steroids - J-Stage

1

Journal of Pharmacological Sciences

©2006 The Japanese Pharmacological Society

Critical Review

J Pharmacol Sci 100, 93 – 118 (2006)

The σ1 Protein as a Target for the Non-genomic Effects

of Neuro(active)steroids: Molecular, Physiological, and Behavioral

Aspects

François P. Monnet1 and Tangui Maurice2,*

1Unité 705 de l’Institut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche 7157 du Centre National de la Recherche Scientifique, Université de Paris V et VII, Hôpital Lariboisière-Fernand Widal, 2, rue Ambroise Paré, 75475 Paris cedex 10, France2Unité 710 de l’Institut National de la Santé et de la Recherche Médicale, Ecole Pratique des Hautes Etudes, Université de Montpellier II, cc 105, place Eugène Bataillon, 34095 Montpellier cedex 5, France

Received December 15, 2005

Abstract. Steroids synthesized in the periphery or de novo in the brain, so called ‘neuro-

steroids’, exert both genomic and nongenomic actions on neurotransmission systems. Through

rapid modulatory effects on neurotransmitter receptors, they influence inhibitory and excitatory

neurotransmission. In particular, progesterone derivatives like 3α-hydroxy-5α-pregnan-20-one

(allopregnanolone) are positive allosteric modulators of the γ-aminobutyric acid type A (GABAA)

receptor and therefore act as inhibitory steroids, while pregnenolone sulphate (PREGS) and

dehydroepiandrosterone sulphate (DHEAS) are negative modulators of the GABAA receptor and

positive modulators of the N-methyl-D-aspartate (NMDA) receptor, therefore acting as excitatory

neurosteroids. Some steroids also interact with atypical proteins, the sigma (σ) receptors. Recent

studies particularly demonstrated that the σ1 receptor contributes effectively to their pharmaco-

logical actions. The present article will review the data demonstrating that the σ1 receptor binds

neurosteroids in physiological conditions. The physiological relevance of this interaction will be

analyzed and the impact on physiopathological outcomes in memory and drug addiction will be

illustrated. We will particularly highlight, first, the importance of the σ1-receptor activation by

PREGS and DHEAS which may contribute to their modulatory effect on calcium homeostasis

and, second, the importance of the steroid tonus in the pharmacological development of selective

σ1 drugs.

Keywords: Neuro(active)steroid, σ1 receptor, neurotransmission, neuronal plasticity,

learning and memory

1. Neurosteroids and σ receptors ....................................................... 01.1. Neuro(active)steroids biosyntheses1.2. Neurosteroids play a role in the excitatory / inhibitory balance

in the brain1.3. The σ receptor, an atypical neuromodulatory system1.4. The σ1 receptor acts as an intracellular amplifier of signal

transduction system involved in the formation and recomposition of membrane lipid microdomains

2. Neurosteroids and σ drugs apparently share the same binding sites................................................................................................ 0

2.1. Steroids and neurosteroids bind both σ1 and σ2 sites in the central nervous system

2.2. Steroids and neurosteroids bind both σ1 and σ2 sites in peripheral tissues

2.3. Do neurosteroids also bind atypical σ-receptor subtypes?

3. Physiological aspects of σ receptor and neurosteroid functions ... 03.1. Does σ binding protein interfere with (neuro)steroid synthesis?3.2. Neurosteroids and σ drugs share modulatory functions at both

pre- and post-synaptic levels3.2.1. Do neurosteroids and σ drugs exhibit similar effects

on neuronal firing?3.2.2. Neurosteroids and σ drugs affect neuronal excitability

induced by the NMDA receptor3.2.3. Neurosteroids and σ drugs may have a similar impact

on neurotransmitter release4. Behavioral effects of σ-receptor ligands and neurosteroids .......... 0

4.1. Neurosteroids and σ drugs affect learning and memory processes4.1.1. Pro-mnesic effects of neurosteroids and σ drugs4.1.2. Anti-amnesic effects in cholinergic models of amnesia4.1.3. Anti-amnesic effects in NMDA-receptor-dependent

amnesia4.1.4. Anti-amnesic effects of neurosteroids and σ drugs

during aging4.2. Neurosteroids and σ drugs may influence abused drug intake4.3. Cross-influences between neurosteroids and σ receptor

5. Conclusions ................................................................................... 0

*Corresponding author. [email protected]

Published online in J-STAGE:

DOI: 10.1254 / jphs.CR0050032

Invited article

FP Monnet and T Maurice2

1. Neurosteroids and σ receptors

The first description of a biological activity for

steroids, the anesthetic properties of progesterone, dates

from the 1941 report by Selye (1). The subsequent

characterization of central effects exerted by steroid

hormones, and their syntheses in the nervous system

defining the concept of neurosteroids, dates from 25

years ago. Neuroactive steroids include both steroids

from the periphery, which are transported through the

blood-brain-barrier and act within the brain, and locally

synthesized neurosteroids. Their physiological actions,

demonstrated from embryogenesis through adult life,

involve genomic actions, mediated by steroid receptors

translocating into the nucleus, and non-genomic neuro-

modulatory actions affecting directly several ion chan-

nels, neurotransmitter receptors, and second messenger

systems. The syntheses and effects of these neuro

(active)steroids and their physiopathological conse-

quences have been extensively reviewed (2 – 13). In

particular, the mechanisms by which they act as allos-

teric modulators of the γ-aminobutyric acid type A

(GABAA) receptor and N-methyl-D-aspartate (NMDA)

type of glutamate receptor is now extensively docu-

mented. They also affect acetylcholine systems through

direct and indirect actions. More atypical is their re-

ported interaction with the sigma1 (σ1) receptors (14 –

17). In the present review article, we will detail the

molecular, physiological, and behavioral data support-

ing the concepts that not only certain neurosteroids

interact at physiological concentration with σ1 receptors

and may represent their endogenous ligands but also that

the neurosteroid /σ1 receptor interaction may present

major physiopathological consequences. We will parti-

cularly illustrate their involvement in memory processes

and vulnerability to drug addiction.

1.1. Neuro(active)steroids biosyntheses

Steroids are synthesised in adrenal glands and gonads

and exert their hormonal effects in peripheral organs and

the brain. The identification of pools of steroids whose

levels were higher in the brain than in plasma, indepen-

dently of peripheral sources led to the concept of ‘neuro-

steroids’ (18 – 20). These neurosteroids are synthesized

locally in the mitochondria of glial cells, such as oligo-

dendrocytes and astrocytes, and neurons. Fifteen days

after removing the sources of circulating steroids by

adrenalectomy and gonadectomy (AdX /CX) in rodents,

no difference in the brain neurosteroid levels could be

measured as compared to non-operated animals (18, 19,

21). Cholesterol is transported into the mitochondrion by

the peripheral type of benzodiazepine receptor and then

converted into pregnenolone (PREG) by a cytochrome

P450 side-chain cleavage (P450scc) enzyme, a conver-

sion that constitutes the rate-limiting step in steroido-

genesis (3). PREG could then be metabolized into

progesterone by a 3β-hydroxysteroid dehydrogenase

(3βHSD). PREG could also be converted into 17α-

hydroxypregnenolone by a cytochrome P450c17, which

leads to dehydroepiandrosterone (DHEA) and andros-

tenedione by the scission of the c17,20 bond. These

two pathways lead to the formation of pregnanes and

androstanes steroids, respectively, and the most highly

expressed steroids in the brain are PREG, DHEA, their

sulphate esters (PREGS and DHEAS), progesterone,

and 3α-hydroxy-5α-pregnan-20-one (allopregnanolone)

among the tetrahydroprogesterone isomers. The expres-

sion, distribution, and ontogeny of most of the

steroidogenic enzymes and synthesized steroids have

been determined in the brain (3) and similarities with

the peripheral synthetic pathway were identified. Most

of the enzymes, including P450scc, P450c17, 3βHSD,

and 5α-reductase, which convert PREG into progester-

one, are co-expressed in limbic structures such as the

hippocampus, caudate putamen, hypothalamic nuclei,

cortex, olfactory bulb, and cerebellum (3).

Various physiological and pathological conditions are

associated with changes in neurosteroid levels. Neuro-

steroid syntheses significantly vary during acute and

chronic stress, pregnancy, neural development, and

normal and pathological aging. In humans, plasma levels

of DHEAS decline with age (22, 23). In rodents,

significant decreases of PREGS levels in aged Sprague

Dawley rat brain were reported to correlate with

impaired memory functions (24). Aged C57BL /6 mice

or senescence-accelerated (SAM) mice also show

important decreases in the brain levels of PREG and

PREGS (PREG /S), DHEA and DHEAS (DHEA /S),

or progesterone (25, 26). Moreover, brain structural

abnormalities related to Alzheimer’s disease, like both

β-amyloid deposits and neurofibrillary tangles, which

result from the aggregation of pathologic τ proteins,

affect brain neurosteroid levels. Brown et al. (27)

reported that β1–42-amyloid protein increased DHEA

levels in a human glia-derived cell line, after a 24 h

application, a synthesis interpreted as a neuroprotective

response to the β-amyloid-induced toxicity. Neuro-

steroid levels were measured in two in vivo models of

β-amyloid toxicity, the intracerebroventricular injection

of aggregated β25–35-amyloid peptide in mice and the

chronic infusion during a 2-week period of β1–40-amyloid

protein in rats. Decreased levels of PREG /S, DHEA /S,

and progesterone were identified in the hippocampus,

cortex, and cerebellum as compared to the control

animals (28, 29). Neurosteroid levels were also mea-

sured post-mortem in individual brain regions of

Neurosteroid Action at σ1 Receptor 3

Alzheimer’s disease patients and aged non-demented

controls, including the hippocampus, amygdala, frontal

cortex, striatum, hypothalamus, and cerebellum (30),

supporting a general trend towards decreased levels of

all steroids observed in brain regions of Alzheimer’s

disease patients compared to controls. PREGS levels

were also significantly lower in the striatum, and

cerebellum, as were DHEAS levels in the hypothalamus,

striatum, and cerebellum. In contrast, progesterone and

allopregnanolone levels were reduced, but non-signifi-

cantly, brain structures, including the hypothalamus,

striatum, frontal cortex, or amygdala. Finally, a signifi-

cant negative correlation was found between the levels

of cortical β-amyloid peptides and those of PREGS in

the striatum and cerebellum and between the levels of

phosphorylated τ proteins and DHEAS in the hypo-

thalamus (30). Since high levels of key proteins

implicated in the formation of plaques and neuro-

fibrillary tangles were correlated with decreased brain

levels of PREGS and DHEAS, the authors, in agreement

with Brown et al. (27), supported the concept of a

possible neuroprotective role of these neurosteroids in

Alzheimer’s disease.

1.2. Neurosteroids play a role in the excitatory/ inhi-

bitory balance in the brain

These neurosteroids, as well as circulating steroids

crossing the blood-brain-barrier and penetrating the

brain, can influence neuronal functions. Among neuro-

steroids, allopregnanolone and 3α,5α-tetrahydrodeoxy-

corticosterone (3α,5α-THDOC) can be oxydized into 5α-

dihydroprogesterone or 5α-dihydrodeoxycorticosterone,

respectively, which have the ability to bind to the cyto-

solic progesterone receptor and subsequently activate

transcription factors, regulate gene expression, and

stimulate protein synthesis (10 – 12, 31, 32). Other

neurosteroids bind to membrane-bound receptors to

exert rapid, non-genomic effects by binding to or indi-

rectly modulating the activity of neurotransmitter recep-

tors or ion channels. Accordingly, two types of neuro-

steroids can be differentiated in the brain on the basis of

their pharmacological actions: excitatory steroids, which

include mainly PREG /S and DHEA /S, the sulphated

forms being more active than the free steroid; and

inhibitory steroids, which include mainly progesterone

and its reduced metabolites 3α/β,5α/β-tetrahydro-

progesterone, with allopregnanolone presenting the

highest brain levels.

