
their blockade on adrenergic and dopaminergic pathways, but
results have been variable and sometimes contradictory (for ago-
nists and antagonists), possibly due to opposing actions of 5-HT6
receptors expressed via GABAergic versus glutamatergic neu-
rons (Kendall et al., 2011; King et al., 2008; Schechter et al.,
2008). Finally, 5-HT7 receptor antagonism has been evoked as a
potential antidepressant strategy (alone and coupled to SERT
blockade) and this may reflect enhanced 5-HT release due to
actions expressed in raphe nuclei, directly and in interaction with
GABAergic interneurons, but data remain limited (Bonaventure
et al., 2007; Kusek et al., 2015; Mnie-Filali et al., 2011).
Needless to say, one should not forget the inhibitory autore-
ceptor roles of 5-HT1B and 5-HT1D receptors (De Groote et al.,
2003; Gardier et al., 1996; Gobert et al., 1998; Gobert and Millan,
1999a; McDevitt and Neumaier, 2011), and there must be a final
word on the 5-HT1A receptors which have been studied in breadth
and depth for decades. While blockade of 5-HT1A autoreceptors
enhances the influence of SSRIs on dialysis levels of 5-HT,
5-HT1A receptor agonists and partial agonists, alone and co-
administered with SSRIs/SNRIs, enhance extracellular levels of
DA and NA in FCX, actions linked to antidepressant properties
(Blier and El Mansari, 2013; Gobert et al., 1998, 1999; Millan
et al., 2000a; Millan, 2006). A role for postsynaptic 5-HT1A
receptors (presumably located on GABAergic interneurons) has
been evoked in the influence of 5-HT1A receptor agonists/partial
agonists on DA/NA release. Nonetheless, on balance, it appears
that recruitment of 5-HT1A autoreceptors is mainly involved
leading to downstream removal of 5-HT2C receptor-mediated
excitation of GABAergic interneurons (Gobert et al., 1998;
Lejeune and Millan, 1998; Santana and Artigas, 2016; Sanchez
et al., 2015). An important point relevant to drug design is that
postsynaptic 5-HT1A receptors are, in addition to 5-HT1A autore-
ceptors, involved in the mediation of antidepressant properties,
including those triggered by inhibition of 5-HT reuptake (see
below) (McDevitt and Neumaier, 2011; Millan, 2006; Samuels
et al., 2016; Sanchez et al., 2015). As regards these postsynaptic
sites, it has been suggested that they influence the activity of
ascending monoaminergic pathways by virtue of feedback loops
running to their cell bodies (Hajós et al., 2003).
Finally, trace amine associated receptors 1 (recruited by trace
amines like tyramine and phenylethylamine) have emerged to
modulate the activity of monoaminergic neurones and the libera-
tion of monoamines in the FCX and sub-cortical regions. They
influence, in particular, dopaminergic and serotonergic transmis-
sion, including the response to drugs suppressing reuptake and/or
impacting mechanisms of release (Di Cara et al., 2011; Ledonne
et al., 2010). In addition, like dopamine D3 and other classes of
autoreceptor, trace amine associated receptors 1 physically and
functionally interact with monoamine transporters, thereby
unveiling a novel dimension to the fine-control of extracellular
levels of monoamines in frontocortical and subcortical structures
(Castro-Hernández et al., 2015; Miller, 2011). Trace amine
associated receptors 1 are also localized postsynaptically to
monoaminergic projections (Pei et al., 2016). They are intrigu-
ing candidates targets for novel classes of drugs for treating
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depression and psychosis but clinical feedback is awaited (Lam
et al., 2015; Revel et al., 2012).
To summarize, the monoaminergic framework articulated by
Millan et al. (2000a) has not, in the past 15 years, been radically
corrected or modified, although the roles of multiple 5-HT recep-
tor subtypes are now better characterized. Further, a modulatory
influence for trace amines has been established, and interactions
between monoaminergic autoreceptors and transporters have
become important. The monoaminergic framework of 2000 has
then continued to provide a coherent schema for devising new
classes of drugs for treating depression, and especially those pos-
sessing actions at novel combinations of monoaminergic receptor
and reuptake site. Nonetheless, things have evidently moved on
and a major emphasis today is the identification of drugs for
treating depression and psychosis that act primarily via mecha-
nisms that are not solely – or at all – monoaminergic. Some
insights into the above strategies and their interrelationships with
monoaminergic transmission are given below.
From monoamine reuptake inhibitors
to novel multi-target classes of
antidepressant
Coupling 5-HT1A receptor activity to
inhibition of 5-HT reuptake: vilazodone and
vortioxetine
One of the earliest ideas of how to accelerate antidepressant onset
of action was based on the somatodendritic localization of the
5-HT1A subtype and its inhibitory influence on serotonergic neu-
rons. It was reasoned that 5-HT1A autoreceptors must brake and
slow the actions of SSRIs by a negative feedback mechanism,
leading to efforts to develop 5-HT1A receptor antagonists and to
associate SERT inhibition and 5-HT1A autoreceptor blockade
either by drug combinations or by their integration into a single
chemical entity (Blier and De Montigny, 1994; Celada et al.,
2004; Gardier et al., 1996; Hjorth et al 2000; McDevitt and
Neumaier, 2011; Millan, 2014a; Millan et al., 1995). Later, as
remarked above, it was realized that postsynaptic 5-HT1A recep-
tors contribute to the antidepressant actions of SSRIs (Millan,
2006; Samuels et al., 2016), so this appeared to be a case of gain-
ing on the roundabout yet losing on the swings. Indeed, 5-HT1A
antagonist/SSRI association studies ultimately failed to improve
on the efficacy of SSRIs and, despite considerable efforts, no
combined 5-HT1A autoreceptor antagonist/SERT blocker has
proven clinical efficacy and reached the market (Blier and El
Mansari, 2013; Celada et al., 2004; Millan, 2006, 2009, 2014a;
Sanchez et al., 2015).
A subtle shift in emphasis led to the notion of coupling 5-HT1A
receptor partial agonist properties to 5-HT reuptake inhibition: this
should permit some postsynaptic efficacy yet also – it is claimed
– realize the more rapid 5-HT1A autoreceptor desensitization com-
pared to a SSRI alone (Celada et al., 2004, 2013; McDevitt and
Neumaier, 2011; Millan et al., 2000a). In fact, it is not so easy to
see why partial agonist properties (drug) should more effectively
desensitize 5-HT1A autoreceptors than SSRI-elevated levels of
5-HT (a full agonist) itself. Nonetheless, 5-HT1A autoreceptor par-
tial agonists/SERT blockers are easier to find (often by accident)
than pure 5-HT1A antagonists/SERT blockers so this was all rather
Millan et al. 13
convenient. Moreover, 5-HT1A partial agonism enhances fronto-
cortical release of NA and DA alone and in association with SSRIs
(Gobert et al., 1998, 1999) and, in contrast to a pure 5-HT1A antag-
onist, one would expect anxiolytic properties from partial agonist
