The role of cholinergic sysTem in neuronal
plasTiciTy: focus on visual corTex and muscarinic
n. origlia1, n. KuczewsKi2, e. pesavenTo2, e. azTiria2
and l. domenici1 3
1 institute of neuroscience (c.n.r.), 56100 pisa, italy; 2 international school for advanced studies (i.s.a.s.-
s.i.s.s.a.), 34014 Trieste, italy; 3 department of Biomedical sciences and Technology (s.T.B.), university of
l’aquila, 67010 l’aquila, italy
i n T r o d u c T i o n
This review is focused on the basal forebrain (BfB) cholinergic system, cholinergic
receptors and cholinoceptive target areas such as the neocortex, all of which are intimately
involved in high cognitive functions and synaptic plasticity. The neurons of the BfB synthe-
size acetylcholine (ach) whose action is mediated by two subclasses of receptors, namely
nicotinic and muscarinic receptors. using the visual system as a model, the aim here is to
integrate and discuss the current knowledge on anatomy, ontogeny and function of the BfB
cholinergic system. This signaling system represents the anatomo-functional basis of ach
action on neuronal network, neuronal plasticity and cognitive functions. cholinergic system
role on higher brain functions has received increasing attention since the first observation
of a. alzheimer (1907) reporting dramatic changes of the BfB cholinergic neuro-anatomy
in one of the most devastating neurodegenerative diseases of adult brain, i.e. alzheimer’s
disease (ad). in addition to this observation, later work demonstrated its participation in
deep re-arrangements of brain connectivity such as the regulation of neuronal plasticity
during maturation of cortical sensory maps, in adult and aged brain.
B a s a l f o r e B r a i n c h o l i n e r g i c s y s T e m
cholinergic neurons of BfB include a series of small contiguous or interconnected
groups of cells with similar properties in mammalian animal species; cholinergic nuclei
project topographically to cholinoceptive target areas. for example, in rodents the nucleus
basalis magnocellularis (nBm) is the equivalent of meynert nucleus in the primates and
send projections to several areas including cerebral cortex and amygdala while the innerva-
tion of hippocampus is provided by the medial septum-diagonal band complex (ms-dB)
corresponding author: luciano domenici, institute of neuroscience (cnr), via moruzzi 1, 56100 pisa, italy. Tel.
+39 050 3153182. fax +39 050 3153220. e-mail: firstname.lastname@example.org
Archives Italiennes de Biologie, 146: 165-188, 2008.
N. Origlia et al.
in addition to cholinergic neurons, in the BfB region there are other projecting neurons
synthesizing different neurotransmitters such as gamma-aminobutyric acid (gaBa; ).
rye et al. (145) showed that 30% of the BfB neurons projecting to the cortex are choliner-
gic. gaBaergic neurons are intermingled with the cholinergic neurons in the BfB region
and account for another 30% (55, 110).
in the rat, BfB cholinergic neurons spread from the anterior medial septal nucleus to the
rostral portion of the lateral hypothalamus caudally. The BfB cholinergic neurons are from
medium to large sized, long axoned, and multipolar. most of the cell bodies have an oval or
fusiform shape. They display intense immunoreactivity for acetylcholine esterase (ache),
choline acetyltransferase (chaT), and vesicular acetylcholine transporter (vachT), as well
as staining for chaT and vachT mrna (151, 82). Based on specific projection areas, the
BfB cholinergic neurons in rat give rise to at least four different pathways: 1) the basalo-
olfactory bundle, projecting to the olfactory bulb and associated nuclei; 2) the basalo-hip-
pocampal bundle originating in ms-dB and innervating the hippocampal formation and
nearby limbic cortex; 3) the medial cortical pathway, originating mainly in the vertical
and horizontal limbs of dB, nBm, and partially in the magnocellular preoptic area and
substantia innominata. These nuclei project to medial cortical regions, including the medial
frontal, cingulate, retrosplenial and medial occipital cortices; 4) the lateral basalo-cortical
and basalo-amygdala tracts, supplying afferents to the remaining allocortex, to the lateral
isocortex, and to the amygdala (16, 151). it is proposed that cholinergic neurons in the BfB
provide the majority of the cholinergic innervation to the telencephalon (145).
in addition, intrinsic cortical cholinergic neurons characterized by a bipolar shape have
been described in different mammalian species such as the rat, although the real cholinergic
nature and function of these cells are still debated (78, 44, 108, 165).