The first report of an interaction between neuro-

steroids and the GABAA receptor is from Harrison and

Simmonds (33) and likely corresponded to the Selye’s

observation in 1941. They demonstrated that alphaxa-

lone increased GABA action when the latter was applied

to rat cuneate nucleus slices and enhanced the binding of

the GABAA-receptor agonist [3H]muscimol. Extensive

electrophysiological studies have been performed, parti-

cularly using recombinant GABAA receptors (for a

review, see ref. 34). Progesterone, its metabolites

3α,5α-tetrahydroprogesterone (allopregnanolone) and

3α,5β-tetrahydroprogesterone (alloepipregnanolone), and

3α,5α-THDOC are potent stereoselective positive

allosteric modulators of the GABAA receptor. Patch-

clamp studies showed that these steroids had no effect on

the single-channel conductance of the receptor, but

greatly promoted the open state of the GABA-gated ion

channel (35). At physiologically relevant concentra-

tions, that is, below 100 nM, these steroids directly

activated the GABAA receptor–channel complex (35,

36) and exerted a GABAmimetic effect sufficient to

suppress excitatory neurotransmission (36). Their

effects affect synaptic as well as non-synaptic GABAA

receptors and a clear heterogeneity of their interaction

with GABAA receptors has been observed, the

selectivity depending on individual brain regions and

even population of neurons (34). Therefore, progester-

one, allopregnanolone and its stereoisomers, and 3α,5α-

THDOC are potent inhibitory neurosteroids. DHEAS

has been reported to antagonize the GABAA receptor by

interacting with the barbiturate site. In particular,

DHEAS decreased the potency of pentobarbital to

potentiate the [3H]flunitrazepam binding and inhibited

GABA-induced currents in neurons (37). High micro-

molar concentrations of DHEAS inhibited [3H]musci-

mol and [3H]flunitrazepam binding to rat brain mem-

branes, primarily by reducing the binding affinities.

DHEAS also produced a concentration-dependent

blockade of GABA-induced currents in cultured neurons

from ventral mesencephalon (38). DHEAS acts as a

non-competitive modulator of the GABAA receptor.

Indeed, DHEAS blocked the GABAA receptor in a

primary culture of ventral midbrain neurons of fetal rats

by accelerating the desensitization and not by acting on

the conductance of the chloride channel (39).

PREGS and DHEAS positively modulate several

NMDA-receptor-mediated responses, and thus appear to

be excitatory neurosteroids. In the nanomolar range,

PREGS specifically enhanced the NMDA-gated cur-

rents in spinal cord neurons (40), intracellular Ca2+

fluxes mediated through NMDA-receptor channels in

cultured rat hippocampal neurons or chick hippocampal

neurons (41, 42), convulsant potency of NMDA in mice

(43), and NMDA-induced phasic firing of vasopressin

neurons in the rat supraoptic nucleus (44). DHEAS

potentiated the intracellular Ca2+ fluxes mediated

through NMDA-receptor channels in mouse neocortical

neuronal cultures (45). However, several evidences


show that PREGS and DHEAS markedly differ in their

effect on the NMDA neurotransmission. PREGS, but

not DHEAS, enhanced the NMDA-induced phasic firing

of vasopressin neurons in the rat supraoptic nucleus (44).

On the contrary, DHEA /S, but not PREG /S, potentiated

the NMDA-evoked catecholaminergic release (15) and

firing activity of CA3 hippocampal neurons (16). More-

over, the NMDA-stimulated [3H]norepinephrine release

is inhibited by PREGS (15). Each neurosteroid thus

acts differently on the NMDA receptor complex.

PREGS behaves also as a positive modulator of NMDA

receptors, whereas epipregnanolone sulphate behaves as

a negative modulator by inhibiting NMDA-induced

conductances. These two types of steroids act at specific,

extracellularly directed sites that are distinct from one

another and from the spermine, redox, glycine Mg2+,

PCP, and arachidonic acid sites (46, 47). Studies using

recombinant receptors showed that the NR2A subunit

controls the efficacy of the neurosteroids enhancement,

but not inhibition, which suggests in addition that

potentiating and inhibitory neurosteroids act at distinct

sites on the NMDA receptor (48). In other respects, there

is still no convincing evidence that DHEAS act directly

at the NMDA receptor.

Progesterone was reported to attenuate by itself the

excitatory neuronal responses to local application of

quisqualate, kainite, and NMDA of cerebellar Purkinje

cells (49). It appeared however that two classes of neuro-

steroids could be distinguished, based on their effects on

the NMDA neurotransmission, namely excitatory neuro-

steroids: PREGS and DHEAS, which potentiate the

NMDA-receptor activation through a direct or indirect

mechanism, and inhibitory neurosteroids: epipreg-

nanolone sulphate and progesterone, which inhibit the

NMDA-receptor activation.

1.3. The σ receptor, an atypical neuromodulatory system

The σ receptor represents a unique binding site in

mammalian brain and peripheral organs, distinct from

any other known transmitter receptors. Historically, the

σ receptor was identified by Martin et al. (50) as one of

the subtypes of opiate receptors, differentiating in the

chronic spinal dog, the unique psychotomimetic effects

induced by N-allylnormetazocine (SKF-10,047) (σ-

syndrome), from the effects induced by morphine (µ-

syndrome) and ketocyclazocine (κ-syndrome). However,

subsequent studies established that σ sites possess

negligible affinity for naloxone or naltrexone and that

certain behaviors elicited by SKF-10,047 were resistant

to the blockade by classical opiate receptor antagonists

such as naloxone or naltrexone (51). A complete

distinction between the non-opiate σ binding sites and

the classical µ-, δ-, and κ-opiate receptors was therefore

established (52). At the same time, σ sites were also

demonstrated to be different from the high affinity

phencyclidine (PCP) binding sites, located within the

ion channel associated with NMDA receptors (52). The

lack of selectivity between the σ and PCP binding

sites showed by several compounds, including benzo-

morphans or PCP derivatives, led to a confusion that was

cleared up by the availability of new highly selective

drugs. Among them, the reference PCP non-competitive

antagonist (+)MK-801 maleate (dizocilpine) fails to

displace radioligands labeling the σ sites and selective σ

agonists like 1,3-di-O-tolylguanidine (DTG), (+)N-

cyclopropylmethyl-N-methyl-1,4-diphenyl-1-ethyl-but-

3-en-1-ylamine hydrochloride (JO-1784, igmesine), 2-

(4-morpholino)ethyl-1-phenylcyclohexane-1-carboxylate

hydrochloride (PRE-084), and 1-(3,4-dimethoxy-

phenethyl)-4-(3-phenylpropyl)piperazine dihydrochloride

(SA4503) do not bind to the NMDA-receptor-associated

PCP site. These compounds are now reference com-

pounds in terms of selectivity between σ and PCP

receptors.

Multiple criteria are now used to identify σ receptors,

especially their ability to bind several chemically unre-

lated drugs with high affinity. These drugs include

psychotomimetic benzomorphans, PCP and derivatives,

cocaine and derivatives, amphetamine, certain neuro-

leptics, many atypical antipsychotic agents, anticonvul-

sants, cytochrome P450 inhibitors, monoamine oxidase

inhibitors, histaminergic receptor ligands, peptides from

the neuropeptide Y (NPY) or calcitonin gene-related

peptide (CGRP) families, substance P, and neuroactive

steroids. The σ binding sites could be labeled by

various specific radioligands, including [3H](+)SKF-

10,047, [3H](+)3-(3-hydroxyphenyl)-N-(1-propyl)-piperi-

dine ([3H](+)3-PPP), [3H]haloperidol, [3H]DTG, and

[3H](+)pentazocine. Pharmacological structure /activity

studies led to the definition of two subclasses of σ sites,

named σ1 and σ2 (53). The two sites were distinguished

based on their different drug selectivity patterns and

molecular weights. The σ1 site shows a stereoselectivity

with high affinity for the dextrogyre isomers of benzo-

morphans, whereas σ2 sites show the reverse stereo-

selectivity with a lower affinity range (54). DTG, (+)3-

PPP, and haloperidol are non-discriminating ligands

with high affinity on both subtypes. In addition, several

biochemical features were proposed to be selectively

observed with σ1 receptors such as an allosteric modu-

lation by phenytoin (55) and sensitivity to pertussis toxin

or G-protein modulators (56, 57). The σ1 receptor is a 29-

kDa single polypeptide that has been cloned in several

animal species and humans (58 – 60). The ligand bind-

ing profile for the cloned σ1 receptors were similar as

described in brain homogenates studies. The 223 amino


acid sequence of the purified protein is highly preserved,

with 87% – 92% identity and 90% – 93% homology

among tissues and animal species. The protein appeared

identical in peripheral tissues and brain and also shares

a similarity, 33% identity and 66% homology, with a

sterol C8 – C7 isomerase (see paragraph 3.1), but no

homology was evidenced with any other mammalian

protein, outlining the unicity of the σ1 receptor as

compared with any other known receptor. The gene,

located on chromosome 9 in human and 2 in rodents, is

7-kbp-long and contains four exons and 3 introns (61).

Exon 2 codes for the single transmembrane domain,

identified at present, but two other hydrophobic regions

exist and one of them may putatively constitute a second

transmembrane domain (62). The σ1-receptor sequence

contains a 22 amino acid retention signal for the

endoplasmic reticulum at its N-terminal region and two

short C-terminal hydrophobic amino acid sequences that

were suggested to be involved in sterol binding (58).

Amino acid substitutions in the transmembrane domain,

S99A, Y103F, and LL105,106AA, did not alter the

expression levels of the protein but suppressed ligand

binding activity (62), suggesting that these amino acids

belong to the binding site pharmacophore located within

the transmembrane domain. In addition, anionic amino

acid residues were identified, D126 and E172, that also

appeared critical for ligand binding (63). The promoter

region sequence of the σ1 receptor contains several

consensus sequences for the liver-specific transcription

factors nuclear factor (NF)-1 /L, activator protein (AP)-

1, AP-2, IL-6RE, NF-GMa, NF-GMb, NF-κB, steroid

response element, GATA-1, Zeste, and for the xeno-

biotic responsive factor called the arylhydrocarbon

receptor (61, 64).

The σ1 protein is widely distributed in peripheral

organs, including the heart, lung, kidney, liver, intes-

tines, and sexual and immune glands. In the brain, the σ1receptor is expressed in neurons, ependymocytes, oligo-

dendrocytes and Schwan cells (65 – 68). It is particularly

concentrated in specific areas throughout limbic systems

and brainstem motor structures. The highest levels of σ1immunostaining can be observed in the granular layer of

the olfactory bulb, hypothalamic nuclei, and pyramidal

layers of the hippocampus (25, 65). Among other areas

exhibiting intense to moderate σ1 immunostaining are

the superficial cortical layers, different striatal areas

including the caudate putamen and nucleus accumbens

core and shell, the midbrain, the motor nuclei of the

hindbrain, Purkinje cells in the cerebellum, and the

dorsal horn of the spinal cord. At the subcellular level,

the σ1 receptor was found to be mostly present within

neuronal perikarya and dendrites, where it is associated

with microsomal, plasmic, nuclear, or ER membranes

(25, 65).

The σ2 site is not yet cloned. It was first characterized

in pheochromocytoma PC12 cells (69). It presents low

affinity for (+)benzomorphans and has an apparent

molecular weight of 18 to 21 kDa (54). Some selective

and high affinity σ2 site ligands are now available such as

1'-(4-(1-(4-fluorophenyl))-1H-indol-3-yl)-1-butyl)spiro

(isobenzofuran-1(3H),4'piperidine (Lu 28-179) (70), N-

[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(1-pyrrolidi-

nyl)ethylamine (BD1008) (54), or ibogaine (71). Several

functions have been proposed for σ2 sites: regulation of

motor functions, induction of dystonia after in situ

administration in the red nucleus (72), regulation of ileal

function (73), blockade of tonic potassium channels

(74), potentiation of the neuronal response to NMDA in

the CA3 region of the rat dorsal hippocampus (75), or

activation of a novel p53- and caspase-independent

apoptotic pathway, distinct from mechanisms used by

some DNA-damaging, antineoplastic agents and other

apoptotic stimuli (76).

1.4. The σ1 receptor acts as an intracellular amplifier of

signal transduction system involved in the formation

and recomposition of membrane lipid microdomains

Acute activation of the σ1 receptor results in a direct

modulation of intracellular calcium ([Ca2+]i) mobiliza-

tions. Selective σ1 agonists, (+)pentazocine and also

PREGS, potentiate the bradykinin-induced increase in

[Ca2+]i, mediated by activation of inositol-1,4,5 tris-

phophate (InsP3) receptors in neuroblastoma cells (77).