properties in the absence of disruptive side-effects (Millan, 2003;
Millan et al., 1995). To cut a long story short, this manner of
exploiting monoaminergic networks was eventually vindicated
with the launching of vilazodone, a partial 5-HT1A autoreceptor
partial agonist/SERT blocker for depression (Ashby et al., 2013;
Hughes et al., 2005; Sahli et al., 2016). Vilazodone robustly ele-
vates FCX levels of 5-HT and possesses antidepressant and anxio-
lytic actions in preclinical models. Further, upon chronic
administration, it results in dorsal raphe 5-HT1A autoreceptor
desensitization yet an enhancement in the activity of postsynaptic
5-HT1A receptors on CA3 neurones in the hippocampus – mimick-
ing other classes of antidepressant (El Mansari et al., 2015; Sahli
et al., 2016). For reasons that remain unclear, it does not seem to
increase FCX release of NA and DA: how this unexpected (and
presumable) drawback influences therapeutic efficacy for treating
depression (and symptoms of anxiety) compared with SSRIs
remains to be clarified (El Mansari et al., 2015; Hughes et al.,
2005; Sahli et al., 2016; Thase et al., 2014).
Another recently-launched antidepressant, vortioxetine, like-
wise emerged from studies of monoaminergic neurochemistry in
the FCX, and the question as to how a SSRI profile might be
upgraded by concomitantly manipulating discrete classes of
5-HT receptor. Initially, this project was again oriented around
SERTs and antagonist actions at 5-HT1A autoreceptors (Sanchez
et al., 2015). However, it was redirected (or redirected itself to
the gratification of astute pharmacologists and chemists) towards
a broader and multi-target profile that embraced not only 5-HT1A
receptor agonist properties, but also partial agonism at 5-HT1B
receptors as well as antagonist properties at 5-HT1D, 5-HT3 and
5-HT7 receptors (Bang-Andersen et al., 2011; Mørk et al., 2012;
Pehrson et al., 2013; Riga et al., 2016; Sanchez et al., 2015).
5-HT1D and 5-HT7 receptor blockade may help promote seroton-
ergic transmission and participate in antidepressant properties
(see above) (Mnie-Falili et al., 2011). Further, the most potent
action of vortioxetine, 5-HT3 blockade, apart from opposing
nausea during treatment, may offer some modest benefit in treat-
ing symptoms of anxiety (Baldwin et al., 2016; Millan, 2003).
Further, 5-HT3 blockade was suggested to participate in the facil-
itation by vortioxetine of FCX levels of monoamines, possibly by
blocking excitatory 5-HT3 receptors on GABAergic interneurons
(Pehrson and Sanchez, 2014; Riga et al., 2016). This GABAergic
action may also lead to the disinhibiton of glutamatergic trans-
missionand hence favour synaptic plasticity and cognitive func-
tion (Pehrson and Sanchez, 2014; Riga et al., 2016). However, an
induction of frontocortical ACh and histamine release by vortiox-
etine – triggered by its distinctive serotonergic properties – offers
a complementary substrate for pro-cognitive properties in pre-
clinical models (Pehrson et al., 2013; Sanchez et al., 2015). In
line with these observations, there are encouraging findings with
vortioxetine and cognitive performance both in preclinical mod-
els and in patients, notably elderly subjects (Katona et al., 2012;
McIntyre et al., 2016). However, it remains to be seen in further
direct and rigorous comparative trials just how the influence of
vortioxetine on depressed mood, anxious symptoms and impaired
cognition compares with other classes of antidepressant (Baldwin
et al., 2016; Thase et al., 2016).
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Dialling in DA as well as 5-HT and NA
transporters
Compromised dopaminergic transmission in both the FCX and
mesolimbic regions is implicated in the mood, reward, cognitive
and even sexual deficits of depression, and all clinically-effective
antidepressants either elevate FCX levels of DA and/or reinforce
postsynaptic DA signalling in limbic regions (reviewed in Millan,
2006). Hence, there has been interest in adding DA reuptake inhi-
bition to blockade of SERTs and NATs. This is chemically quite
straightforward – indeed, it not infrequently happens without any
conscious effort at all (Lane, 2015; Millan 2009, 2014a).
Accordingly, a whole range of “triple inhibitors” have been
described (Lane, 2015; Millan, 2009). However, rigorous clinical
demonstration of antidepressant properties at well-tolerated
doses is awaited and several – like tesofensine – have been re-
routed to other indications like the treatment of addiction and
neurological disorders. Furthermore, direct elevation of extracel-
lular levels of DA in limbic structures incurs the risk of abuse,
dependence and psychosis, while “triple inhibitors” may also
have autonomic/cardiac-cardiovascular side-effects and cause
insomnia (Millan, 2009; Lane, 2015).
Blockade of α2-adrenoceptors to disinhibit
NA and DA release in FCX
Another way of reinforcing the influence of antidepressants on
DA – as well as NA – levels in FCX would be to block tonically-
active α2-ARs on the terminals of dopaminergic and adrenergic
fibres innervating the FXC (vide supra): since α2-ARs do not in
this way control mesolimbic dopaminergic pathways, there is – in
contrast to triple inhibitors – no risk of excess DA release in sub-
cortical regions (Gobert et al., 1999; Hertel et al., 1999; Millan
et al., 2000a, 2000b). Mirroring the blockade/desensitization of
5-HT1A autoreceptors, pharmacological co-antagonism of α2-ARs
should rapidly reproduce the progressive desensitization of pre-
synaptic α2-ARs seen upon long-term exposure to antidepressants
that elevate extracellular levels of NA (Gobert et al., 1998, 1999;
Millan et al., 2000a, 2000b; Sanacora et al., 2004; Serres et al.,
2012). Further, α2-ARs blockade should counter sexual dysfunc-
tion and, via enhanced ACh release, favour cognitive performance
(Coupland et al., 1994; Millan, 2006). This reasoning led several
groups to develop mixed α2-ARs/inhibitors of 5-HT and/or NA
reuptake (Andrés et al., 2007; Cordi et al., 2001; Gobert et al.,
2002; Serres et al., 2012). One such agent is the spiroimidazoline
S35966, a member of a small family of compounds that have high
affinity for both SERTs and NATs, yet negligible affinity for
DATs, together with high affinity for α2-ARs. The only other
activity of significance is, interestingly enough – and resembling
vortioxetine – antagonist properties at 5-HT3 receptors (pKi 7.5
for S35966) (Cordi et al., 2001; Gobert et al., 2002). As shown in
Figure 6, this receptor profile is distinctive as compared with a
broad range of antidepressants. This includes mianserin that
blocks α2-ARs (and 5-HT3) receptors but not SERT and which, in
contrast to S35966, potently blocks α1-ARs and histamine H1
receptors risking autonomic and sedative side-effects (Millan,
2006). The accompanying Figure 7 shows that venlafaxine
inhibits NA neurons due to release of NA onto α2-ARs (Millan
et al., 2001a),whereas S35966 is auto-protected from feedback-
inhibition and preserves locus coeruleus firing by virtue of its
14 Journal of Psychopharmacology