The BfB cholinergic system is typically defined as a diffusely projecting system since
almost all brain areas receive a cholinergic projection from it. however, cholinergic projec-
tions innervate relatively small regions, especially in sensory cortical areas. The sensory
cerebral cortices can be divided into cytoarchitectonic regions that are constrained by the
sensory modality of their thalamic inputs, and in turn each sensory modality is organized in
a point to point representation. These modules are innervated by discrete groups of cholin-
ergic cells residing in BfB region. Thus, activation of discrete cholinergic groups of neu-
rons may induce functional changes in their respective sensory modules in the neocortex.
cortical cholinergic varicosities have been described diffusely throughout different
cortical layers. remarkably, only a small fraction of ach varicosities in the cerebral
cortex displayed membrane synaptic specialization (164, see ). specifically in the rat
parietal cortex at postnatal day 38 (p38), i.e. at an age when the cholinergic fibers have
already assumed an adult-like shape, synaptic junctions represent only 17% of whole vari-
cosities (118). usually, symmetric cholinergic synaptic contacts are primarily made with
dendritic branches and less frequently with spines and cell bodies of either glutamatergic
or gaBaergic neurons (168, 79). on the other hand most of the cholinergic varicosities
terminate free in the extracellular matrix without being associated with any synaptic spe-
cialization (164, 124). non-synaptic varicosities seem to be randomly distributed in rela-
the rOle Of chOliNergic system iN NeurONal plasticity
tion with the surrounding elements and show an incessant movement of translocation and
re-shaping along the fibres (36). This motility may constantly modify the exact position of
ach releasing sites in relationship to the targets and hence their functional influence. such
characteristic suggests that ach may act in a paracrine fashion on cortical elements, with
ach diffusing to the extracellular space and then influencing a large number of cortical ele-
ments (36). This modality of release is consistent with the concept of volume transmission
of neurotransmitters proposed by fuxe and agnati (61). The concept of volume transmis-
sion is supported by the evidence of a small number of BfB projecting cholinergic neurons
compared with that of cortical neurons subjected to cholinergic modulation (120).
cholinergic nuclei receive afferent inputs from the cortex, striatum, hypothalamus and
brainstem (152, 153, 32, 75; for a review see ). afferent projections can be classified
according to the neurotransmitter that is being released and these are basically glutamate,
gaBa and noradrenaline. glutamatergic afferents of the basal forebrain arise primarily
from cortical and amygdala areas. in addition, a prominent projection from pre-frontal
cortex to BfB has recently received great attention for its important role in the modulation
of executive and attentive functions (134, 64). Brainstem regions, including the pedunculo-
pontine tegmental nucleus (ppT), may also send glutamatergic projections to the BfB.
Telencephalic projections, including afferents from the amygdala, converge on BfB neu-
rons and modulate their excitability; in particular, the substantia innominata is reciprocally
connected to the basolateral amygdale (Bla) region (94, 84; reviewed in ). several
studies have focused on the regulation of cortical ach efflux by gaBaergic inputs to basal
forebrain cholinergic neurons, presumably originating in the nucleus accumbens (ac).
These studies show that basal forebrain cholinergic neurons express gaBaa receptors and
respond to gaBaergic agonists and antagonist modifying cortical ach release. a promi-
nent projection is represented by noradrenergic afferents to the BfB cholinergic neurons
that originate in the locus coeruleus (lc) particularly in the a5 group of the ventrolateral
tegmentum. BfB cholinergic neurons receive a particularly dense noradrenergic input and
the distal segments of their dendrites are repeatedly contacted by noradrenergic fibers.
moreover, BfB nuclei receive brainstem cholinergic projections from peduncolo-pon-
tine and laterodorsal tegmental (ppT/ldT) cholinergic nuclei (157). ppT/ldT neurons
appear to activate BfB nuclei through glutamate (143), which co-localizes with choline
acetyl transferase (chaT) in these cells (102).