A similar observation was also carried out in primary

culture of hippocampal neurons by both (+)SKF-10,047

and (+)pentazocine (78). After depletion of intracellular

Ca2+ from endoplasmic reticulum (ER) stores, the

depolarization-induced increase in [Ca2+]i in the cells

could also be modulated by σ1 agonists. Both effects

were blocked by an antisense oligodeoxynucleotide

targeting the σ1 receptor (77). Therefore, activation of

the σ1 receptor resulted in a complex, bipolar modulation

of calcium homeostasis. At the ER level, the σ1-receptor

activation facilitates the mobilization of InsP3 receptor-

gated intracellular calcium pools and at the plasma

membrane level, the σ1-receptor activation modulates

extracellular calcium influx through voltage-dependent

calcium channels. A co-immunoprecipitation study

further revealed that the σ1 receptor could regulate the

coupling of the InsP3 receptor with the cytoskeleton

via an ankyrin B anchor protein, a cytoskeletal protein

originally attached to ER membranes (79). Activation of

the σ1 receptor dissociated ankyrin B from InsP3 receptor

in NG-108 cells, and this dissociation correlated with

the efficacy of each ligand in potentiating the Ca2+ efflux

induced by bradykinin. These results, coherent with the


σ1-receptor subcellular localization (65, 80), showed

that the σ1 receptor might act as a sensor /modulator for

the neuronal intracellular Ca2+ mobilizations and con-

secutively for extracellular Ca2+ influx.

After the observation using classical immuno-

precipitation and subcellular fractionation techniques

that activation of the σ1 receptor resulted in its trans-

location from the ER, Hayashi and Su (81 – 83) used

confocal fluorescence microscopy to examine the

protein dynamics in NG108 cells and primary oligo-

dendrocytes over-expressing tagged σ1 receptors. They

observed that endogenously expressed σ1 receptors

localize on the ER reticular network and nuclear enve-

lope. They are seen particularly as highly clustered

unique globular structures associated with the ER

(80 – 82). These σ1-receptor-enriched globules contain

moderate amounts of free cholesterol and neutral lipids

(81).

Therefore, on the one hand, σ1 receptors translocate

from the ER lipid droplets to plasmalemma membranes

when stimulated by agonists. The translocation of σ1receptors, associated with the ankyrin B protein,

consequently affects Ca2+ mobilization at the ER (78,

79). On the other hand, lipid droplets are formed by

coalescence of neutral lipids within the ER membrane

bilayer and may, when reaching a critical size, bud off to

form cytosolic lipid droplets, serving as a new transport

pathway of lipids between the ER and Golgi apparatus or

plasma membrane (83 – 85). Indeed, Hayashi and Su

(81) observed that when functionally dominant negative

σ1 receptors, which can not target ER lipid droplets and

can not translocate, are transfected into NG108 cells, a

large amount of neutral lipids and cholesterol is retained

in the ER, causing the pathological aggregation of the

ER and decreases of cholesterol in the Golgi and plasma

membrane. Therefore, σ1 receptors on the ER may play a

role in the compartmentalization of lipids into the ER

lipid storage sites and in the export of lipids to

peripheries of cells (81). Lipid rafts play a role in a

variety of cellular functions including vesicle transport,

receptor clustering and internalization, and coupling of

receptor with proteins involved signal transduction (86).

Over-expression of functional σ1 receptors increased

cholesterol contents and altered glycosphingolipid

components in lipid rafts of NG108 or PC-12 cells (83,

87), suggesting that up-regulation of σ1 receptors

potentiates lipid raft formation. Since glycosylated

moieties of gangliosides have been proposed to play a

role in regulating, for instance, the localization of

growth factor receptors in lipid rafts (86), chronic

activation of σ1 receptors may present substantial

consequences in cell viability and differentiation.

2. Neurosteroids and σ drugs apparently share the

same binding sites

2.1. Steroids and neurosteroids bind both σ1 and σ2sites in the central nervous system

The initial link between steroids and σ sites was

suggested by Su et al. (14) given the discovery that in

guinea-pig brain progesterone was the most active

inhibitor of [3H](+)SKF-10,047 binding to σ receptors,

with a Ki value of 268 nM in membrane extracts. It is

noteworthy that this Ki value is close to the physiological

concentration of the steroid during pregnancy. In

addition, the variation of the Bmax for the σ1 binding site

within the rodent brain from 100 (striatum, cingular

cortex, globus pallidus) to 600 fmol /mg of tissue

(cranial nerve nuclei and cerebellar Purkinje cells)

parallels that of progesterone in the rodent brain (for a

review, see ref. 72). The Bmax in the presence or absence

of progesterone remained between 700 – 770 fmol /mg

of protein, indicating that all σ1 sites available could

bind the steroid.

2.2. Steroids and neurosteroids bind both σ1 and σ2sites in peripheral tissues

Simultaneously with their study in the central nervous

system, Su et al. (14) showed that human peripheral

blood lymphocytes and rat spleen, ovaries, testis, and

pituitary contain high densities of [3H](+)SKF-10,047-

sensitive σ1 receptors. The rationale for this outstanding

study was based on the observations that gonadal and

adrenal steroids share molecular weights, non-peptidic

nature, and influence both humoral and cell-mediated

immunity with σ-active brain material obtained by

partial purification from male guinea pigs. In their

binding study performed with crude membrane fractions

of rat spleen, Su et al. (14) showed that progesterone

was by far the most active inhibitor of [3H](+)SKF-

10,047 binding to σ receptors, with a Ki value of 376 nM

in the spleen, a value being close to the physiological

concentration of the steroid during pregnancy. The Bmax

in the presence or absence of progesterone remained

within the same order of magnitude of that in brain

(≈ 600 – 800 fmol /mg of protein), indicating that all

σ1 sites available could label the steroid. In addition,

Scatchard analysis indicated that the steroid was acting

in a competitive manner. Testosterone, desoxycortico-

sterone, and PREGS were about equipotent, Ki values

within the micromolar range, while PREG and estro-

genic hormones (estriol, estrone and estradiol) remained

inactive. The notion that the potent ligands for the

progesterone receptor 11β-hydroxy-progesterone,

promegestone, and RU-27987 failed to modify

[3H](+)SKF-10,047 binding to σ1 sites underlined the


specificity of the labeling for progesterone to the σ1binding site.

Simultaneously with the initial work of Su et al. (14),

linking steroids and σ-binding protein from brain and

spleen extracts, Wolfe et al. (88) have demonstrated

such a relationship in the immune and endocrine

systems. Indeed, they have shown the existence of high

affinity binding sites for both σ-receptor subtypes, that

is, labeled with [3H](+)pentazocine for the σ1 subtype or

[3H]DTG or [3H]haloperidol for the σ2 subtype, in

lymphocytes and thymocytes (88, 89). In addition,

hypophysectomy increased σ binding in the adrenal

gland and testis (88), indicating that in immune and

endocrine tissues, the interplay between steroids and σ

sites may also exist. Inasmuch as kinetic and pharmaco-

logical characteritics of σ receptors were similar to those

obtained with steroids in the endocrine and immune

systems, it was thus logical to propose that at least some

physiological effects of steroids in the endocrine and

immune systems might be mediated through σ1 recep-

tors. Here also, as in the brain and spleen, the rank order

for steroids was progesterone > 5α-dihydrotestosterone

> testosterone > corticosterone > estradiol ≈ cholesterol.

Further support for this notion was provided by the

observations that ovarian maturing follicules, which are

under the control of progesterone, are the richest

region for σ sites where hypophysectomy depleted σ

binding, indicating the strength of the link between the

σ1 receptor and endocrine tissues. The hypothalamic-

pituitary-adrenal (HPA) axis may possibly also be under

the control of distinct σ-receptor subtypes or distinct

intracellular regulations triggered by the same σ-receptor

subtype. Evidence for a positive control of the HPA

axis by σ drugs was provided by the observations

that (±)SKF-10,047, (±)pentazocine, (±)3-PPP, and

(±)butaclamol dose-dependently and stereoselectively

stimulate adrenocorticotropic hormone release in vivo.

In addition, the σ-receptor-mediated modulation of the

HPA axis has been demonstrated to be mimicked by

steroids, since (+)SKF-10,047 and (+)pentazocine

increase, as did progesterone (after estrogen priming),

testosterone and desoxycorticosterone, whereas (+)3-

PPP and (−)butaclamol decrease prolactin release (90,

91). This was however not the case in vitro for corti-

cotrophin-releasing-factor-evoked adrenocorticotropic

hormone release from primary culture cells of the

anterior pituitary (90, 92).

Splenic lymphocytes and B-enriched lymphocytes

possess higher amounts of σ receptors than T-enriched

lymphocytes, mesenteric node lymphocytes, or thymo-

cytes (89). Both (+)pentazocine and DTG have been

reported to suppress mitogen (ConA, PWM)-stimulated

T- and B-lymphocyte proliferation and LPS-stimulated

B-lymphocyte proliferation (88, 89). More recently,

Casellas et al. (93) have shown that SR-31747 inhibits

mitogen (ConA, PWM)-stimulated human T-lympho-

cyte proliferation with a similar efficacy to cyclosporin

A; interfere with the production of proinflammatory

cytokines IL-1, IL-6, and TNF-α; and also inhibit

experimental acute graft-versus-host disease by sup-

pressing the production of IFN-γ by Th1 CD4+ T-cells

(94, 95). Conversely to most σ drugs, haloperidol

affected only the ConA-mediated response, suggesting

that σ receptors might differentially regulate T- and B-

cell-mediated proliferation. The non-selectivity of both

haloperidol and DTG for differentiating σ1 and σ2 sites

and the discrepant modulation of lymphocyte prolifera-

tion induced by haloperidol and DTG do not permit any

conclusion to be drawn concerning the subtype of σ

receptor involved in both effects. Further assessing the

role of σ ligands in the immune system, Ganapathy et al.

(95) have shown that [3H]haloperidol-sensitive σ1receptor is expressed in the Jurkat cell, a human CD4+

T-cell line able to release IL-2 and IFN-γ in response to

stimuli. Although (+)dextromethorphan and (+)SKF-

10,047 exhibited Ki values in the low micromolar (0.2 –

0.5) range, Northern blot analyses and RT-PCR using

poly(A)+ RNA with σ1-receptor-specific primers, have

supported the involvement of the receptor. These

authors also showed that progesterone inhibited the

Jurkat cell [3H]haloperidol-sensitive σ1-binding in a

competitive manner (with a Ki value of 93 nM and a Bmax

of 5.7 pmol /106 cells). The cDNA-induced pro-

gesterone binding was however unaffected by R-5020, a

steroid competitor of progesterone against the classical

nuclear progesterone receptor. In addition, the Kd values

calculated directly from binding of progesterone were

88 nM against [3H]haloperidol-sensitive σ1 sites and

69 nM against [3H](+)pentazocine-sensitive σ1 sites.

This would signify that whatever the phase of the

menstrual cycle, the T-cell-mediated impact of pro-

gesterone would likely be dependent at least on the σ1receptor.

Among peripheral tissues, the liver is the richest tissue

in σ binding sites. The concentration of binding protein

being appreciatively 20 – 100 times higher than that

found in the brain, with Kd values within the low

nanomolar range and Bmax values ≈ 10 pmol /mg of

protein (69, 96 – 98). The similar order of binding

potency for σ ligands in inhibiting [3H](+)SKF-10,047

binding suggests that central and hepatic σ sites might be

identical (72, 99, 100). However, in liver microsomes,

the [3H]haloperidol-sensitive σ site might also resemble

a variant of the σ1-receptor subtype that, conversely

to the classical σ1 receptor, exhibits high affinity for

4-(4-chlorophenyl)-α-4-fluorophenyl)-4-hydroxy-1-pipe-


ridinebutanol (reduced haloperidol) and arylethylene

diamine-related compounds, such as (+)cis-N-methyl-

N-[2-(3,4-dichlorophenyl)ethyl]-2-(1-pyrrolidinyl)cyclo-

hexylamine (BD737), but low affinity for DTG and the

benzomorphans (+)SKF-10,047 and (+)pentazocine, that

is, Kd values between 100 and 1000 nM (98).

Linking further steroids and σ sites, McCann and Su

(97) assessed whether [3H]progesterone binds to the σ

receptor from rat liver membranes. Unexpectedly, no

receptor binding could be detected using acutely isolated

cells. However, when a solubilized receptor preparation

from liver was used, in lieu of a membrane preparation,

[3H]progesterone was found to compete with the σ drugs.