Figure 6.  Overall binding profile of a novel class of mixed α2-AR antagonists/SNRIs in comparison to other classes of antidepressant agent.
S35966 – and highly-related “S” ligands – behave as inhibitors of 5-HT and NA reuptake (pInhibitory Concentration50 values shown for rat synaptosomes) and, in contrast
to venlafaxine, as antagonists at α2-ARs (pKb value shown for blockade of Gαi/o-protein coupling at hα2A-ARs). The comparable pKi values for S35966 at these sites and
for a dozen other sites underlying beneficial and deleterious actions of antidepressants (Gobert et al., 2002; Millan, 2006) were used to construct (by principal com-
ponent analysis) a dendrogramme: this formally revealed the distinctive overall receptor-binding profile of S35966 and its relatives compared to venlafaxine and other
“categories” of antidepressant. Note that, like venlafaxine and other SNRIs and in contrast to the other classes of antidepressant, S35966 had low affinity (pKi, < 5.5) for
histamine H1 receptors, α1-ARs and muscarinic M1 receptors eliciting autonomic/cardiovascular side-effects (Gobert et al., 2002; Millan, 2006).

antagonist properties at α2-AR autoreceptors. Correspondingly,
S35966 induces a profound increase in NA release in FCX (and
hippocampus) even greater than co-administration of venlafaxine
and a α2-AR antagonist. Likewise, S35966 is more effective than
venlafaxine in elevating DA levels in FCX (Gobert et al., 2002).
Conversely, the efficacy of S35966 and venlafaxine for increasing
5-HT levels in FCX is similar, possibly since α2-ARs on 5-HT
neurones are not tonically active – S35966 does not block 5-HT1A
or 5-HT1B/1D autoreceptors. However, this remains to be clarified.
Compared to venlafaxine, the robust neurochemical profile of
S35966 translates into marked antidepressant properties, for
example, in a forced swim test. It is also associated with the more
rapid alteration of biomarkers of onset of antidepressant efficacy
upon chronic administration to rats: down-regulation of 5-HT2A
receptors, up-regulation of BDNF, and modulation of neuroplasti-
city genes like Arc and Shank (Serres et al., 2012). Moreover,
S35966 can be distinguished from venlafaxine by its induction of
ACh release in FXC and precognitive properties in preclinical
models (Gobert et al., 2002).
In view of the rather striking profile of S35966 (Figure 7), it
might not unreasonably be asked why it has not become available
to patients, notably for those resistant to SSRIs and SNRIs. In
fact, it was not even evaluated in humans owing to concerns of
unacceptable cardiovascular side-effects, in particular hyperten-
sion due to blockade of α2-ARs on sympathetic terminals. Thus,
despite this preclinical proof of concept, α2-AR autoreceptor
blockade for reinforcing the efficacy of SERT-NAT blockers has
not (yet) been translated into the clinic.
Blockade of 5-HT2C receptors for improving
antidepressant efficacy
As mentioned above, blockade of excitatory 5-HT2C receptors
on GABAergic interneurons inhibitory to dopaminergic and
adrenergic pathways elevates release of DA and NA in the
FCX. Accordingly, selective antagonists and inverse agonists at
5-HT2C receptors, despite not changing 5-HT release in FCX,
possess antidepressant properties and – probably reflecting
blockade of 5-HT2C sites in limbic regions like the hippocampus
and amygdala – they also display anxiolytic actions (Dekeyne
et al., 2008; Di Giovianni and De Deurwaerdère, 2016; Millan,
2005b, 2006). However, no selective antagonist or inverse agonist
has ever been clinically developed. One factor that hampered
clinical progress was the persistent lack of a positron emission
tomography (PET) ligand to verify 5-HT2C receptor engagement

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Millan et al. 15

Figure 7.  Overview of the influence of S35966 compared to venlafaxine on monoaminergic transmission in the FCX, and antidepressant properties in
the forced-swim test in rats.
Panel A, Activity of adrenergic cell bodies in the locus coeruleus: The α2-AR antagonist, atipamezole, blocks the inhibitory influence of venlafaxine which releases NA
onto α2-AR autoreceptors – intrinsically blocked by S35966 itself. Panel B, Extracellular levels of NA in FCX: atipamezole blocks α2-AR feedback inhibition by venlafaxine
to potentiate increases in levels of NA which are even more pronounced for S35966 alone – active dose-dependently from 0.16–10.0 mg/kg, s.c. (not shown). Panel C,
Extracellular levels of DA in FCX: S35966 has a more pronounced action than venlafaxine which via NA indirectly activates α2-ARs on dopaminergic terminals. Panel D,
Extracellular levels of 5-HT in FCX: no difference between the drugs, possibly since α2-ARs on serotonergic terminals are not tonically active. Panel E, Extracellular levels
of ACh in the FCX: S35966 elevates ACh by blocking inhibitory α2-ARs on their terminals. Panel F, Reduction of immobility in a forced-swim test of antidepressant proper-
ties: S35966 at equivalent doses to those increasing FCX levels of monoamines is more potent than venlafaxine and has a greater maximal effect. Drugs given i.v. for
Panel A, 3 min pre-measure; for Panels B to E, drugs were given s.c. as shown by arrows; and for Panel F, drugs were given s.c. three times, 24, 1 h and 30 min pre-test.
All data are means ± SEMs.*P < 0.01 for drug to vehicle, and # for S35966 versus Venlafaxine, in Newman–Keuls test following ANOVA. N = 6–8 per value.

in human subjects. In the case of S32006, its non-development (1-(1-phenylethyl)-spiro[indole-3,4′-piperidin]-2(1H)-one)

was also partly due to the pre-existence of agomelatine and the
decision to explore other avenues for exploiting 5-HT2C recep-
tors. For example, the union of α2-AR and 5-HT2C antagonist
properties into a single compound. By doing so, it was hoped to
mimic the beneficial effects of mianserin and mirtazapine,
which block both these sites, while avoiding their detrimental
actions due to off-target effects at, as mentioned above, α1-ARs
and histamine H1 receptors as well as, albeit less markedly,
muscarinic M1 receptors.
This was realized with the discovery of S32212, which
revealed a promising preclinical profile in terms of its induction
of FCX levels of NA and DA and robust actions in tests of anti-
depressant, anxiolytic and pro-cognitive properties, yet it had to
be abandoned owing to unanticipated actions at ion channels
that may have triggered cardiac side-effects (Dekeyne et al.,
2012; Millan et al., 2012b). Another potential, dual-acting
antidepressant based on 5-HT2C antagonism was S42141
which was designed to retain the mixed 5-HT2C/SNRI-like prop-
erties of older agents like amitriptyline yet ditch their unfavour-
able actions at Na+/Ca++ channels, histamine H1 and muscarinic
receptors (Millan, 2006). The appropriate binding profile was
achieved without undue difficulty (pKi values for SERT/NAT/5-
HT2C receptors 8.2/7.9/7.4 respectively versus less than 6.0 for
histamine H1 receptors, muscarinic M1 receptors and α1-ARs.
Further, S42141 robustly induced frontocortical release of NA,
DA and 5-HT, was active in tests of antidepressant activity in
rodents to the same degree as amitriptyline and – in contrast to
the latter – displayed anxiolytic properties (Millan et al., unpub-
lished observations). While S42141 escaped the fate of S32212
at ion channels, it was discarded for what is euphemistically ref-
ered to as “strategic” considerations.
Thus, it proved possible to design and validate new 5-HT2C
receptor-based agents for manipulating monoaminergic trans-
mission in FCX, and to demonstrate using microdialysis their

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16 Journal of Psychopharmacology

Figure 8.  A conceptual framework for the design of antidepressant agents acting either selectively or at pairs of monoaminergic and/or non-
monoaminergic target.
The figure highlights a selection of mechanisms that have been directly examined in the lab of the authors. Drugs in violet are selective and those in pink are dual-
acting. The latter include agomelatine which has been launched for the clinical treatment of depression. S32212 and S41744 have been extensively described in the
literature, S47445 has also recently been documented, while S35966 and S42141 are both outlined herein. All the actions shown would be expected to improve mood in
depression, though they may not be sufficient alone. In green are indicated some additional benefits of the non-monoaminergic property in question though there may
be others, and the actions indicated are not necessarily exclusive. For example, melatoninergic mechanisms may also reduce anxiety, while positive allosteric modulation
(PAM) of AMPA receptors may influence circadian rhythms.