Thus, BfB neurons can be primarily activated by connections from prefrontal cortex
(top-down) and by an ascending reticular system mainly via noradrenergic projections
neurogenesis of the rat cholinergic neurons begins at caudal level on embryonic day
12 (e12) and is completed at the most rostral level by e17 (152). chaT, the rate limiting
enzyme leading to ach synthesis, is already detected at early embryonic stages of develop-
ment (41, 66). By using an anterograde tracer method, BfB axons projecting to the cortex are
detected in the white matter under the occipital cortex at postnatal day 0 (p0). at this age the
occipital cortex comprises the subplate, layer vi, an emerging layer v, an undifferentiated
cell dense cortical plate, and the marginal zone. at p3, when layer iv starts to differentiate
N. Origlia et al.
from the cortical plate, BfB axons are present in layers vi and v, while by p4 they reach
layer iv. at p6, all layers of the occipital cortex are differentiated and the BfB axons are
seen throughout all layers, although the vast majority of labeled axons are still confined in
the infragranular layers. during the second postnatal week the axons continue to develop till
reaching a pattern of mature distribution by p11 (17). marked changes occur by p14 when
the morphological features of the BfB neurons approach an adult pattern (41, 18).
chaT immunoreactivity in the BfB reaches an intensity comparable to that of an adult
by the end of the second postnatal week (41). in adult rat somatosensory cortex, chaT
immunoreactive fibers with periodic varicosities appear to form a loosely organized net-
work throughout all cortical layers. chaT terminals are found in association with dendrites
of pyramidal neurons and somata of non-pyramidal neurons (79). chaT activity in the rat
visual cortex cannot be detected until p8. Thereafter, the level of chaT activity increases
during the second postnatal week reaching an adult-like level; this first peak was followed
by a decline and subsequently by a slow increase towards adult levels (51). in contrast
with data obtained by measuring chaT activity cortical release of ach elicited by electri-
cal stimulation was low during the first postnatal week increasing until the end of the first
postnatal month (135). interestingly, varicosities and synaptic junctions assume adult-like
features already at p8 when cholinergic innervation is installed (118).
in adulthood, the pattern of chaT activity in the rat visual cortex shows no statistical
difference among layers; however the peaks of activity were observed in layer v, i and to a
less degree of intensity in layer ii-iii and iv (115). overlapping results were obtained using
immunohistochemistry for chaT (117): layer v was the highest densely innervated by chaT
immunopositive fibres while layer iv was one of the lowest in parietal and occipital cortex.
another possible marker of cholinergic fiber distribution is represented by ache activ-
ity. at the end of the second postnatal week, ache activity was shown to peak in layers i,
and iv and the deeper part of layer iii. in the third week, a peak of activity also appears in
layer v. in the adult, across the thickness of primary somatosensory and visual cortex (s1
and v1), ache reaction product is distributed throughout all cortical layers with peaks in
layer i, deep layer iv, and deep layer v (119). in particular, in v1 ache reaction product
shows the highest density in layer iv with smaller peaks in layer i and v. Thus ache
cortical distribution does not clearly matches the laminar distribution of chaT axons and
varicosities (117), possibly reflecting a cortical innervation from several sources. lesions of
either the lateral geniculate nucleus (lgn) or the BfB reveal that in the first three weeks of
postnatal development, the peaks of ache activity are due to the lgn projecting neurons,
with a gradual shift towards a BfB origin between the fourth postnatal week and the adult-
hood (72). The latter findings suggest that, since the geniculocortical pathway develops
before the cholinergic projections, the peaks of ache activity could reflect a role of the
thalamic input in the formation of cholinergic synapses.
in summary, the results on cortical areas suggest that: i) BfB cholinergic neurons com-
plete their development by the third postnatal week, and this time course is paralleled by
ach release and chaT immunoreactivity; ii) the BfB cholinergic terminals densely dis-
tribute in layers i, v and to a lesser degree in layer iv with distinct differences between the
different cortical areas; iii) ache distribution throughout cortical layers does not closely
match the distribution of cholinergic terminals during early stage of development.
the rOle Of chOliNergic system iN NeurONal plasticity
3. Release of Acetylcholine.
The cortical release of ach is almost entirely due to the activity of BfB cholinergic
cells. evoked activity (24, 21, 99) in the BfB region induces an increase of cortical cholin-
ergic release respect to the basal rate in motor, sensory and visual cortices. phasic and tonic
release of ach have been described in relation with phasic and tonic firing of BfB cholin-
ergic neurons. for example, BfB cholinergic neurons receive noradrenergic projections
from lc being predominantly depolarized via α1 receptors upon noradrenaline release.
noradrenaline, then, drives cholinergic cells into a tonic mode of firing increasing their rate
of repetitive spike discharge (55, 110).