Ross (101) confirmed this observation by showing that

the binding of [3H]SKF-10,047, [3H](+)3-PPP, and

[3H]haloperidol was inhibited by progesterone with

apparent IC50 values of 442, 640, and 146 nM, respec-

tively, in rat brain membranes, and 31, >10000, and

146 nM, respectively in rat liver membranes. Additional

evidence further supports the link between steroids and

σ sites in liver since [3H]progesterone binding, which

is saturable with a Kd value of 31 nM and Bmax <6

pmol /mg protein (98), exhibits a similar rank order of

affinity for several high affinity and selective σ drugs to

[3H]haloperidol binding. Interestingly, a similar profile

for an atypical σ-related receptor has also been shown in

the brain, where progesterone was acting as a powerful

antagonist (102). In addition, in the liver, 5β-pregnane-

3,20-dione, 5α-dihydrotestosterone, testosterone, and

5α-pregnane-3,20-dione exhibited similar affinity to

[3H]haloperidol (i.e., IC50 values between 270 and

600 nM) and [3H]progesterone binding, contrarily to

progesterone that shows different affinities for both

sites (i.e., IC50 values between 25 and 150 nM), whereas

21-hydroxyprogesterone, 3α-hydroxy-5α-pregnan-20-

one, and 17β-estradiol had micromolar affinities. In

this latter study, typical σ drugs showed a tendency to

attain the uppermost limit at the level of 60% – 70%

inhibition of the [3H]progesterone binding, whereas

progesterone and other steroids increased inhibition in a

concentration-dependent manner with 85% inhibition at

their maximal concentration used (10 µM). A further

step supporting the functional link between steroids and

σ receptors concerns the membrane-bound progesterone

binding site purified from liver. Most interestingly,

Falkenstein et al. (103) described that part of the

immunostaining for the [3H]progesterone-sensitive

binding protein was localized to the endoplasmic

reticulum and Golgi apparatus, which is quite unusual

for the classical steroid binding proteins.

Human placental syncytiotrophoblast and choriocar-

cinoma have also been considered with respect to the

link between steroids and σ receptors. [3H]Haloperidol

was found to bind to purified membranes from the

placental brush border with an apparent Kd of 3.5 nM

and Bmax of 1.16 pmol /mg protein (104). Similar results

were obtained with choriocarcinoma cells, with Kd value

within the nanomolar range and Bmax ≈ 2.5 pmol /mg

protein, indicating that σ proteins might play important

roles in this endocrine tissue whose main hormonal

function is to secrete the progesterone mandatory for the

maintenance of pregnancy. The σ nature of the binding

was ascertained by the effectiveness of DTG, (+)3-PPP,

and dextromethorphan, but the dopaminergic antagonist

spiperone or the serotonergic antagonist ketanserin did

not affect [3H]haloperidol binding. Homogenates from

placental syncytiotrophoblasts as well as from chorio-

carcinoma cells exhibited similar binding values to those

obtained from fresh border cells and choriocarcinoma

extracts, indicating that the cell distribution of σ sites,

that is, within microsomes, mitochondria, nucleus, and

plasma membrane, are similar in placental cells and

brain. It also suggested that the σ protein present in

placenta is most likely of the σ1 subtype. However, it

seems that this σ1-receptor subtype might differ slightly

from the brain σ1 site since phenytoin and cocaine bound

with less affinity in placenta than in the nervous system.

Following the characterization of the placental σ protein,

Ramamoorthy et al. (104) assessed whether steroids

might affect the σ binding in placental tissue. In their

study, progesterone and testosterone at a concentration

of 1 µM were indeed capable of reducing [3H]halo-

peridol binding by 40% and 24%, respectively, with

IC50 values of 1.2 and 3.8 µM, respectively, whereas in

placental syncytiotrophoblasts, the Ki values were 310

and 970 nM, respectively. 17β-Estradiol, estrone, deoxy-

corticosterone, 5-α/β-pregnan-3α/β-ol–20-one deriva-

tives, or DHEA were at least two orders of magnitude

less active than the two previous steroid hormones. The

same order of potency was found in choriocarcinoma

cells against [3H]haloperidol, with either progesterone,

testosterone, or 17β-estradiol, which exhibited Kd values

of 70 nM, 120 nM, and 15 µM, respectively. Finally,

Scatchard analysis of the binding indicated that a single

binding site was present in both human placental

syncytiotrophoblast and choriocarcinoma and that

progesterone was a competitive inhibitor of [3H]halo-

peridol binding in both tissue membranes.

Primary breast carcinomas have also been used to

assess the putative interrelationship between the σ1 re-

ceptor and human sterol isomerase. Simony-Lafontaine

et al. (105) have indeed found a close positive correla-

tion between σ1 protein expression and progesterone

receptor status, but found an inverse one between the σ1receptor and human sterol isomerase in 95 patients

using immunochemical analysis.


2.3. Do neurosteroids bind atypical σ-receptor sub-

types?

Using chromatographic procedures designed to purify

the σ proteins on a DAPE-containing column, Tsao and

Su (106) proposed that [3H](+)SKF-10,047 might also

bind to another protein bearing certain similarities to

the σ receptor sensitive to (±)SKF-10,047 but distinct

from the classical opiate-insensitive receptor since the

labeling appeared to be sensitive to naloxone, the proto-

typic opiate antagonist. Furthermore, its Kd value for

[3H](+)SKF-10,047 was 165 nM with Bmax values of

8,064 pmol /mg. It was however underlined that pro-

gesterone exhibited a 537 nM affinity for the CHAPS-

solubilized liver preparation, versus [3H](+)SKF-10,047,

while the steroid exhibited a much weaker competitive

activity against the affinity-purified protein labeled

with [3H](+)SKF-10,047, as shown by the drop in Kd

value to 18 µM (106). Although the significance of

this atypical [3H](+)SKF-10,047-binding protein has

remained elusive, these data further link steroids and σ

drugs in the liver.

Meyer et al. (107) have clearly established, with pig

liver crude membrane preparations and solubilized

fractions, that haloperidol, (+)3-PPP, DTG, and rimca-

zole compete with progesterone for [3H]progesterone

binding, with a Ki value of 20, 290, 310, and 510 nM,

respectively, while (±)pentazocine, (+)SKF-10,047,

and phenytoin exhibited low micromolar affinity. In

this study, the most active steroids were progesterone

> corticosterone ≈ testosterone > cortisol >>17β-estradiol.

The only cytochrome P450 inhibitor active against the

[3H]progesterone binding was SKF-525A, with a Ki

value of 140 nM, while methyrapone and cimetidine

remained inactive even at concentrations >35 µM. The

unusual rank order of potency for these drugs does not fit

completely with the typical order proposed for either the

σ1- or σ2-receptor subtype (53). This atypical binding

protein might thus correspond to that found by Tsao

and Su (106), who reported a [3H](+)SKF-10,047-sensi-

tive σ protein with a molecular mass of 31 kDa, that is,

similar to the cloned 28-kDa σ1 protein (58), which was

preferentially sensitive to dextrorotary benzomorphans

and naloxone but not to DTG or (+)3-PPP and exhibited

only high micromolar affinity for progesterone. It is thus

tempting to speculate that in the liver but likely also in

other tissues, atypical σ proteins might exist with

specific binding activity and specific physiological

properties.

Apart from the cell surface central benzodiazepine-

associated chloride channel and the nuclear gluco-

corticoid receptor, DHEA binds to both the peripheral

benzodiazepine receptor and the σ1 protein. The benzo-

diazepine binding protein is the one that cooperates with

sterol transporters to translocate cholesterol from the

outer to the inner mitochondrial membrane, with a Ki

value of 4 nM and a Bmax of 20 – 50 pmol /106 cells (108,

109). The [3H](+)SKF-10,047-sensitive σ1 site, for

which DHEA and progesterone exhibit a Ki values of

0.4 – 4 µM and a Bmax of 10 – 15 pmol /106 cells (98), is

that associated with limiting plasma membranes, mito-

chondria, synaptic vesicles, and endoplasmic reticulum

membranes (65).

In addition, DHEA and its sulphate derivative as well

as pregnenolone sulphate have been shown to compete

in a concentration-dependent manner with (+)pentazo-

cine for stimulating the [35S]GTPγS binding from

synaptic membranes from mouse frontal cortex. The

notion that this action was mediated by the σ1 receptor

was provided by the following: i) the inhibition of

neurosteroid-induced effects by the prototypic σ1antagonist NE-100 as well as by progesterone, and

ii) the fact that inactivation of Gi /o proteins by ADP-

ribosylation with pertussis toxin has prevented this

action.

We previously mentioned that Ganapathy et al. (95)

demonstrated the presence of [3H]haloperidol-sensitive

σ receptor in the Jurkat cell, which exhibits Ki values in

the low micromolar range (0.2 – 0.5 µM). Nevertheless,

Northern blot analysis and RT-PCR using poly(A)+

RNA with σ1-receptor-specific primers, have supported

the involvement of a splice variant of the cloned σ1receptor characterized by the deletion of 31 amino acids.

3. Physiological aspects of σ receptor and neuro-

steroid functions

3.1. Does σ binding protein interfere with (neuro)steroid

synthesis?

Thereafter, Klein and Musacchio (110) also focused

on the putative link between steroids and [3H](+)3-PPP-

and [3H]dextromethorphan-sensitive σ sites, using for

this purpose guinea-pig brain membranes. Progesterone,

deoxycorticosterone, testosterone, 17α-hydroxy-pro-

gesterone, 5α-androstane-3,17-dione, 4-androstene-3,17-

dione, and dehydroisoandrosterone were the most active

compounds with an IC50 of 0.65, 3.5, 4, 5, 7, 9, and

15 µM against [3H]dextromethorphan and 0.26, 0.68,

0.45, 5.6, 0.7, 8.5, and 3.7 µM against [3H](+)3-PPP,

respectively. Conversely to Su and co-workers, Klein

and Musacchio suggested that the steroids were labeling

a σ2-receptor subtype or that they bind to a σ1-receptor-

like site with no affinity for dextromethorphan. This

notion also emerges from the displacement experiments

of [3H](+)3-PPP by steroids. Interestingly, pregnenolone

and its 17-hydroxy-derivative, which were reported to be

totally inactive in the study of Su et al. (14), displaced


[3H]dextromethorphan with IC50 values over 50 µM.

However, they questioned whether σ1 binding proteins

might interfere with steroidogenesis. Having tested

both metabolites and precursors of their most active

steroids (progesterone, testosterone, and corticosterone),

Klein and Musacchio (110) found that the former ones

lacked affinity for the σ-related binding sites, thus

concluding that σ-binding proteins were unlikely to be

functionally associated with the steroidogenic enzyme

responsible for C17 hydroxylation. This was nevertheless

reconsidered by Moebius et al. (111), who cloned the

σ1 protein from guinea-pig liver microsomes. They have

indeed suggested that the σ1 protein corresponded to a

variant or mutant of a sterol isomerase having a role in

postsqualene sterol biosynthesis. Focusing particularly

on the yeast ERG2 gene, which encodes the C8-C7

isomerase of ergosterol biosynthesis pathway that

catalyzes a shift of the C8(9) double bond in the B-ring

of sterols to the C7(8) position, whose equivalent in

mammals is the emopamil binding protein that catalyzes

the reductases involved in the biosynthesis of cholesterol,

Moebius et al. (111) claimed that the σ1 protein was a

sterol enzyme. This assertion was based on i) a similar

binding profile of the σ drugs haloperidol, ifenprodil,

and opipramol for the cloned σ1 protein, the fungal

ERG2 protein and the emopamil binding protein; ii) a

30% homology of the gene of the σ1 protein with those

of the fungal ERG2 gene product and the emopamil

binding protein; iii) from identical transmembrane

topologies of the mammalian σ1 protein, the emopamil

binding protein, the fungal ERG2 protein, as well as

their similar aminoterminal membrane anchor in the

membrane of the endoplasmic reticulum. This anchor

was indeed composed of two additional stretches of

hydrophobic residues involved in the substrate binding

by proteins. Moreover, Moebius et al. (112) have shown

that both the [3H]haloperidol-sensitive σ1 receptor and

the ERG2 binding site were affected by (+)benzomor-

phans, although (+)pentazocine elicited highly different

potencies for both bindings, with Ki values being 1.7 nM

for the σ1 site and 1 µM for the ERG2 protein, respec-

tively. It is noteworthy that in mammalian cells,

conversely to yeast, sterol signaling is not restricted to

the nucleus but also involves the endoplasmic reticulum

membrane. This provides further interest to the hypo-

thesis of Moebius and co-workers. A functional comple-

ment to this hypothesis was recently provided by

Simony-Lafontaine et al. (105). Indeed, these authors

have failed to show any correlation between the expres-

sion and the human sterol isomerase from human

primary breast carcinomas. They did report however a

positive correlation between the σ1 status and both

estrogen and progesterone receptor densities which both

correlated negatively with the human sterol isomerase

protein. Although the conclusion that the σ1 receptor

represents a sub-form of sterol isomerase still remains in

question, it is noteworthy that Hayashi and Su (81 – 83)

have very recently proposed that the σ1 protein might

take up cholesterol from the intracellular medium to

carry it to the plasma membrane and even outside the

cell. Steroids have been known for years to activate the

HPA axis.