pronounced reinforcement of frontocortical monoaminergic
transmission. However, as so often in “R and D” for psychiatric
disorders, they fell either at the intimidating hurdle of safety
pharmacology or failed to gel with current development priori-
ties. More cheerfully, as noted below, greater luck was had with
a further 5-HT2C-plus ligand, agomelatine.
Coupling non-monoaminergic properties to
monoaminergic actions
As discussed elsewhere (Millan 2006, 2009, 2014a), a further
and inherently rich strategy for improving the clinically-vali-
dated profiles of monoaminergic antidepressants, largely –
though not only – articulated around blockade of SERTs,
NATs and 5-HT2C receptors, is to integrate actions at non-
monoaminergic classes of receptor (Figure 8). One major exam-
ple is provided by the combined melatonin MT1 and MT2 recep-
tor agonist/5-HT2C receptor neutral antagonist, agomelatine,
launched for the treatment of depression in Europe (De Barardis
et al., 2015; De Bodinat et al., 2010; Kamal et al., 2015; Millan
et al., 2003, 2005, 2011; Soumier et al., 2009). This agent has
unfortunately been handicapped by the need to control for the
risk of rare hepatic side-effects (Perlemutter et al., 2016) but
remarkably it is still the only currently-available antidepressant
with a non-monoaminergic component of action. In addition to
the advantages of 5-HT2C receptor antagonism (see above),
melatonergic properties were conceived to promote sleep and
resynchronize depressed patients with insomnia and disrupted
circadian rhythms (Catena-Dell’Osso et al., 2012; De Berardis
et al., 2015; De Bodinat et al., 2010). Further, reflecting its
unique and synergistic mechanism of action, agomelatine
improves symptoms of anxiety as well as depressed mood
(Stein et al., 2013).
Several other novel concepts have been explored for the rein-
forcement of monoaminergic transmission in the FCX by graft-
ing additional actions onto a core and validated monoaminergic
mechanism. For example, mixed neurokinin (NK1) receptors
antagonists/5-HT reuptake inhibitors where blockade of NK1
receptors moderates (by analogy to 5-HT autoreceptor blockade)
the impact of 5-HT reuptake suppression on extracellular levels
of 5-HT in FCX – and in parallel increases the frontocortical
release of NA and DA (Gobert et al., 2009; Guiard et al., 2006;
Millan et al., 2010). These and related agents have not yet
reached patients, but this still appears to be a promising avenue
for progress.
In this light, as recently analyzed elsewhere (Millan, 2014a),
an important question is whether to either: (1) employ associa-
tions of two separate drugs or (2) integrate dual activity into a
single compound. On balance, the latter approach is the more
promising, but the former is well adapted to drug reorientation.
The most appropriate multi-target strategy will depend upon drug
availability, the mechanism of action sought and the patient pop-
ulations to be treated. Whatever the strategy, microdialysis evalu-
ation of the impact on frontocortical release of monoamines is
likely to be a pivotal step in preclinical characterization.
Coupling regulation of ACh release to
strategies for improving cognition
As mentioned above, several monoaminergic mechanisms
(directly and indirectly) engaged by clinically-employed antide-
pressants can promote release of ACh in the FCX, including

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Millan et al. 17
5-HT1A partial agonism, 5-HT4 agonism and blockade of α2-ARs
(Gobert et al., 2003; Millan, 2006; Millan et al., 1995; King et al.,
2008; Peñas-Cazorla and Vilaró, 2015; Sanchez et al., 2015).
This should promote performance in certain cognitive tasks, an
issue of importance since cognitive deficits in depression have
been neglected relative to schizophrenia, yet they can sometimes
be quite disabling (Marazitti et al., 2010; Millan et al 2012a).
In fact, α2-AR antagonist properties (possessed by mianserin
for example) may be a double-edged sword since frontocortical
populations of postsynaptic α2-AR (probably on pyramidal
neurons) promote attention, working memory and certain other
components of cognitive function (Coupland et al., 1994; Millan,
2006; Millan et al., 2015; Ramos and Arnsten, 2007; Robbins
and Arnsten, 2009; Sallee, 2010).
Further, it remains difficult to extrapolate from pro-cognitive
observations in rodent tests to real-world function in human
patients (see below), and no antidepressant has yet been given a
market authorization for restoring cognitive performance in
patients (Millan et al., 2012a; Rosenblatt et al., 2015). In this
light, a particularly interesting compound to pursue will be vorti-
oxetine which, as outlined above, releases ACh (as well as DA,
NA and histamine) via serotonergic mechanisms without block-
ing muscarinic or other classes of receptor that may compromise
potential pro-cognitive properties, and which has shown positive
effects on cognition in rodents and patients (Katona et al., 2012;
McIntyre et al., 2016; Pehrson et al., 2013; Rosenblatt et al.,
2015; Sanchez et al., 2015).
One class of receptor that vortioxetine does not interact with
is the 5-HT6 receptor. Antagonists at this site have been designed
for the improvement of cognition primarily in Alzheimer’s dis-
ease and schizophrenia, at least partially due to their enhance-
ment of cholinergic transmission, and they are still in active
development, at least for the former indication (De Bruin and
Kruse, 2015; Hirst et al., 2006; King et al., 2008; Loiseau et al.,
2008; Meffre et al., 2012). Indeed, they possess strikingly robust
patterns of pro-cognitive properties in preclinical models (op.
cit.) but, as pointed out above, the neurochemistry of 5-HT6
receptors is complex and actions at GABAergic and glutamater-
gic as well as catecholaminergic pathways are relevant to the
influence of 5-HT6 receptor ligands upon cognition (De Bruin
and Kruse, 2015; Kendall et al., 2011; King et al., 2008; Schechter
et al., 2008).
We have found a facilitatory influence of 5-HT6 antagonists
upon FCX (and hippocampal) release of DA and NA (Figure 9),
and inhibition of GABAergic interneurons may be at least
partially responsible for this observation (De Bruin and Kruse
2015; Schechter et al., 2008). On the other hand, and rather sur-
prisingly, the influence of 5-HT6 antagonists upon FCX levels of
ACh (De Bruin and Kruse 2015, Hirst et al., 2006) has proven to
be significant yet quite modest under the rigorous dialysis condi-
tions (no cholinesterase inhibitors) that we have developed. Thus,
by analogy to multi-target agents for relieving depressed mood,
the question arises as to whether 5-HT6 antagonism might best be
coupled to complementary pharmacological properties for fur-
ther strengthening the induction of ACh release in FCX and,
accordingly, pro-cognitive properties.
One promising candidate would be blockade of dopamine D3
receptors (Saavedra et al., 2016) since: (1) this mechanism
consistently reinforces frontocortical (though not hippocampal)
cholinergic transmission; (2) in contrast to antagonism of D2
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receptors which denigrates cognitive performance, blockade of
D3 receptors in the FCX is associated with a remarkably broad-
based and robust pattern of pro-cognitive activity in rodent and
primate models of pro-cognitive properties: moreover, pro-cog-
nitive actions are at least partly expressed in the FCX and (3) the
potential antipsychotic properties of D3 receptor blockade would
be of utility in the treatment of both schizophrenia and
Alzheimer’s disease, as well as psychotic depression (Gross
et al., 2013; Loiseau and Millan, 2009; Millan and Brocco, 2008;
Millan et al., 2007a; Nakajima et al., 2013; Watson et al., 2012).
It is, thus, of interest that sub-maximal and just effective doses of
5-HT6 and D3 receptor antagonists cooperatively elevated levels
of ACh in the FCX (Figure 9). Both 5-HT6 and D3 receptor antag-
onists are effective alone in rat models of social and object recog-
nition – expressing their actions in the FCX itself (Loiseau and
Millan, 2009; Loiseau et al., 2008; Watson et al., 2012). Mirroring
their neurochemical interaction, the association at sub-active
doses of 5-HT6 and D3 receptor antagonists “synergistically”
improved performance in a model of social recognition. This
interaction was confirmed by use of two different 5-HT6 antago-
nists (Figure 9).
Although we have failed to find an influence of 5-HT6 antago-
nists upon extracellular levels of glutamate (Rivet, unpublished
observations), this may reflect technical and kinetic limitations
(see above), so an influence on glutamatergic transmission should
not be excluded. Indeed, pro-cognitive properties of 5-HT6
antagonists were abolished by the NMDA receptor antagonist,
dizocilpine, suggesting dependence on functionally-intact
NMDA receptors that are known to modulate social cognition,
likely by actions in the FCX (De Bruin and Kruse 2015;
Shimazaki et al., 2010). Interestingly, pro-cognitive properties of
D3 antagonists expressed in the FCX are dependent upon the
release of d-serine from astrocytes onto the co-agonist site of
NMDA receptors in FCX (Dallerac et al., 2014). This provides a
fascinating example of (D3/5-HT6 receptor driven) molecular
convergence in the control of cognition via glutamatergic/d-ser-
ine signalling at FCX-localized NMDA receptors.
Dual dopamine D3/5-HT6 receptor antagonists are just one
example of an unjustly neglected yet potentially important pal-
ette of designer multi-target drugs for promoting release of ACh
and other transmitters controlling cognition in the FCX, and
hence countering cognitive dysfunction in depression and other
CNS disorders.
Influence of potential glutamatergic
drugs for treating depression on
extracellular levels of monoamines
and amino acids in FCX
The pervasive contribution of glutamatergic mechanisms to fron-
tocortical networks controlling mood, cognition and other func-
tions disrupted in depressed states, and evidence for a perturbation
of glutamatergic signalling in depression, has kindled interest in
potential “glutamatergic” drugs for treating depression. In this
regard, it is useful to recall the notion of a push/pull (accelerator/
brake) influence of glutamatergic projections on monoaminergic
pathways: that is direct, excitatory actions counterbalanced
and finely-tuned by indirect, GABAergic interneuron-mediated
inhibition. This can explain some at first sight contradictory
18 Journal of Psychopharmacology