The use of microdialysis techniques to measure the levels of ach release in living ani-
mals allowed relating different behavioral and functional states of the brain with the varia-
tion of local ach concentrations. experimental evidence has shown that brain ach levels
can vary from few percentage points up to 800% over basal ach release (reviewed by
). in absolute value, basal endogenous ach is shown to vary in the range of concentra-
tion of hundred picomolar to nanomolar (basal ach; ). cortical ach increase is asso-
ciated with spontaneous and acquired behaviors such as exploration of a new environment
(160, 63), locomotor activity (33, 23), visual attention and arousal (2, 133), chronic stress
(122), sensory stimulation (50, 3), working memory and spatial learning (76,48), operant
behavior (129), visuo-spatial attentional task (33) and contextual fear conditioning (125).
variations in ach levels in the cortex as well as in the hippocampus are also associated
with the wake-sleep cycle. indeed, an increase of cholinergic release is observed during
active waking as compared to slow waves sleep when the levels of ach decrease to less
than one third to rise again during the rem sleep (83, 86). Beyond the association of ach
changes with behavioral states it has been shown that patterned visual stimulation is able
to elicit significant increases in acetylcholine release in the visual cortex of anaesthetized
animals (101). These authors showed that the degree of cholinergic system activation is
strictly related to the level of visual activity. Thus, activation of BfB cholinergic system by
electric stimulation or behavioral tests may modulate the release of ach in cholinoceptive
areas respect to the basal rate.
The variation of ach endogenous level together with the type (tonic versus phasic) and
the cellular organization (synaptic versus extrasynaptic) of release concur to define the
cholinergic flexibility that is necessary to accomplish for the numerous actions exerted by
ach in different cortical areas.
c h o l i n e r g i c r e c e p T o r s
The cholinergic receptors can be divided into two families: the ionotropic or nicotinic
receptors (nachrs) and the metabotropic or muscarinic receptors (machrs). The neuronal
nicotinic receptors (nachrs) comprises 12 subunit genes with a common ancestor, includ-
ing α2 through α10 and β2 through β4. These receptors appear as pentameric ligand-gated
ion channel with low selectivity for cations and able to gate high relative amounts of cal-
cium (56). The majority of neuronal nachrs fall into two main classes: 1) the homomeric
or heteromeric α-bungarotoxin (αBgtx) sensitive receptors made up of α7, α9, α10 or
N. Origlia et al.
α9/α10subunits, 2) αBgtx insensitive heteromeric receptors made up of α2-α6 and β2-
β4 subunits in different combinations. The α4β2 subtype was the first neuronal nicotinic
receptor to be biochemically characterized and the most widely distributed in mammalian
brain (176, 167). nicotinic receptors are permeable to monovolent na+ and K+ ions and to
divalent ca++; permeability and ion selectivity changes as a function of subunits composi-
tion (131, 26, 39). nicotinic receptors modulates the release of various neurotransmitters
including ach, glutamate, dopamine, noradrenaline and gaBa acting at pre-synaptic sites
(177). nicotinic receptors are also capable of modulating the excitability of neurons acting
at post-synaptic sites particularly in cortical interneurons (178). α4β2 nachrs account
for > 90% of the high-affinity nicotinic receptors in the brain and bind epibatidine while
homomeric α7 receptors are the second most represented subtype in different regions of
the brain and bind alpha-bungarotoxin, (175). given the focus of the present review, we
will not discuss in details the expression and physiological impact of all nicotinic receptor
subunits in different brain areas (for recent review see ). a brief overview, however, is
necessary to give some insights on the principal nicotinic subunits and receptors present in
the visual system with particular emphasis on visual cortex areas. visual system develop-
ment and functionality requires the activity of nicotinic subunits. in particular, the β2 subu-
nit is required in the formation of eye-specific layers at thalamic level depending on retinal
waves of spontaneous activity that rely on its activation (144, 25, 67). indeed β2 Ko mice
show altered retino-fugal projections that do not segregate into eye-specific areas, both in
the lgn and in the superior colliculus. The idea has been raised that β2 (possibly making
part of a nicotinic receptor with a (α4)3(β2)2 stoichiometry) would have a role in the nerve
cell function essential for the correct wiring of the visual system during early development
even before the photoreceptors are present (for a review see ). at the visual cortex level
it has been observed that αBgtx insensitive heteromeric receptors that bind epibatidine are
involved in the regulation of visual cortex responses (132). similar data have been reported
in other sensory cortices such as auditory and somatosensory cortex (136). a second
widely represented nicotinic receptor in the visual system contains α7subunits (possibly
homomeric). The α7receptor exhibits an extraordinary permeability to calcium ions and
is particularly enriched in the cortex (for a review see ). Both α4and α7 subunits are
distributed across all layers in the visual cortex of rat as demonstrated by antibody labeling
(8). it is largely accepted that nicotinic receptors regulate thalamo-cortical synaptic trans-
mission acting at pre-synaptic site on glutamatergic synapses. nicotinic receptors are also
expressed at post-synaptic sites as observed in somatosensory cortex (108) contributing to
excitation of pyramidal and gaBaergic interneurons.