Although very abundant in liver, the functional

importance of the σ receptor in this tissue still remains

unexplored. This aspect reinforces the “σ Enigma”,

especially in the liver, and the exact nature and role

of the receptor have been questioned. A decade ago,

taking into consideration that the sub-cellular distri-

bution of the σ binding protein was predominant in the

microsomal fraction, representing 48% of [3H](+)SKF-

10,047 or [3H]dextromethorphan binding; most xeno-

biotics metabolized in the liver bind to σ sites; and

several cytochrome P450 inhibitors compete with σ

binding, Musacchio and co-workers (110, 113) have

thus proposed that σ binding sites might represent

microsomal enzymes related to the cytochrome P450

hemoproteins. They showed that SKF-525A, the

classical inhibitor of liver cytochrome P450 oxygenases;

l-lobeline, GBR-12909, and sparteine, which exhibit

cytochrome P450 inhibitor activities; or cytochrome

P450 substrates, such as the β-adrenergic antagonist

bufaralol, perhexiline, miconazole, or ajmalicine,

displaced [3H]dextromethorphan, [3H](+)3-PPP, and

[3H]DTG from the σ-binding proteins with very similar

affinities, ranging from 2.4 to 3.7 µM for SKF-525A.

This observation, reproduced by Ross (101) with

[3H](+)SKF-10,047 and [3H]haloperidol, supported the

notion of close interrelationships between cytochrome

P450 and σ-binding proteins. The binding was equally

distributed between σ1- and σ2-receptor subtypes. Since

[3H](+)3-PPP and [3H]dextromethorphan bindings were

not affected by either phenobarbital or phenytoin, known

to bind to σ1 sites (72) and to recruit cytochrome P-450B

isoforms, Klein and Musacchio (110) preferred to pro-

pose a preferential association of σ proteins with non-B

isoforms of the enzymes. Interestingly, haloperidol, the

prototypic σ1 antagonist (114, 115), was acting in human

liver microsomes as a competitive inhibitor of 1'-

hydroxybufaralol formation with a Ki of 1.2 µM,

suggesting that the drug was binding to the active site of

the enzyme. However, this could not definitively allow

the conclusion that σ proteins exhibit enzymatic activity.

In this regard, Klein and Musacchio (110) also focused

on the purported link between steroids and cytochrome

P450 /σ sites. Their attention was stimulated following

reports that most steroidogenic enzymes belong to the


cytochrome P450 superfamily of oxidases and favored

the notion that steroids were active on σ sites rather than

on cytochrome P450-related binding proteins. Simulta-

neously, but conversely to Klein and Musacchio (110),

Yamada et al. (98), working with liver microsomes,

excluded the possibility that cytochrome P450 iso-

enzymes or other steroid /drug-metabolizing enzymes

participate directly in the progesterone /σ binding since

they failed to show any correlation between the

inhibitory action of the σ drugs as well as that of SKF-

525A and GBR-12909 on the binding and oxidative

metabolism of liver microsomes. Further support for

doubting that σ sites may be solely linked with

cytochrome P450 isoenzymes are their distinct cellular

distribution since the former but not the latter are close

to the plasma and nuclear membranes, as suggested by

their sensitivity to 5'-nucleotidase, a plasma membrane

marker (116), their coupling to G proteins (114), and as

shown by both confocal microscopy (80) and electronic

microscopy (65). Altogether, this has ruined the asser-

tion that σ-binding proteins represent cytochrome P450

isoforms or co-factors. Additional interest for the inter-

relationships between liver and σ-binding proteins has

been suggested by the observation that microsomal anti-

estrogen-binding proteins bound arylethylene-diamine-

related compounds (117). In fact, microsomal anti-

estrogen-binding proteins share a high affinity for BD

compounds with the emopamil-binding protein. How-

ever, the microsomal antiestrogen-binding protein,

unlike the emopamil protein, lacks affinity for 17β-

estradiol and other estrogens.

Liver and brain mitochondria are known to contain

high levels of σ-binding sites as well as steroidogenic

enzymes (118). Although it has long been known that

cytochrome P450scc, which converts cholesterol into

pregnenolone, is located on the outer membrane of the

mitochondria, it is only recently that Klouz et al. (118)

have demonstrated that a [3H](+)pentazocine-sensitive

σ1 protein is present on the outer membranes of purified

rat liver and brain mitochondria. The analogous distri-

bution of both the steroidogenic precursor enzyme and a

σ-binding protein on the outer mitochondrial membrane

would be compatible with their interaction, although

definitive data are still lacking. The observation that

progesterone modulates the mitochondrial binding of

[3H](+)pentazocine further strengthens the intimate links

between steroids and the σ receptor. The physiological

significance of such binding is emphasized by the

importance of mitochondria in the regulation of the

intracellular calcium homeostasis, the ATP production,

as well as the initial phases of the steroid metabolism

from cholesterol.

3.2. Neurosteroids and σ drugs share modulatory func-

tions at both pre- and post-synaptic levels

3.2.1. Do neurosteroids and σ drugs exhibit similar

effects on neuronal firing?

The initial statement that σ ligands potentiate the

NMDA neuronal response originates with Monnet et al.

(119, 120) who showed that only drugs active on σ

receptors exhibited this ability to affect the NMDA-

induced neuronal activation. Therefore, the paradigm of

in vivo microiontophoretic application of NMDA and σ1drugs has been extensively used in the rat hippocampus

to recognize the agonistic or antagonistic profile of

action of the ligands. Accordingly, σ drugs that aug-

mented the excitatory action of NMDA were denoted as

agonist, whereas those devoid of effect on micro-

iontophoretic application of NMDA but capable of

preventing the action of σ agonists, for example, the

antipsychotic haloperidol, were denoted as σ antagonists

(120). Subsequently, whole-cell patch clamp recordings

confirmed these modulatory actions of σ drugs showing

that NMDA-induced currents were modified in a similar

manner as neuronal firing while non-NMDA responses

were only slightly affected by huge doses of the σ drugs

(121).

Bergeron et al. (16) extended this observation using

the in vivo microiontophoretic approach by showing

that DHEA potentiated the NMDA response in a pro-

gesterone-, testosterone-, and haloperidol-sensitive

manner. However, in this paradigm, neither PREG nor

PREGS were acting as σ agonists or antagonists (16,

122). To further complement their study, Bergeron et al.

(16) have also investigated the electrophysiological

behavior of σ drugs in spayed rats (i.e., progesterone-

free rats). Following a 2 – 3-week ovariectomy, the

prototypic σ agonist DTG further potentiated the NMDA

response in the hippocampal CA3 pyramidal layer. In

addition, in pregnant rats and 3-week progesterone-

treated rats, the σ1 agonists were ten times less effective

on the NMDA response than in control conditions.

Furthermore, at day five post-partum, the neuronal

response to NMDA following σ1 agonist administration

was not only restored but again enhanced (123), indicat-

ing that σ1 receptor was most likely tonically inhibited

by endogenous progesterone (16, 123). Altogether, these

in vivo data supported the initial statement that pro-

gesterone was acting as a σ1 antagonist (15). Farb et al.

(124), using whole cell recordings from voltage-

clamped spinal cord neurons, have observed no enhanc-

ing effect of DHEAS (at concentrations up to 10 µM) on

the basal transmembrane potential, and the spontaneous

firing activity and no modulatory effect of DHEAS on

the neuronal response to NMDA, that is, a direct

modulatory action. However, Meyer et al. (125) showed


that DHEAS, in the concentration range of 10 – 100 µM,

weakly facilitated the activation of CA1 neurons in

hippocampal slices after stimulation of the Schaffer

collaterals. This enhancement was related however to a

concomitant antagonistic activity of the neurosteroid on

GABA-mediated inhibitory postsynaptic potentials as

well as an indirect augmentation of the glutamatergic

excitatory postsynaptic potentials.

PREG and PREGS have no effect on spontaneous fir-

ing (124), but allosterically potentiate at micromolar

concentrations NMDA-evoked currents in rat hippo-

campal neurons in culture (see above; and refs. 40 – 42,

124, 125). More recently, Partridge and Valenzuela

(126) have shown that PREGS, acting on both NMDA

and AMPA ionotropic receptors, enhances paired-pulse

facilitation of EPSPs with an EC50<1 µM, giving support

to the notion that the neurosteroid acts presynaptically

to modulate neuronal excitability.

Since the initial studies assessing the intracellular

impact of σ drugs on the membrane potential, potassium

conductance was considered as the prominent target.

Indeed, from rat cortical synaptosomes, C6 glioma cells

(74) or NCB-20 cells (127), DTG, (+)3-PPP, and

haloperidol have been shown to facilitate hyperpolari-

zation with a reversal potential corresponding to that of

K+ or to block tonic outward K+ currents. In rat neuro-

hypophysis, Wilke et al. (128) have confirmed this

notion by providing evidence that (+)pentazocine and

(+)SKF-10,047 as well as DTG and haloperidol, but

not DHEAS or progesterone, elicited a marked inhibi-

tion of K+ currents. This latter study then suggested that

σ drugs and steroids may act distinctly at the molecular

level. Pursuing the elucidation of the molecular target

of σ drugs, Soriani et al. (129, 130) have characterized

that σ1 ligands affected at least K+ conductance of IA,

Ca2+-activated K+ current, and IM types using perforated

patches of frog melanotropic cells. Using reconstituting

responses in transfected Xenopus oocytes, Aydar et al.

(131) documented that σ1 receptor, activated by

(+)benzomorphans and inactivated by antisense con-

structs, modulated voltage-gated K+ channels (Kv1.4

and Kv1.5) depending on the presence or absence of σ

ligands. According to their results, σ1 protein likely

forms a stable complex with the K+ channels, acting as a

co-allosteric modulatory protein with variable function-

alities, depending on the presence or absence of labeling.

Interestingly, Wang et al. (132), using Chinese hamster

ovary cells, found that PREGS as well as 3α-hydroxy-

5α-pregnan-20-one and 3α-hydroxy-5β-pregnan-20-one

increased current amplitude and decreased time con-

stants for the voltage-gated K+ channels Kv1.1 and

Kv2.1.

Several groups have investigated the neuromodula-

tory effect of neurosteroids using the intracellular and

patch-clamp techniques. Bowlby et al. (133) were the

first to demonstrate in outside-out and cell-attached

configurations the facilitatory role of PREGS on the

NMDA-mediated current from primary culture of rat

hippocampal neurons. This action was concentration-

dependent and rapidly reversible, supporting a co-

allosteric modulation (47, 133). The PREGS-induced

increase corresponded to a facilitation of open pro-

bability attributed to an increase in both the frequency of

opening and mean open time of the NMDA receptor.

PREGS was the most active neurosteroid and its effect

appeared selective to the NMDA receptor (133), as soon

as the NMDA- subunit NR2A was constitutively or

transiently expressed in the cell (48, 134).

3.2.2. Neurosteroids and σ drugs affect neuronal excit-

ability induced by the NMDA receptor

Although the capacity of σ receptor ligands to modu-

late NMDA-mediated glutamatergic neuronal firing in

the mammalian central nervous system has been docu-

mented for a decade, the exact nature of this interaction

remains still elusive, biochemical paradigms supporting

their close interrelationships. In particular, the selective

σ ligands igmesine and DTG potentiated and inhibited,

respectively, the NMDA response in a concentration-

dependent manner, using the in vivo approach combin-

ing microiontophoresis and extracellular recordings of

hippocampal pyramidal neurons (120). Haloperidol,

which also displays high affinity for σ1-binding sites, but

not spiperone, another butyrophenone devoid of such

affinity, prevented the effects of igmesine and DTG. The

ability of progesterone to bind both [3H](+)SKF-10,047-

and [3H]haloperidol-sensitive σ1 sites under equilibrium

binding conditions on rat brain (see above) prompted us

to assess whether neurosteroids might mimic σ drugs,

that is, modulate NMDA-evoked [3H]noradrenaline

([3H]NE) overflow via action on σ1 receptors. Accord-

ingly, DHEAS, at nanomolar concentrations and in a

concentration-dependent manner, potentiated the release

of [3H]NE induced by NMDA (15). The lowest effective

concentration of DHEAS (30 nM) enhanced the

response by NMDA by 37%. Conversely, PREGS

inhibited the NMDA-induced release also in a concen-

tration-dependent manner. The lowest effective concen-

tration of PREGS (100 nM) induced a 60% inhibition

of the NMDA response. Haloperidol and 1-[2-(3,4-

dichloro-phenyl)ethyl]-4-methylpiperazine (BD1063)

(100 nM), but not spiperone, completely prevented both

the potentiating effect of DHEAS and the inhibitory

effect of PREGS. Conversely to sulphated steroids,

progesterone, DHEA, PREG, and allopregnanolone

did not affect NMDA-evoked [3H]NE release in the


concentration range of 10 nM to 1 µM. In addition,

progesterone concentration-dependently inhibited (in

the 10 nM to 1 µM range) both the potentiation and

inhibition of NMDA-evoked release induced by DHEAS

and PREGS but also by DTG, respectively. At 100 nM,

progesterone decreased the enhancing effect of DHEAS

by 69% and abolished the reducing effect of PREGS.