Figure 9.  Complementary 5-HT6 and D3 receptor antagonist properties for promoting cognitive performance: a neurochemical and behavioural
characterization in rats.
Panel A, Influence of the selective 5-HT6 receptor antagonist, SB271,046, upon extracellular levels of monoamines in the FCX. Panel B, Influence of SB271,046, and of
the selective D3 receptor antagonist, S33084, alone and together upon extracellular levels of ACh in the FCX. Panel C, Influence of combined administration of SB271,046
and S33084 upon social recognition in rats. Both these drugs are dose-dependently (0.16–10.0 and 0.01–0.63 mg/kg respectively) active in this procedure (Loiseau
et al., 2008; Loiseau and Millan, 2009; Millan et al., 2007): they were combined at sub-active doses to test for synergy. Panel D, Influence of combined administration
of another selective 5-HT6 receptor antagonist, SB258,585 plus S33084, upon social recognition. Panel E: Blockade of the pro-cognitive properties of S271,046 in the
social recognition test by the NMDA receptor channel blocker, dizocilpine. Drugs given s.c. For Panels A and B, arrows indicate times of drug administration. For Panels
C to E, T2 (test two) − T1 (test one, 2 h earlier) is the difference in exploration time during a 5 min text by the treated adult rat of the same conspecific. A minus score
indicates good retention in the second test (low exploration rate as familiar). In control experiments when a new rat is presented (not shown), T2 − T1 is close to zero
(see Figure 10 and Loiseau and Millan, 2009). The drugs were given 1 min after T1. All data are means ± SEMs. *P < 0.01 for SB271,046 to vehicle in Panel A. For Panels
B to D, #P < 0.01 for SB271,046/S33084 versus SB271,046 or S33084 alone in Newman–Keuls test following ANOVA. In Panel E, P < 0.01 for Dizocilpine/SB271,046 vs
Vehicle/SB271,016. N = 6–8 per value.

observations such as why both a NMDA receptor antagonist and
antagonist may activate dopaminergic and adrenergic transmis-
sion (Table 2) (Celada et al., 2013; Millan, 2005a).
While potential antidepressant actions of mGluR2 receptor
antagonists and mGluR7 receptor agonists have been evoked
(Campo et al., 2011; Millan, 2014a; Palucha et al., 2007), a larger
body of evidence has focussed on blockade of mGluR5 receptors
as a strategy for treating depression (Hughes et al., 2012;
Lindemann et al., 2015; Pałucha-Poniewiera et al., 2013;
Pomierny-Chamioło et al., 2010) This focus partly reflects their
functional coupling to NMDA receptors, but also compound
availability since mGluR5 antagonists have been intensively
studied in Fragile X syndrome and, more recently, Alzheimer’s
disease (Dölen et al., 2010; Kumar et al., 2015; Millan 2013).
Antidepressant actions of mGluR5 antagonists have been
reported in a variety of rodent models, and it has been suggested
that serotonergic mechanisms may be involved in their effects
(Fukumoto and Chaki, 2015). However, not all studies agree and
we have seen no influence of mGluR5 antagonists like “MPEP”
upon 5-HT levels in dialysates of FCX (Table 2). We likewise
observed little influence on extracellular levels of DA and NA in
FCX, supporting a mechanism of action distinct from conven-
tional antidepressants, observations well aligned with the work of
Lindemann et al (2015) on another mGluR5 antagonist. As
shown in Table 2 and consistent with actions downstream of glu-
tamatergic terminals, MPEP exerted negligible influence upon
dialysate levels of most amino acids, including taurine and PEA,
underpinning the benign influence of mGluR5 antagonists on
overall cellular function and integrity (Inta et al., 2012). Indeed,
the only effect seen was a modest decrease in GABA levels, pre-
sumably reflecting relief on an excitatory mGluR5 tone on
GABAergic neurones (Sarihi et al., 2008).
As mentioned above, mGluR5 and NMDA receptors are func-
tionally coupled, and there is evidence that an interference with
activity at NMDA receptors is involved in the antidepressant
actions of mGluR5 antagonists (Fukumoto and Chaki, 2015;