The muscarinic receptor family of ach receptors comprises five different genes (m1-
m5), and is pharmacologically defined as m1-m5 subtypes, see ). The m1-m4 receptors
are the most abundant machrs in the cortex and hippocampus (for a review see ).
These receptors can be grouped according to the type of g proteins that they activate.
indeed, while m2 and m4 machrs are coupled to the pertussis toxin-sensitive gi and go
proteins, leading to the inhibition of adenylyl cyclase, the m1 and m3 machrs preferentially
interact with the pertussis toxin-insensitive gq/11 and g13 proteins, leading to the activation of
phospholipases c and d (6, 22; for a review see ). The affinity of ach is higher for m2
and m4 than for the m1 or m3 receptors (103 130). in the neocortex, the relative abundance
the rOle Of chOliNergic system iN NeurONal plasticity
of machrs subclasses is m1 > > m2 > m4 > m3 (106, 105, 22). in addition the highest
levels for m1 are detected in layers ii/iii, and vi, where virtually all pyramidal neurons are
stained. on the other hand the m2 labeling is denser in layers iva, ivc and at the border of
layers v/vi with its immunoreactivity concentrated in spines and small dendrites of mostly
interneurons (106, 123, 77, 161). The m4 immunoreactivity, is low if compared to that of
the m1 or m2 receptor subtypes, with the highest staining in neuropil of supragranular layers,
layer v, and patches in layer iv (106, 77, 161). equivalent results were obtained by using
quantitative receptor autoradiography in the rat visual cortex (150). The laminar distribution
of the different receptor subtypes in the rat brain correlates well with the cellular localization
of their respective mrnas (172).
Besides exhibiting multiple post-synaptic localizations machrs can also be found at
the pre-synaptic sites. pre-synaptic receptors have been described on cholinergic fibres (i.e.
autoreceptors) and serve to inhibit the release of ach. pre-synaptic cholinergic receptors
are also reported for noradrenergic, dopaminergic and glutamatergic fibres. pre-synaptic
localization of m1-m4 subtypes have been reported in monkey neocortex with m2 being
prominent in axons versus dendrites of the primate occipital cortex (42). in agreement with
this observation, the selective deletion of m2 in knock-out mice increases ach release by
reducing autoreceptor function in cerebral cortex and hippocampus (180).
There is a great body of evidence supporting that in the cortical areas, both excitatory
pyramidal neurons and inhibitory interneurons express machrs and are modulated by ach
(159, 114, 113, 12, 73, 171, 46). using electron microscopy disney et al. (42) showed that
excitatory neurons in the visual cortex of monkeys express mainly m1 in dendrites while the
soma of gaBaergic neurons is the preferred subcellular site for m2. furthermore, in the rat
somatosensory cortex muscarine affects different subclasses of layer v inhibitory neurons
(178). interestingly, there are inter-areal differences in machr cell expression also within
regions subserving the same sensory modality. indeed, while machrs are equally expressed
in gaBaergic interneurons across primary (v1) and secondary visual cortices their expres-
sion in glutamatergic cells varies being higher in the secondary visual cortex (42).