The pre-treatment with pertussis toxin, injected in the

dorsal hippocampus 3 to 11 days prior to sacrifice,

totally abolished the effects of both DHEAS and PREGS

on NMDA-evoked release of [3H]NE, indicating that the

neurosteroids were most likely acting via the σ1-receptor

subtype. From that initial observation, progesterone

has been thereafter proposed as a potential endogenous

σ1-receptor antagonist. Consistently, the σ1-receptor-

mediated antagonist-like activity of progesterone has

since been supported by in vivo experiments showing

that stereotaxically administered, progesterone, inactive

on induced neuronal activation in the CA3 dorsal

hippocampus, counteracted the DTG-induced facilita-

tion of the response of pyramidal neurons to NMDA

(16). However, in the release experiments, the addition

of haloperidol (100 nM) partially reversed the inhibitory

effect of PREGS (in the concentration range of 0.1 to

1 µM) on NMDA-evoked [3H]NE overflow and induced

a robust potentiation of the NMDA response following

Gi /o protein inactivation. In such conditions, the

occurrence of an inhibitory effect of PREGS most likely

allows the conclusion that there is an indirect σ1-

receptor-mediated modulation of the NMDA response

since haloperidol and BD1063 blocked the PREGS-

mediated response. Thus, PREGS would exert two

opposite effects on NMDA-induced neuronal activation:

the direct potentiation of NMDA-receptor interaction

and an indirect σ1-receptor-mediated inhibition of the

NMDA response, which seems to predominate under

physiological conditions. In the light of the data of

Farb et al. (124) on isolated neurons, the data obtained in

the NMDA-evoked [3H]NE overflow paradigm with

hippocampal slices point to an indirect effect of DHEAS

on the NMDA response, that is, via the σ1-receptor

subtype. Although, the affinity of DHEAS for σ1-binding

sites is within the micromolar range, the effect of

BD1063 and haloperidol, which interact with the σ1-

binding sites but not with the NMDA receptor indicate

that DHEAS most likely acted on σ1 receptors at such

nanomolar concentrations depending when the drug

application lasted 20 min.

Monnet (135) has recently shown that the co-super-

fusion of either DHEAS or PREGS, with a D2 dopamine

receptor antagonist, spiperone or sulpiride, produced a

facilitation of the KCl-evoked [3H]NE release from rat

hippocampal slices, which can be suppressed by both L-

and N-type voltage-sensitive calcium channel (VSCC)

blockers, supporting a contribution of both neurosteroids

into the Ca2+ modulation during the neurotransmitter

release. According to Hayashi et al. (77), the involve-

ment of the σ1 receptor could however be excluded from

this action of neurosteroids since PREGS inhibits via the

σ1-receptor-mediated KCl-induced increase in Ca2+,

supporting the previous observation of Monnet et al.

(15), who have demonstrated that NMDA-evoked

release of [3H]NE was reduced in the presence of nano-

molar concentrations of PREGS.

3.2.3. Neurosteroids and σ drugs may have a similar

impact on neurotransmitter release

Murray and Gillies (136) have shown that DHEAS,

but not PREGS, concentration-dependently (1 pM –

10 nM) stimulated endogenous dopamine release from

in vitro perfused rat hypothalamic cell cultures. Further-

more, intracerebral microdialysis of PREGS (100 – 400

pmol) enhanced in vivo meso-accumbens dopamine

release from dopaminergic terminals, and further

enhanced morphine-induced dopamine release (137) as

well as acetylcholine release from rat hippocampus

(138). Recently, Meyer et al. (139) reported that PREGS

(10 – 50 µM), DHEAS (0.1 – 1 µM) as well as (+)penta-

zocine (5 – 50 µM) caused a robust and transient

enhancement of the frequency and amplitude of AMPA-

mediated miniature excitatory postsynaptic currents in

primary mixed hippocampal cell cultures. This effect

involved σ-receptor activation per se since haloperidol

and BD1063 blocked the PREGS-induced increase in

miniature excitatory postsynaptic current frequency.

Moreover, the σ1-receptor subtype was most likely

involved since pre-treatment with pertussis toxin pre-

vented the PREGS response. They also showed with

single pre-synaptic elements that the miniature excita-

tory postsynaptic currents, corresponding to the most

elementary forms of synaptic transmission due to

postsynaptic responses to action potential-independent

spontaneous glutamate release, was sensitive to the

modulation of [Ca2+]i with BAPTA-AM, suggesting that

neurosteroids as well as σ1 ligands may interfere with a

membrane-bound receptor that initiates a signal

transduction cascade involving a desensitization process

that occurs simultaneously with the translocation of

σ1 proteins and activation of the Gi /o protein-

phospholipase C (PLC)-protein kinase C (PKC) cascade,

as Morin-Surun et al. (80) have shown.

4. Behavioral effects of σ-receptor ligands and neuro-

steroids

The neuromodulatory effects of neurosteroids and


σ drugs on excitatory and inhibitory neurostransmission

have been linked to several physiopathological behav-

ioral responses. In particular, exogenous, systemic as

well as central, administration of steroids influences

response to stress, depression, anxiety, sleep, epilepsy,

and memory formation (6 – 13). Some drugs, including

σ ligands and the selective serotonin reuptake inhibitor

fluoxetine, were also demonstrated to affect neuro-

transmission systems by modifying intracerebral neuro-

steroid levels (140, 141). Systemic administration of

selective σ1-receptor agonists and antagonists has also

notable effects on response to stress, depression, anxiety,

drug addiction and memory formation. A crossed

pharmacology between both systems was clearly

evidenced, reinforcing the concept that an interaction

with the σ1 receptor is a main component in the rapid

neuromodulatory effect of neurosteroids. We will focus

more specifically on two behavioral aspects, learning

and memory and drug addiction, which are particularly

representative of the recent achievements in the field.

The reader is encouraged to refer to recent and

exhaustive reviews covering similar aspects relevant to

depression and mood disorders (142, 143).

4.1. Neurosteroids and σ drugs affect learning and

memory processes

4.1.1. Pro-mnesic effects of neurosteroids and σ drugs

The involvement of neurosteroids in learning and

memory processes has been demonstrated, first, by the

observation of pro-mnesic and anti-amnesic effects of

exogenously administered neuroactive steroids and,

second, by a parallel decrement between cognitive

functions and neurosteroid levels during normal and

pathological aging. Neurosteroids have been shown to

affect memory performances by themselves in control

animals and to alleviate the deficits in several pharmaco-

logical models of amnesia. For instance, DHEA, PREG,

and their sulphate esters enhanced, after central admin-

istration, memory retention in an active avoidance

learning task in mice after central or oral administration

(144 – 146). PREGS appeared as the most potent

compound and its long-duration effect suggested that

PREG may serve as a precursor for the formation of

other different steroids, ensuring a near-optimal modula-

tion of transcription of immediate-early genes required

for the facilitation of the plastic changes in memory

processes (145). PREGS also enhanced memory forma-

tion when administered after the first training session in

an alternation task in rats (147) or in an appetitive

reinforced Go-No go visual discrimination task in mice

(148). The rapid effect of the sulphated steroid clearly

evokes its non-genomic interaction with both the

GABAA and /or NMDA receptors and suggests that the

learning-induced activation of physiological responses

could be enhanced by an increase in neuroactive steroid

levels. PREGS also enhanced performances in a spatial

recognition memory task after post-acquisition intra-

cerebroventricular infusion (138). Interestingly the

active dose, 12 nmol, induced a mild increase in extra-

cellular acetylcholine (ACh) level measured by micro-

dialysis, whereas higher doses (up to 192 nmol) pro-

voked robust increases in ACh levels without behavioral

improvement. DHEAS, administered immediately after

training systemically or centrally or given in the

drinking water during two weeks, facilitated memory

retention in a step-down passive avoidance test in

mice but did not improve acquisition (149). Moreover,

when administered subcutaneously or intracerebroventri-

cularly, the steroid improved the percentage of correct

responses in a delayed alternation task in a Y maze and

improved the performances in a water-maze task as

compared to vehicle (oil)-treated rats (150). Therefore,

memory enhancing effects have been demonstrated for

both PREGS and DHEAS.

In most of the studies examining the anti-amnesic

potentials of σ1-receptor agonists in either pharmaco-

logical or pathological models of amnesia, a putative

pro-mnesic effect was examined in short-term or long-

term memory tests, by injecting the drugs alone.

Consistently, none of the compounds, (+)SKF-10,047,

(+)pentazocine, PRE-084, igmesine, SA4503 or DTG,

tested in large dose range, facilitated learning in

control animals (for reviews, see refs. 6 – 8). Activation

of the σ1 receptor failed to improve learning capacities

in control animals. Conversely, σ1 antagonists such as

α-(4-fluorophenyl)-4-(5-fluoro-2-pyrimidinyl)-1-pipera-

zine butanol (BMY-14,802), haloperidol, N-[2-(3,4-

dichlorophenyl)ethyl]-N,N',N'-trimethylethylenediamine

(BD1047), or N,N-dipropyl-2-[4-methoxy-3-(2-phenyl-

ethoxy)phenyl]ethylamine monohydrochloride (NE-100)

failed to show any amnesic effect. Moreover, down-

regulation of the σ1-receptor expression using in vivo

antisense strategies also failed to affect the learning

ability of mouse submitted to a passive avoidance test,

confirming the lack of involvement of the receptor in

normal memory functions (151, 152). The behavioral

phenotyping of σ1-receptor knockout animals (153) is

expected to confirm this observation. The lack of

consequence of σ1-receptors blockade on learning pro-

cesses is presently understood as a consequence of its

neuromodulatory role. As usually observed in most of

the physiological or pharmacological tests used to

evidence the σ1-receptor pharmacology, σ1 compounds

are devoid of effect alone, but exert some action only

when the transmission is perturbed. Therefore, the direct

effects of neurosteroids on learning capacities may not


exclusively involve their interaction with the σ1 receptor,

but also imply their modulatory actions at GABAA and

NMDA receptors. Indeed, both receptors are expressed

on cholinergic neurons of the medial septum and

diagonal band projecting into the hippocampal forma-

tion and glutamatergic and GABAergic responses regu-

late the activity level of cholinergic inter-neurons.

Cholinergic systems sustain the learning-induced plas-

ticity and cholinergic deficiency, particularly during

neurodegenerative diseases, is directly responsible for

learning deficits (24). Therefore, the cholinergic activity

of neurosteroids, shown on physiological studies (138),

is directly responsible for their anti-amnesic properties

(12, 13).

4.1.2. Anti-amnesic effects in cholinergic models of

amnesia

The beneficial effects of neuroactive steroids were

tested in experimental models of amnesia. Several

studies examined the effects of steroids on amnesia

induced in rodents by the cholinergic muscarinic antago-

nist scopolamine. PREGS and DHEAS reversed its

amnesic effects in mice submitted to a foot-shock active

avoidance test (149), step-through type passive avoid-

ance test (154), Go-No go visual discrimination task

(148), and spontaneous alternation and place learning in

a water-maze (155). The beneficial effects induced by

both sulphated steroids could be related to their acetyl-

choline release properties resulting from negative

modulation of GABAA receptors and /or positive

modulation of NMDA receptors. However, an involve-

ment of the σ1 receptor could not be definitively

excluded. The learning impairment induced by scopol-

amine, measured using spontaneous alternation, passive

avoidance, or water-maze procedures, could be attenuated

or reversed by σ1 agonists. This was indeed described for

DTG, (+)3-PPP, (+)SKF-10,047, pentazocine, igmesine,

or SA4503 (155 – 160). These effects could be fully

blocked by σ1 antagonists or an in vivo antisense strategy

(152). In addition, administration of SA4503 has also

been reported to attenuate the learning impairment in

rats with cortical cholinergic dysfunction, such as

ibotenic acid injection of the basal forebrain, that is,

lesioning cholinergic ascending pathways, in the passive

avoidance and water-maze tests (161). In parallel,

physiological studies showed that (+)SKF-10,047,

pentazocine, DTG, igmesine or SA4503 potentiated

[3H]acetylcholine release from hippocampal slices in

vitro or extracellular acetylcholine levels, measured in

vivo by microdialysis, in the rat frontal cortex and

hippocampus (162 – 164). Therefore, activation of the

σ1 receptor either directly affects cholinergic systems or,

through a positive modulation of the NMDA-receptor

activation, indirectly potentiates acetylcholine release.