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Millan et al. 19
Hughes et al., 2012; Pałucha-Poniewiera et al., 2013; Pomierny-
Chamioło et al., 2010). This renders comparisons with the
NMDA channel blocker ketamine of interest. Ketamine is cur-
rently enjoying something of renaissance since it rapidly allevi-
ates depressed mood upon i.v. (and possibly intranasal)
administration to humans, and also has pronounced antidepres-
sant actions in rodents (Park et al., 2015). In contrast to the
mGluR5 antagonist, MPEP, ketamine enhances frontocortical
release of NA and DA – even at lowish doses. A prompt elevation
in frontocortical DA and NA release may be related to the rapid-
onset antidepressant actions of ketamine, though a role for sero-
tonergic transmission should not be excluded, especially at
higher yet clinically-relevant doses (Du Jardin et al., 2016).
The decrease in extracellular levels of GABA seen with keta-
mine (Table 2) – albeit at a high dose – agrees with previous
studies suggesting that it suppresses tonic NMDA receptor
(NR2B subtype) excitation of GABAergic interneurons. As a rule
of thumb, NMDA receptor channel blockers like ketamine have
more robust actions than competitive NMDA receptor antago-
nists like CPP which did not affect levels of GABA (Table 2).
Nonetheless, in this light, it is apposite to mention “off-target”
actions of ketamine independent of NMDA receptors which
should not be neglected in interpreting its actions (Abdallah
et al., 2015; Gobert et al., 2011; Park et al., 2015; Zhou et al.,
2015). At a high dose, ketamine elevated levels of glutamate and
aspartate in FCX, confirming previous work with NMDA recep-
tor channel blockers, and presumably reflecting a loss of
GABAergic inhibition (Table 2) (Abdallah et al., 2015; Park
et al., 2015; Zhou et al., 2015). It is thought that glutamate
released by ketamine downstream of GABAergic neurones
recruits AMPA receptors that themselves stimulate BDNF and
mammalian target of rapamycin (mTOR) signalling, leading to
expression of antidepressant properties (Abdallah et al., 2015;
Park et al., 2015). Interestingly, independently of glutamate
release, ketamine metabolites also exert antidepressant properties
via recruitment of AMPA receptors, though it is not clear exactly
how they exert their actions (Zanos et al., 2016).
The presumptive role of AMPA receptors in the antidepressant
actions of ketamine (though it is not certain that they are sufficient
rather than just necessary) has rekindled interest in AMPA recep-
tors themselves as targets for antidepressant agents (Farley et al.,
2010; Li et al., 2003; Nations et al., 2012; Park et al., 2015; Zanos
et al 2016). For example, the AMPA receptor positive allosteric
modulator (PAM), S47445, is under development for the treat-
ment of cognitive and mood deficits in Alzheimer’s disease
(Bretin et al., 2015). In rat procedures of antidepressant proper-
ties, AMPA receptor PAMs like LY392,098 are active at doses
corresponding to those expressing their pro-cognitive properties
(Figure 10). As for the underlying neurochemical mechanisms,
LY392,098 did not affect NA or 5-HT levels in FCX dialysates,
though it did exert a mild increase in DA levels suggesting that
dopaminergic mechanisms in the FCX might be involved in the
antidepressant actions of AMPA receptor PAMs: this awaits con-
firmation. LY392,098 exerted little influence on amino acids
(Table 2), in line with the notion that it acts downstream of
the NMDA receptor/GABAergic interneurone/glutamatergic axis
engaged by ketamine. Furthermore, the lack of influence of
LY392,098 upon taurine and PEA supports a lack of excitotoxic-
ity. Finally, AMPA receptor PAMs may enhance the actions of
SSRIs and, as depicted in Figure 8, the notion of their association
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is attractive, either as “dual-acting ligands” or as a combinatin of
drugs (Abdallah et al., 2015; Fitzpatrick et al., 2016; Li et al.,
2003; Millan, 2014a).
Influence of glycine reuptake
inhibitors, glycine and d-serine, upon
dialysis levels of amino acids and
monoamines in FCX
Inhibitors of glycine reuptake (Gly-RI) elevate extracellular lev-
els of glycine and hence indirectly promote glycine-mediated
activation of the co-agonist glycine B site on NMDA receptors
(Millan, 2005a). It has been suggested – based on preclinical
observations – that they may be useful in the treatment of depres-
sion (Pałucha-Poniewiera et al., 2013; Park et al., 2015). In this
regard, it is of note that the Gly-RI, sarcosine, exerts partial ago-
nist actions at NMDA receptors but it is unclear how this action
relates to potential antidepressant properties (Chen et al., 2015;
Park et al., 2015). Conversely, the modest increase by sarcosine
and another Gly-RI, ORG 24598, of extracellular levels of DA in
FCX may more readily be assimiliated into a positive influence
upon depressed mood (Table 2) (Alberati et al., 2012; Nagy et al.,
2010).
It is worth stressing the selectivity of the actions of sarcosine
inasmuch as levels of d-serine and glutamate were unchanged at
doses that elevated dialysis levels of glycine (Figure 11 and Table
2). Though sarcosine provoked an increase in taurine levels, phe-
nylethanolamine was not – in contrast to NMDA – affected,
underscoring the more benign impact of sarcosine on cellular
integrity (Table 2). Collectively, these data support the selectivity
of sarcosine for glycine transporters and for increasing glycine
levels at doses equivalent to those used clinically as an adjunct
to antipsychotics – albeit with mitigated success (Chue, 2013;
Heresco-Levy and Javitt, 2004; Kantrowitz and Javitt, 2010;
Millan, 2005a). These findings are not trivial since, to our knowl-
edge, they comprise the first comprehensive characterization of
its neurochemical profile – decades after initiation of clincical
trials! (Gobert et al., 2011; Kantrowitz and Javitt, 2010).
As regards ORG 24598 (Brown et al., 2001), it likewise
exerted little influence on amino acids other than glycine, again
supporting the safety and selectivity of Gly-RIs (Figure 11)
(Alberati et al., 2012). Findings in schizophrenia are not to our
knowledge available for ORG 24598, despite preclinical and
clinical support with bitopertine (as monotherapy) for the hypoth-
esis that Gly-RIs may improve negative and cognitive symptoms
(Alberati et al., 2012; Bugarski-Kirola et al., 2014; Heresco-Levy
and Javitt, 2004; Kantrowitz and Javitt, 2010; Liem-Moolenaar
et al., 2010; Umbricht et al., 2014). Less positively, bitopertine
seems to have an inverted dose–response curve and, despite
encouraging Phase II observations, Phase III findings were
ambivalent (Bugarski-Kirola et al., 2014; Umbricht et al., 2014).
Nonetheless, neurochemistry was crucial in getting the drug to
patients, and it was elegantly shown by measuring glycine in
human CSF that the doses of bitopertine used in clinical trials
were equivalent to those yielding positive neurochemical and
behavioural effects in animals (Alberati et al., 2012; Bugarski-
Kirola et al., 2014; Hofmann et al., 2016; Umbricht et al., 2014).
By analogy to the Gly-RI, sarcosine, glycine itself has
been extensively evaluated as an adjunct in schizophrenia
20 Journal of Psychopharmacology

Figure 10.  Influence of AMPA receptor positive allosteric modulators upon monoaminergic transmission in the FCX in comparison to antidepressant
and pro-cognitive properties in rats.
Panel A, Influence of the AMPA receptor PAM, LY392,098, on extracellular levels of 5-HT in FCX in comparison to the SSRI, citalopram. Panel B, Influence of LY392,098
on extracellular levels of NA in FCX in comparison to the NARI, reboxetine. Panel C, Influence of LY392,098 on extracellular levels of DA in FCX in comparison to the DA
reuptake inhibitor, bupropion. Panel D, Facilitation by LY392,098 of the recognition of a familiar conspecific in a social recognition procedure. For a description of the
procedure, see legend to Figure 8 and Loiseau et al. (2008). Panel E, Influence of LY392,098 on immobility in a forced swim test of antidepressant properties. Drugs were
given 24 h, 1 h and 30 min prior to the test. Panel F, Influence of another AMPA PAM, LY404,187, on immobility in a forced swim test employing the same procedure.
Drugs were given i.p., except for Panel F. All data are means ± SEMs. *P < 0.01 for drug versus vehicle in Newman–Keuls test following ANOVA. N = 6–8 per value.

(Heresco-Levy and Javitt, 2004; Kantrowitz and Javitt, 2010),
yet its neurochemical profile has never been thoroughly charac-
terized. Using our novel dialysis system, it was found that doses
comparable with those used clinically elicited a colossal increase
in dialysis level of glycine, dwarfing the effects of Gly-RIs
(Figure 11) (Alberati et al., 2012; Gobert et al., 2011; Kantrowitz
and Javitt, 2010). It is unclear whether this observation suggests
that Gly-RIs do not attain levels sufficiently high for robust clini-
cal effects or, more likely, that the effect of glycine itself on
extracellular glycine – assuming that extrapolation to humans is
valid – is extremely high. Also striking is the influence of glycine
upon a broad range of amino acids, including a decrease in d-
serine release. This may be compensatory for the elevation in
glycine levels/enhanced NMDA receptor engagement, and a
reduction in d-serine levels was also seen upon administration of
NMDA (Table 2). By analogy to NDMA, the diminution in d-serine
release induced by glycine was blunted by the competitive
NMDA receptor antagonist, CPP, suggesting partial and media-
tion by NMDA receptors (Gobert et al., 2011; Rivet, unpublished
observations). It should also be mentioned that the decline in
d-serine levels was specific in that l-serine was not affected
(Gobert et al., 2011). Interestingly, glycine provoked a significant
increase in extracellular levels of DA and NA in FCX. Though
we have not formally analyzed the underlying mechanisms, this
reinforcement in frontocortical dopaminergic transmission – which
mirrors the effects of some mixed dopaminergic/serotoninergic
drugs for treating psychosis (Millan et al., 1998a,b) – might be
interpreted as supporting the utility of Gly-RIs and glycine B
ligands for control of the cognitive and negative symptoms of
schizophrenia (Bugarski-Kirola et al., 2014; Kantrowitz and
Javitt, 2010; Millan, 2005a; Umbricht et al., 2014; Yang and
Svensson, 2008). However, this remains to be proven and clinical
data with glycine in schizophrenia have been (as for the glycine
B site partial agonist, d-cyloserine) ambivalent (Heresco-Levy
and Javitt, 2004; Kantrowitz and Javitt, 2010).
Finally, in contrast to this broad pattern of changes seen with
glycine, d-serine – which has also yielded chequered results as an
adjunct in schizophrenia (Heresco-Levy and Javitt, 2004;
Kantrowitz and Javitt, 2010) – was remarkably selective in exclu-
sively increasing dialysate levels of d-serine (without affecting
l-serine) (Gobert et al., 2011). This suggests that these two agents
should not be considered as interchangeable, in line with their

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Millan et al. 21

Figure 11.  Influence of glycine, d-serine and two glycine reuptake inhibitors upon extracellular levels of DA, NA, glycine and d-serine in FCX in rats.
Panels A and B, Dose-dependent influence of glycine upon dialysis levels of DA and NA. (5-HT was not affected (not shown). Panel C, Dose-dependent influence of
glycine compared to d-serine and the two glycine reuptake inhibitors, sarcosine and ORG 24598, upon dialysis levels of glycine in FCX. Panel D, Dose-dependent influence
of d-serine compared to glycine, sarcosine and ORG 24598, upon dialysis levels of d-serine in FCX. Note that glycine decreases the levels of d-serine whereas d-serine does
not affect levels of glycine. All data are means ± SEMs. *P < 0.01 for drug versus vehicle in Newman–Keuls test following ANOVA. N = 6–8 per value.

contrasting patterns of action at NMDA receptor subtypes and
functional profiles (Danysz and Parsons, 1998; Fossat et al.,
2012). Moreover, these data are reassuring as regards the safety of
d-serine as an agent for probing glycine B receptor function. On
the other hand, d-serine should not be regarded as entirely benign
inasmuch as over-production and over-activation of endogenous
pools has been implicated in the pathophysiology of Alzheimer’s
disease and may, together with Aβ42 and Tau, serve as a useful
biomarker for this disorder (Madeira et al., 2015).
Collectively, the above observations underpin the importance
of microdialysis: (1) for evaluating the influence of glutamater-
gic agents upon FCX function and determining their potential
therapeutic pertinence and (2) for measuring extracellular levels
of amino acids and exploring their relevance to the pathogenesis
and treatment (by other drug classes) of CNS disorders.
Translating insights from FCX
neurochemistry into humans: a work
in progress
“Translational” has become something of a catchword in recent
years, and it is sometimes used for almost anything and every-
thing that is done pre-clinically in terms of its ostensible rele-
vance to – and pursuit by – clinical studies. More precisely, the
term refers to the use in animals of procedures and readouts,
models and measures that are as close to and as relevant as pos-
sible to those used in humans in order to optimize the transition
from drug discovery to clinical drug development (Hendriksen
and Groenink, 2015; Kas et al., 2011; Millan, 2008; Pratt et al.,
2012). Arguably, this is exactly what pharmacologists and
clinicians have been trying to do since the very advent of drug
discovery – though the trendy buzz-word “translational” was
only invented much later. “Translational research” is not, then, a
conceptual revolution at all, but rather a procedural revision inas-
much as we have, thanks to technological progress, a broader
range of possibilities available than several decades ago – and
indeed in 2000. There is also far greater understanding of the
neural substrates of CNS disorders and of the mechanisms of
action of therapeutics - information which helps guide transla-
tional studies. Finally, partly due to many failures in the clinical
development of drugs for treating CNS disorders, the mindset has
changed. There is a much wider perception of the importance of
reciprocally bridging animal and human studies, and of the need
for two-way traffic: human studies driving preclinical designs
and vice versa (Bespalov et al., 2016; Millan et al., 2015).
This is epitomized by the “MATRICS/CNTRICS” initiative to
harmonize preclinical and clinical measures of cognitive perfor-
mance and its modulation by potential medication (Buchanan
et al., 2010; Millan and Bales, 2013; Young et al., 2009). Further,