At the behavioral level, a crossed pharmacology

between neurosteroids and σ1-receptor ligands was

reported for this amnesia model. DHEAS and PREGS

attenuated the the scopolamine-induced learning impair-

ments in mice submitted to a series of spatial and

contextual tests. These effects could be blocked by the

selective σ1-eceptor antagonist NE-100 or progesterone

(155). Similar crossed pharmacology studies have been

performed in amnesia models induced by blockade of

the NMDA-receptor activation.

4.1.3. Anti-amnesic effects in NMDA-receptor-depen-

dent amnesia

Impairment of the NMDA-receptor activation, by

either competitive or non-competitive antagonists, also

results in marked learning deficits. Administration of

PREGS dose-dependently reduced the learning deficit

and motor impairment induced by pre-training admin-

istration of the competitive antagonist 3-((±)2-carboxy-

piperazin-4-yl)-propyl-1 phosphonic acid (CPP) in a

step-through passive avoidance task in the rat (165)

or induced by (−)-2-amino-5-phosphonopentanoic acid

(D-AP5) in an active avoidance test in mice (166). In

addition, administration of PREGS in rats prevented

the cognition deficits induced by dizocilpine, a non-

competitive NMDA-receptor antagonist (167).

The σ1-receptor agonists were extensively studied in

this last amnesia model. (+)SKF-10,047, (+)pentazo-

cine, igmesine, DTG, PRE-084, or SA4503 attenuated

the dizocilpine-induced learning deficits in rats and

mice submitted to mnesic tasks involving spontaneous

alternation, passive avoidance, place learning in the

water-maze, three panel runway, or 8-radial arms maze

(160, 168 – 171). Moreover, the involvement of the σ1receptor in the anti-amnesic effect induced by the

steroids was indicated by the blockade of both DHEA

sulphate and pregnenolone sulphate effects by the

selective σ1-receptor antagonists BMY-14,802 and NE-

100 (17, 172) and the observation that progesterone

behaved as a clear antagonist of the efficacy of the σ1agonists (17, 172).

Interestingly, the in vivo antisense strategy revealed

some discrepancies regarding the anti-amnesic effects

mediated by neurosteroids. The antisense probe treat-

ment led to a complete blockade of the anti-amnesic

effect mediated by DHEAS, confirming that the anti-

amnesic effect of the steroid involves primarily an

interaction with σ1 receptors. On the contrary, PREGS

still induced a potent anti-amnesic effect in antisense-

treated animals. This result must be analyzed in line

with other observations. Both steroids act as negative

allosteric modulators of the GABAA-receptor-mediated


responses (37, 173, 174). They also potentiate several

responses mediated through the NMDA receptor.

However, when PREGS acts through a specific extra-

cellularly directed modulatory site located on the

NMDA-receptor complex, but distinct from either the

spermine, glycine, phencyclidine, arachidonic acid,

Mg2+ or redox sites (47), DHEAS failed to affect the

NMDA receptor through a direct interaction (46, 175).

However, through its interaction with the σ1 receptor,

DHEA /S have been reported to indirectly potentiate

several NMDA-mediated physiologic responses in vitro

and in vivo, and at the behavioral level (15, 16, 155, 160,

176). Morever, PREGS acts as an inverse σ1-receptor

agonist in vitro, when DHEA /S is a full agonist (see

above, and ref. 15). Parallel to this observation, it must

be noted that both steroids differently affected the extent

of excitotoxic insults resulting from over-activation of

the NMDA receptor. PREGS was reported to facilitate

the NMDA-receptor-mediated excitotoxic cell death in

several in vitro models of neurodegeneration (177)

and in mice exposed to a hypoxic insult in vivo (178).

The selective and efficient potentiation of the NMDA-

receptor activation induced by PREGS led to major

consequences in the case of over-activation of this

receptor. Indeed, the steroid potentiated the excitotoxic

neurodegeneration and worsened the resulting behavioral

deficits. DHEAS, on the contrary, prevented or reduced

the neurotoxic effects in primary hippocampal cultures

exposed to NMDA (175, 179, 180) and blocked both

the appearance of neurodegeneration and the resulting

learning deficits in mice exposed to carbon monoxide

(178). This neuroprotective effect seemed only partly

related to the interaction of the steroid with the σ1receptor, since it was poorly sensitive to σ1 antagonists,

NE-100, rimcazole, or BD1063 (178, 179). However,

these different results confirmed and brought physio-

pathological consequences for the differential pharmaco-

logical profiles presented by both steroids.

4.1.4. Anti-amnesic effects of neurosteroids and σ drugs

during aging

Finally, the anti-amnesic effects of neuroactive

steroids and σ1-receptor agonists have been tested

against learning deficits induced by normal or patho-

logical aging. Robel et al. (181) observed a significant

correlation between the PREGS levels in the hippo-

campus of aged rats and memory performances in a

water-maze and a two-trial recognition task; the animals

with better performances had greater levels of PREGS.

Furthermore, Vallée et al. (24) reported significant and

selective decreases of hippocampal PREGS levels in

aged rats as compared with young adults, and a positive

correlation between these levels and cognitive perfor-

mances in a delayed alternation task in the Y-maze and

a place learning task in the water-maze. Administration

of PREGS directly into the hippocampus temporarily

corrected the memory deficits of aged rats when

administered immediately after the acquisition trial (24).

Cholinergic systems in the basal forebrain are known to

be altered during aging and degenerative changes in

cholinergic nuclei are correlated with memory impair-

ment in aged rats. The central administration of PREGS

stimulated acetylcholine release in the adult rat hippo-

campus. Moreover, a correlation between PREGS levels

and learning and memory performance was evidenced in

24-month-old rats (24). In parallel, a single systemic

injection of DHEAS immediately after training im-

proved the impairment of memory in middle-aged and

old mice submitted to a footshock active avoidance

test, up to the levels observed in young mice (149).

Furthermore, the neurosteroid plays a physiological role

in preserving and /or enhancing cognitive abilities in old

animals, possibly via an interaction with the central

cholinergic systems. Such observations suggest that the

neuromodulatory action of PREGS and /or DHEAS

reinforce tonically neurotransmitter systems. Further-

more, since their blood concentrations decrease with

age, neurosteroids might be the rate-limiting endo-

genous substances for correct memory capacities (182).

The σ1-receptor expression and behavioral efficacy of

selective σ1 drugs has been extensively studied in aged

animals. Using radioligand binding, positon-emission

tomography (PET) scan imaging, and immunohisto-

chemical and molecular approaches, a remarkable

preservation of the σ1-receptor expression levels has

been observed in the different brain structures, including

limbic and cortical structures of SAM, aged mice or

aged monkeys (25, 26, 183, 184). In parallel, σ1-receptor

agonists attenuated after both acute and repeated

treatments, the learning deficits in SAM, aged mice or

aged rats (25, 176, 185, 186). Decrease of central levels

of neurosteroids was identified in these studies.

Moreover, a preserved expression of σ1 receptors was

documented by RT-PCR and immunohistochemical

techniques in numerous brain structures, whereas the

behavioral efficacy of σ1 drugs was maintained and

inversely correlated with the decrease in progesterone

contents (25, 26). Although no direct link could be

formalized, these observations are in agreement with the

endocrine manipulations studies detailed in paragraph

4.3.

4.2. Neurosteroids and σ drugs may influence abused

drug intake

The recent demonstration by Romieu et al. (187) that

neuroactive steroids are able to modulate the acquisition


of cocaine-induced conditioned place preference (CPP),

a behavioral procedure measuring the appetitive pro-

perties of drugs, suggest their influence on acquisition

of drug addiction. PREGS and DHEAS potentiated

cocaine-induced CPP acquisition, as do selective σ1-

receptor agonists, like igmesine or PRE-084, and the

effect was blocked by the σ1 antagonist BD1047 (188).

Exogenous administration of progesterone, or its endo-

genous accumulation by a finasteride treatment, blocked

the cocaine rewarding effect (187). This may be

mediated through either GABAA- and NMDA-receptor-

modulation, since mesolimbic dopamine neurons in the

nucleus accumbens are under the control of inhibitory

GABA neurons and excitatory glutamatergic neurons

originating from the frontal cortex or hippocampus.

Indeed, diazepam, a benzodiazepine acting as a positive

GABAA-receptor modulator, reduced the release of

dopamine in the nucleus accumbens measured by in

vivo microdialysis (189) and, in turn, attenuated

cocaine-induced CPP (190). Similarly, pre-synaptic

NMDA receptors facilitate dopamine release and

NMDA-receptor antagonists decreased cocaine-induced

locomotor stimulation, sensitization, or CPP (191, 192).

Since the respective pharmacological profiles of DHEA,

PREG, and progesterone on GABAA and NMDA

receptors are highly consistent with such actions, these

interactions could be involved in their effects on

cocaine-induced CPP. However, most of GABAA- and

NMDA-receptor modulators showed CPP or place

aversion (CPA) by themselves. Indeed, CPP was

observed with the direct GABAA agonist meprobamate,

the GABA metabolite γ-hydroxybutyric acid (GHB), or

several benzodiazepine compounds. Moreover, benzo-

diazepine antagonists or inverse agonists produced CPA

(for an exhaustive review, see ref. 194). It is noteworthy

that 3α-hydroxy-5α-pregnan-20-one (allopregnanolone),

devoid of affinity for the σ1 receptor but acting as a

highly efficient GABAA-receptor positive modulator

induced CPP after exogenous administration (193).

While NMDA-receptor competitive antagonists have

also been reported to induce CPP, more inconsistent

results were reported with noncompetitive antagonists,

particularly dizocilpine and both CPP or CPA has been

described (194). Curiously, neuroactive steroids failed to

induce CPP or CPA alone, with the notable exception of

testosterone (195).

Interestingly, a recent report by Barrot et al. (137)

showed that intracerebroventricular injection of PREGS

increased dopamine efflux measured in the rat nucleus

accumbens by in vivo microdialysis and potentiated the

morphine-induced dopamine release. Consequently, the

authors suggested that this potentiation of mesolimbic

dopamine concentration may explain the involvement of

neurosteroids in mood and motivation.

The involvement of the σ1 receptor in several cocaine-

induced behavioral effects has also been extensively

examined (for a review, see ref. 196). Selective σ1antagonists, like haloperidol, BMY-14,802 or rimcazole,

blocked the locomotor stimulant effect of cocaine after

acute administration (197 – 199) and the development of

behavioral sensitization after repeated cocaine injections

and withdrawal (200). Selective σ1 antagonists, like

BD1047 or NE100, or antisense oligodeoxynucleotide

probes targeting the σ1 receptor also blocked acquisition

or expression of cocaine-induced CPP in mice (188,

201).

It is noteworthy that the modulating effect of neuro-

steroids was less observable on cocaine-induced loco-

motor sensitization and toxicity. Some effects could

however be observed on locomotor sensitization such as

DHEA sensitizing to a delayed injection of cocaine and

progesterone attenuating the short-term sensitization to

cocaine (P. Romieu & T. Maurice, unpublished observa-

tions). However, σ1 antagonists potently prevented

cocaine-induced locomotor stimulation and sensitization

(197, 200) and contribution of the other targets of

neurosteroids, namely, the NMDA and GABAA recep-

tors, but also of genomic effects may also contribute to

an effective modulation of locomotor effects. Observa-

tion of a clear pharmacological response on reward as

compared to other behavioral responses suggests that

the σ1 receptor and the resulting interaction of neuro-

active steroids appear to be particularly sensitive in

specific brain structures and pathways. Indeed, a 4-day

cocaine treatment resulted in high increase of the σ1-

receptor mRNA levels only in the nucleus accumbens, a

key structure for drug reward (188), while self-admin-

istered methamphetamine increased expression of σ1-

receptor mRNA in the hippocampus and decreased its

expression in the frontal cortex, in contrast to yoked

administration (202).