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22 Journal of Psychopharmacology
functional magnetic resonance imaging (fMRI) is being “back-
translated” (albeit laboriously) into mice and rats; it is relatively
easy to undertake parallel electroencephalographic studies in
rodents and humans; “PK/PD studies” of drug exposure are
becoming de rigeur; and PET, imaging and other measures of
target engagement are quasi-mandatory (Millan, 2008; Millan
et al., 2015; Murphy and Mackay, 2011; Sakkalis, 2011;
Whittington et al., 2010; Vyas et al., 2011).
It is not unreasonable then to ask how microdialysis and neu-
rochemistry in rodents can be translationally advanced into, and
inform, clinical characterization of novel chemical entities.
In fact, this continues to present challenges, not least since one
cannot implant probes into the brains of human volunteers or
patients – other than those undergoing neurosurgical procedures
– participating in trials of drug efficacy. Nonetheless, there are
several possibilities for progress.
First, as pointed out above, in elegant and rigorous studies of
the novel Gly-RI, bitopertine, its influence upon extracellular
levels of glycine in the FCX of rodents was compared with its
influence upon their levels in the CSF of humans and compara-
bility established as a foundation for selecting doses for clinical
trials (Bugarski-Kirola et al., 2014; Hofmann et al., 2016;
Umbricht et al., 2014). This general approach, complemented by
measures of the drug itself, can be applied to other biologically-
active molecules impacted by pharmacotherapy. This may well
prove possible in the near future as regards neurotoxic Aβ42 and
Tau which can be reliably measured in human CSF, and by
microdialysis in the interstitial fluid of rodents (see above)
(Brinkmalm et al., 2015; Olsson et al., 2016; Takeda et al., 2016).
Second, PET also has a broader role than just in the definition
of drug target engagement, notably in the extension of neuro-
chemical work in rodents to studies in humans. One example is
provided by PET ligands at D2/D3 receptors: interference with
their binding potential can be used as a surrogate measure of the
availability of endogenous DA and hence its extracellular levels.
This has proven useful in studies of schizophrenia to estimate the
(increased) occupation of sub-cortical D2 receptors (FCX D2/D3
receptor density is too low to be visualized) by endogenous DA
and hence deduce a hyperactivity/hypersensitivity of striatal
dopaminergic pathways (Kegeles et al., 2010). The approach can
also be applied to drugs that release DA such as agents for treat-
ing attention deficit hyperactivity disorder, like methylphenidate,
which acts via actions in the FCX (Robbins and Arnsten, 2009;
Millan et al., 2015; Sallee, 2010).
Third, glutamatergic and GABAergic systems can be exam-
inedby magnetic resonance spectroscopy. While most studies
have focused on a composite “Glx” (glutamate + glutamine or
glutamate + glutamine + GABA) signal, technical advances are
helping facilitate their differentiation, and other metabolites can
be determined concurrently, such as the acetylcholine catabolite,
choline (Poels et al., 2014; Stzrelecki et al., 2015). One drawback
is that results reflect levels in whole tissue, with little or no
distinction between intracellular, intra and extrasynaptic com-
partments: hence it remains unclear to what extent measurements
genuinely reflect neurotransmission and its modulation, in par-
ticular for GABA (Myers et al., 2016; Poels et al., 2014).
Finally, to conclude on a simple note, microdialysis quantifi-
cation of central levels of hormones can be reproduced without
any great difficulty in humans by measures of their levels in
either the CSF or plasma.
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Thus, it is clearly possibly to expand the translation of experi-
mental neurochemistry/microdialysis of the FCX and other cer-
ebral regions, but there is a need for additional work along these
lines.
Progress and perspectives: concluding
comments
Considerable progress has been made, and continues to be made,
in our understanding of the FCX by the application of microdi-
alysis coupled to increasingly refined and broad-based methods
for quantification of biologically-active molecules from neuro-
transmitters to hormones to metabolites to growth factors to sec-
ond messengers to neurotoxic proteins. As outlined here, the
lion’s share of work to date has been devoted, not altogether sur-
prisingly, to neurotransmitters. Nonetheless, neurotoxic proteins
incriminated in the “spreading” of neurological disorders, such as
Aβ42 and tau – as well as other proteins like “homeobox” mes-
sengers (Prochiantz and Di Nardo, 2015) – may well become a
major focus of future work. Cytokines and other pro and anti-
inflammatory mediators will also attract considerable interest in
view of evidence that neuroinflammation is a prominent feature
of both psychiatric and neurological disorders (Millan et al.,
2016; Yamanaka et al., 2015).
A rather new class of intercellular mediators derived from
neurons, astrocytes and microglia, are microRNAs: they are
highly stable in the extracellular environment – since they are
bound to argonaut proteins and/or protected in exosomes (Bartel,
2009; Danborg et al., 2014; Millan, 2011, 2013, 2014b).
MicroRNAs are of considerable interest due to their key roles in
coordinating neuronal function at the cell and circuit level, their
release by neurones and other cell types, and their promise as
potential CSF and plasma biomarkers of depression, Alzheimer’s
and other diseases (Bartel, 2009; Danborg et al., 2014; Millan,
2014b). It is recommended and predicted that miRNAs and other
classes of ncRNA should become important – and translatable –
themes of future research into FCX function using microdialysis
and other approaches. Underpinning this contention, it has now
been reported that microRNAs can be recuperated and quantified
in cerebral microdialysates in humans (Bache et al., 2005).
Incorporation of the above-mentioned and other important
molecules into microdialysis studies will be facilitated by ongo-
ing progress in the sensitivity and precision of methods of detec-
tion and quantification. In this regard, it would be instructive to
undertake studies across the entire cascade of neurotransmission
in time and in space: for example, in determining the influence of
a novel potential antidepressant not only upon extracellular lev-
els of 5-HT and glutamate, but also upon second messengers like
cGMP and cAMP, as well as, in the context of chronic adminis-
tration, BDNF. Such work could be coupled to complementary
studies of intracellular events, like signalling cascades, kinase
recruitment and gene transcription. The question of whether keta-
mine (and related compounds) can achieve sustained as well as
rapid-onset relief of depression is still under discussion (Park
et al., 2015), accentuating the importance of examining the long-
term as well as short-term effects of novel agents; work which is
still insufficiently undertaken today.
As for the practicalities of measurements themselves, while
conventional one to several hours periods of measurement are
Millan et al. 23
well-adapted to studies of pharmacological agents – and models
of CNS disorders where baseline levels are important – it would
be interesting to shorten sample times to have greater insights
into the dynamics of events in the extracellular/peri-synaptic
space. At the other end of the temporal scale, as shown above for
hormones, it would also be important to systematically examine
fluctuations across the daily cycle, (Figure 3) both as regards the
influence of potential medication (which may be administered
either in the mornings and/or evenings (De Bodinat et al., 2010))
and the characteristics of genetically-modified mice and rodent
models for CNS disorders. Two simple examples extracted from
the above discussion are: first, AMPA receptor PAMs, since
AMPA receptors provide a major optic drive to the master pace-
maker in the suprachiasmatic nucleus (Mizoro et al., 2010) and,
second, animal models for depression, bearing in mind the
problem of desynchronization encountered by many patients
(Catena-Dell’Osso et al., 2012).
Another important domain of future progress would be to
better interlink the neurochemistry of the FCX with studies of its
global network function as revealed by electroencephalography
and fMRI (Millan et al., 2012a). As pointed out above, microdi-
alysis is complementary to these strategies which provide little
direct information on the precise neurochemical substrates
underpinning circuit operation, disruption and restitution
(Murphy and Mackay, 2011).
Moving in the opposite direction in terms of scale – though
sticking to networks – a further domain of progress would be to
systematically study neuromodulators in the extracellular envi-
ronment of primary cultures of FCX and especially of cortical
neurons, networks and organoids derived from human (and
murine) induced pluripotent cells. Studies could be undertaken
both in cells from normal subjects and in patients with CNS dis-
orders impacting the FCX; under baseline conditions and upon
exposure to known and potentially novel classes of medication
(Brennand et al., 2015).
Though frontocortical organoids derived from human induced
pluripotent cells are still somewhat esoteric, the more common
and critical use of microdialysis would be in the thorough charac-
terization of animal models for CNS disorders, especially under
the influence of both symptomatic and disease-modifying drugs.
Such studies are reasonably well-established in, say, schizophre-
nia research (Millan et al., 2016), yet they are still very thin on
the ground and urgently need intensification with animal models
forAlzheimer’s disease and other neurological disorders. And not
just Aβ42 and Tau: there is a need to maintain a broad perspec-
tive. For example, it would be naïve in the extreme to imagine
that one should only be interested in dosing neurotoxic proteins
when developing a potential Tau or β-amyloid disease-modifying
therapy for Alzheimer’s disease: just as for symptomatic therapy,
it is critical to look at relevant neurotransmitters from monoam-
ines to ACh to glutamate. This assertion is underpinned by the
crucial insights provided by microdialysis studies of the influ-
ence of vortioxetine on neurotransmitters other than 5-HT
(Sanchez et al., 2015), and of l-DOPA upon neurotransmitters
other than DA (De Deurwaerdère et al., 2016).
Finally, from a pragmatic and economic point of view, where
feasible, the most effective and informative way to pursue micro-
dialysis and neurochemistry is hand in hand with complementary
strategies for model and drug characterization. Feedback is
instantaneous and dynamic when in-house, profiting from
Downloaded from jop.sagepub.com at CORNELL UNIV on October 18, 2016
reciprocal and fruitful exchanges with other experimental (and
clinical) domains. The setting up of a neurochemistry/microdialysis
lab requires expertise, commitment, patience and a significant
financial investment. Nonetheless, in the long-term, the running
of a focussed yet flexible and polyvalent microdialysis unit is not
only instructive and productive, but less expensive and more effi-
cient than the piecemeal farming out of specific studies to outside
organizations.
As amply illustrated above, enormous progress has been
made since the turn of the Millenium (see Millan et al., 2000a,
this journal) in the application of microdialysis to our under-
standing of frontocortical networks, their disruption in CNS dis-
orders, and the characterization of novel therapeutic strategies for
their treatment. However, there is still plenty of progress that can
and should be made. A microdialysis approach to studying the
neurochemistry of the FCX and other cerebral structures remains
a translationally-relevant and crucial pillar of modern neurosci-
ence and drug-discovery for both psychiatric and neurological
disorders.
Acknowledgements
We thank all our many colleagues and collaborators for their excellent
work over the years. In particular, L Cistarelli and R Billiras for their
devotion and their virtuoso performance and analysis of microdialysis
studies in rats and mice. G Bouchez, R Boulanger, B Di Cara, L De
Groote, F Panayi and G Rollin-Jego are also thanked for very fruitful
participation with microdialysis; A Dekeyne, M Brocco, F Lejeune, F
Loiseau, C Mannoury la Cour and A Newman-Tancredi are thanked for
important contributions to the characterization of drugs discussed herein,
and A Cordi, G Lavielle, G de Nanteuil and JL Peglion and colleagues are
thanked for long-term support with excellent medicinal chemistry.
Note this paper has been written in accordance with the new NbN
nomenclature guidelines that Journal of Psychopharmacology now
endorses: http://nbnomenclature.org/_static/docs/NbN_Glossary.pdf
Declaration of conflicting interests
The authors declared the following potential conflicts of interest with
respect to the research, authorship, and/or publication of this article: All
the authors are full-time employees of the Institut de Recherche Servier
and have no other interests to declare.
Funding
The authors received no financial support for the research, authorship,
and/or publication of this article.
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