A recent study extended the behavioral evidences by

showing that neurosteroids, administered at very low

doses also modulate cocaine’s effects. A modified

passive avoidance procedure was used in mice to

examine whether cocaine induces state-dependent learn-

ing (StD) (203). Cocaine administration, at the low dose

of 0.1 mg /kg before training, produced a chemical state

in the brain that served as an endogenous cue. Animals

must be treated with the same dose of the drug before

retention to ensure optimal retention. Among neuro-

active steroids, pregnanolone and allopregnanolone

sustained StD by themselves. However, steroids also

acting as σ1 agonists (e.g, DHEA and PREG) or as

antagonist (i.e., progesterone) failed to induce StD but

modified the cocaine state. The σ1 agonist igmesine or


antagonist BD1047 also failed to induce StD but

modified the cocaine state. Furthermore, optimal reten-

tion was noted in mice trained with (igmesine or

DHEA) + cocaine and tested with a higher dose of

cocaine or in mice trained with (BD1047 or pro-

gesterone) + cocaine and tested with vehicle. From these

studies, it is concluded that a low dose of cocaine

induces a chemical state /memory trace that sustains

StD. Modulation of σ1-receptor activation is not suffi-

cient to provoke StD, but it alters the cocaine state, in

line with the pure neuromodulatory role of the σ1receptor. Neurosteroids have a unique impact on state-

dependent versus state-independent learning, via

GABAA- or σ1-receptor modulation, and can affect the

cocaine-induced mnesic trace at low brain concentra-

tions.

Finally, similar observations could be presented for

other drugs of abuse, since methamphetamine, mor-

phine, and ethanol also involve σ1-receptor activation

(202, 204 – 206). In particular, morphine-induced

hyperlocomotion and CPP could be modulated by σ1-

receptor ligands (T. Maurice, unpublished observations).

Moreover, the naloxone-precipitated morphine with-

drawal symptoms could be blocked by administration

of the σ1 agonist igmesine (T. Maurice, unpublished

observations) as well as DHEA in a NE-100-sensitive

manner, thus confirming the involvement of the σ1-

receptor subtype, as stated by Ren et al. (207).

4.3. Cross-influences between neurosteroids and σ

receptor

The interaction of endogenous neuro(active)steroids

with the σ1 receptor has direct physiological conse-

quences. The variations of neuro(active)steroids levels,

for example, during pregnancy, stress, mood disorders,

or normal or pathological ageing, directly affects the

efficacy of σ1-receptor agonists in vivo.

The direct demonstration of the physiological impor-

tance of the steroidal tonus was provided by Bergeron

and co-workers (123) who examined the effects of σ1agonists in control female rats, at day 18 of pregnancy

and day 5 post-partum, and in ovariectomized rats

following a 3-week treatment with a high dose of

progesterone. In pregnant rats and following a 3-week

treatment with progesterone, tenfold higher doses of

DTG, (+)-pentazocine, and DHEA were required to

elicit a potentiation of the NMDA response of hippo-

campal neurons comparable to that obtained in control

females. Conversely, at day 5 post-partum and following

a 3-week treatment with progesterone and after a 5-day

washout, the potentiation of the NMDA response

induced by the σ agonist DTG was greater than in

control females (123). These data showed changes in

the function of σ1 receptors during pregnancy and post-

partum, that is, in case of drastic variations of steroid

levels, and suggested that the steroid tonus may impede

the behavioral efficacy of σ1 agonists.

This question was also addressed through a series of

endocrine manipulations using adrenalectomized (AdX)

and castrated (CX) mice, to deplete the peripheral

biosyntheses in circulating steroids, and treated with

trilostane or finasteride, in order to deplete or accumu-

late progesterone in the brain. Accordingly, the in vivo

[3H](+)SKF-10,047 binding levels to σ1 sites in the

mouse forebrain and hippocampal formation appeared

significantly increased in AdX /CX mice and further

increased after the trilostane treatment. Conversely, the

finasteride treatment led to a significant decrease of

binding as compared to AdX /CX animals. The attenuat-

ing effect of PRE-084 against dizocilpine-induced

learning impairment was examined using spontaneous

alternation behavior, step-down passive avoidance, and

contextual latent learning in the elevated plus-maze in

the different endocrine conditions. The learning deficits

induced by dizocilpine were not affected by the treat-

ments. The anti-amnesic effect of PRE-084 was

facilitated in AdX /CX mice and even more after

trilostane treatment since several parameters for (PRE-

084 + dizocilpine)-treated animals returned to control

values. The PRE-084 effect was blocked after finasteride

(208). Moreover, PREGS infusion in AdX /CX rats

was previously reported to prevent the learning deficits

induced by dizocilpine and (−)3-[2-carboxypiperazin-4-

yl]-propyl-1-phosphonic acid (CPPene) (209). It was

also demonstrated that infusion of PREGS markedly

increased the brain contents of PREG, progesterone,

5α-pregnane-3,20-dione, and allopregnanolone (209).

Interestingly, blockade of 5α-reductase activity led to

the disappearance of the PREGS effect, leading the

authors to suggest that an increase in allopregnanolone

level could mediate the observed effect. As an alterna-

tive, 5α-reductase activity inhibition led to sustained

increase in progesterone levels and the steroid, acting as

a σ1-receptor antagonist, which may attenuate the

PREGS effect.

We also observed that an acute swim stress induced

an increase in the level of progesterone and a decrease of

in vivo [3H](+)-SKF-10,047 binding in the hippocampus

of control animals (21). These effects were enhanced

in AdX /CX mice, but completely blocked following

treatment with trilostane. A significant inverse correla-

tion was observed between progesterone increase and

the inhibition of in vivo binding to σ1 sites after stress

among the different endocrine conditions. These

observations not only confirmed that neurosteroids

modulate the efficacy of σ1-receptor agonists in the


response to stress and depression, but also showed that

endogenous neurosteroid levels directly and tonically

regulate the in vivo bioavailability of σ1 receptors.

These observations documented that progesterone

could be a major endogenous effector of the σ1 receptor

and stressed that physiopathological variations of this

neurohormone must be taken into consideration before

designing any pharmacological strategy targeting the σ1receptor. Moreover, a different behavioral efficacy of

σ1-receptor ligands was observed among mouse strains,

that could be related to the endogenous tonus in neuro-

steroids (210). In particular, the C57BL /6 mouse

appears to present the lowest progesterone levels in

basal and stressful conditions, in line with a higher

efficacy of σ1 drugs and a highest vulnerability to

cocaine’s appetitive effects (210). Extrapolated to

humans, patient populations showing important levels in

steroids acting negatively on σ1 receptors may impede

the use of selective σ1 ligands.

On the contrary, physiopathological conditions with

disturbed steroidal tonus may be highly sensitive to σ1-

ligand treatments. This is of importance during ageing

and in Alzheimer’s disease-like neurodegenerative patho-

logies. With this regard, we examined the pharmaco-

logical efficacy of several σ1-receptor agonists in two

nontransgenic rodent models of Alzheimer’s disease:

mice injected acutely and intracerebroventricularly with

β25–35-amyloid peptide (28) and rats infused intra-

cerebroventricularly during 14 days with the β1–40-

amyloid protein delivered using an Alzet minipump

(29). Neurosteroid levels were measured in several brain

structures after β-amyloid injection in mice or infusion

in rats, in basal and stress conditions. The β25–35 mice

exhibited decreased progesterone levels in the hippo-

campus. Progesterone levels, both under basal and

stress-induced conditions, were also decreased in the

hippocampus and cortex of β1–40-amyloid-treated rats.

The levels in PREG /S and DHEA /S appeared less

affected by the β-amyloid infusion (29). The behavioral

efficacy of σ1 agonists is known to depend on the levels

of neuro(active)steroids synthesized by glial cells and

neurons, which are affected by the β-amyloid toxicity.

The antidepressant-like response of animals was

examined using either the forced swim test in mice or

the conditioned fear stress test in rats. The β25–35-peptide-

injected mice developed memory deficits after 8 days in

contrast to the controls injected with scrambled β25–35

peptide or vehicle solution. In the forced swim test, the

β-amyloid treatment failed to affect the immobility

duration, but the antidepressant effect of the σ1 agonists

was facilitated in β25–35-treated mice: igmesine reduced

immobility duration at 30 mg /kg versus 60 mg /kg in

control groups and PRE-084 decreased immobility

duration at 30 and 60 mg /kg only in β25–35-treated mice.

In comparison, desipramine reduced the immobility

duration similarly among the groups and fluoxetine

appeared less potent in β25–35-treated animals (28). In

rats, igmesine and (+)-SKF-10,047 significantly reduced

the stress-induced motor suppression at 30 and 6 mg /kg,

respectively, in β40–1-amyloid-treated control rats. Active

doses were decreased to 10 and 3 mg /kg, respectively,

in β1–40-amyloid-treated rats. The DHEAS effect was

also facilitated, both in dose (10 vs 30 mg /kg) and

intensity, in β1–40-amyloid-treated rats (29). These

results suggested that σ1-receptor agonists, due to their

enhanced efficacy in a nontransgenic animal model, may

be particularly interesting drugs to alleviate not only

Alzheimer’s disease-associated memory impairments

but also depressive symptoms (8, 28, 29, 176, 185).

5. Conclusions

The understanding of the action of steroids within

the brain functions and their inter-relationships must be

currently considered within the double framework of

endocrine mechanisms, as responses elicited by hor-

mones secreted by the gonads and adrenal glands, and

regulation of neural functions by autocrine and /or

paracrine actions of neurosteroids, that is, as responses

elicited by steroids synthesized and metabolized within

the nervous system. Whatever their origins, several lines

of evidence support the notion that apart from their

genomic actions, neuroactive steroids, as well as neuro-

steroids, play a pivotal role in cognition and adaptive

responses to neuronal damage. Moreover, the σ1 receptor

constitutes one of the key targets in their trophic,

neuromodulatory and behavioral effects. Although

partially unveiled using pharmacological, biochemical,

and genetic approaches, experiments outlined above

have hence enlightened and documented the role of this

orphan endoplasmic reticulum-bound receptor in the

regulation of physiologic responses. These include

modulation of transmembrane K+ and Ca2+ fluxes,

NMDA-sensitive glutamatergic, catecholaminergic, and

cholinergic pathways, to the immediate and robust

recruitment of Ca2+-dependent intracellular signaling

and protein (de)phosphorylation. These neuromodulatory

effects accounted for the molecular mechanisms of

interaction between σ1 sites and neuro(active)steroids in

relationship with most of their physiological properties,

detailed here in terms of cognitive and addictive out-

comes. The merely simultaneous impact of σ1-receptor

activation at the level of both cytosol and nuclear and

plasmic membranes, due to its unique translocation

process, provides further insight into the identification

of the commonality of action of σ1 drugs /neurosteroids


involved in these behavioral tests and further points out

the interest for the σ1 receptor in cognition and ageing.

This should facilitate the elaboration of efficient

strategies for preventing and treating cognitive and

memory deficits during ageing, stimulating neural

regeneration and correcting behavioral disorders in

response to stress. These above-mentioned findings

support the notion that the recruitment of membrane-

bound second messenger cascades (i.e., G-protein /PLC

/PKC pathway) via activation of a single transmembrane

domain intracellular receptor constitutes a novel mode

of action for medicines.

Finally, behavioral studies focusing on the impact of

variations in the central level of neurosteroids in

AdX /CX mice demonstrated that progesterone levels in

basal and stressful conditions determine the behavioral

efficacy of σ1-receptor drugs. Endocrine conditions

must therefore be taken into consideration for the

development of σ1-receptor-based therapies. Extra-

polated to humans, patient populations showing impor-

tant levels in steroids acting negatively on σ1 receptors

may impede the use of selective σ1 ligands. On the

contrary, physiopathological conditions with disturbed

steroidal tonus may be particularly responding to σ1ligand treatments.

Indeed, σ1-receptor-based therapies were already

proposed to be of physiological and therapeutic impor-

tance in neurology and psychiatry since the σ1 receptor is

abundant in the brain, where it regulates these functions.

Although this σ1 receptor plays a crucial role in the

adaptative changes mandatory for memory processes or

addiction, it remains however to determine whether and

how these molecular mechanisms involved in learning

and recruited by σ1 drugs operate synergically (most of

them usually proceeding in parallel) and whether the

interplay between cytosolic and membrane-bound

molecular events occurring in response to σ1 drugs is

consistent with the roles of cytoskeleton in mediating

these changes. There is no doubt that the identification

of the nature of the proteins synthesized in response to

σ1 drugs will also need to be addressed soon.

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