interesting insights came from the autoradiographic study on the ontogenetic profile of
muscarinic receptors in the rat brain. The binding sites for the total population of muscarinic
receptors increase steeply between e20 and p21, while a slower increment is detected there-
after. at e20, the relative amount of m1 binding sites in the neocortex is on average 11% of
that in adults. The supragranular layers of the occipital cortex increase the number of bind-
ing sites from e20 to p35, and partially decrease thereafter. The other cortical layers stop
the increment of their binding sites by p21 and decrease slightly thereafter. in the occipital
cortex, the putative m2 binding sites are still very low at p7; from p7 to p60 the density
of binding sites increases steadily in all layers (7). a different temporal development of
muscarinic receptor subtypes was described in mouse forebrain by using subtype specific
antibodies. in this case, while m2, as well as m1, immunoreactivity reaches a staining pat-
tern characteristic of the adult by p14, the intensity of the cortical immunoreactivity for m2
continues to increase until p30 (77). despite these discrepancies, if we consider that, the m2
receptor is assumed to be mainly an autoreceptor at cholinergic presynaptic terminals (105),
the difference in the pace of temporal maturation between m1 and m2 receptors subtypes
could be particularly meaningful.
N. Origlia et al.
altogether the reported results indicate that the different machrs subtypes develop
precise ontogenetic profiles resulting in distinct distribution patterns throughout the differ-
ent cortical layers. additionally, results obtained using electron microscopy suggest that
different machrs are expressed in different subcellular districts (dendrites, cell bodies and
axons) of single cortical neurons with inter-areal differences in cell expression at the level
of primary and secondary visual cortices.
f u n c T i o n o f T h e c h o l i n e r g i c s y s T e m
The activity of the cholinergic system affects several aspects of neuronal function spread-
ing from learning and memory to arousal, attention, plasticity of sensory maps and modula-
tion of neuronal electrophysiological properties (21, 158, 104, 47, 154 146, 33, 29, 109).
in particular damage of the cholinergic BfB can result in global cognitive impairment. for
instance, aneurysms of the anterior communicating artery that injure the basal forebrain
are associated with amnesia and impairment of executive function in humans (34, 40, 1).
moreover, pathological conditions that determine cognitive deficits such alzheimer’s dis-
ease are characterized by alterations in the BfB and the severity of the cognitive deficits
are related to the extent of cholinergic neurons’ impairment in this brain area (138, 13). in
addition, experimental approaches designed to interfere with cholinergic transmission such
as blocking muscarinic receptors with specific antagonist (31, 156, 4), lesions of cholin-
ergic afferent fibres (38) or selective lesion of the BfB cholinergic neurons with the 192
igg-saporin immunotoxin, have confirmed the importance of cholinergic transmission in
memory, attention (104, 116, 169) and cortical synaptic plasticity (98).
The investigations on the physiological action of ach in the cns support the idea that
the cholinergic system influences neuronal functions by exerting a modulatory action at
three different levels: a) neural excitability b) synaptic transmission c) neuronal plasticity.
The following part of this review will be focused on ach action when mediated by mus-
1. Cholinergic modulation of neuronal excitability via muscarinic receptors.
ach, activating machrs, acts as a potent regulator of neuronal activity in many classes
of mammalian neurons. at the neuronal level, ach modifies post-synaptic conductance
resulting in a modulation of intrinsic excitability. in particular, application of ach on
hippocampal and cortical neurons determines membrane depolarization, an increase in
membrane resistance and an increase in the firing rate of the cells (95, 112). for example,
muscarinic activation in neocortical pyramidal cells through a reduction in a slow ca(2 +)-
activated K+ current iahp, and/or a voltage-dependent K+ current, im, results in a decrease
in spike frequency adaptation and increased responsiveness to depolarizing inputs (111).
all these excitatory effects are mediated through machrs and are due to the reduction of
at least three types of potassium conductances:
1) the low-threshold slowly-activating, and non-inactivating KcnQ/m-current potas-
sium channels (15, 170) that is a voltage-dependent outward potassium current activated by
small depolarization close to the firing threshold; this current promotes membrane hyperpo-
larization following a spike episode (96);
the rOle Of chOliNergic system iN NeurONal plasticity
2) m1 activation reduces constitutively active inwardly rectifying K(+)(Kir2) channel
currents in prefrontal cortex (pfc) pyramidal neurons. reduction of Kir2 channel currents
by m1 receptor stimulation significantly increases the temporal summation of excitatory
synaptic potentials (epsps) evoked by repetitive stimulation of layer i. This action was
complemented by m2/4 receptor mediated presynaptic inhibition of the same terminals. as a
consequence of this dual modulation, the responses to a single, isolated afferent volley was
reduced, but the response to a high-frequency afferent burst was potentiated (20);
3) machrs in rat cortex regulate different calcium and voltage-sensitive nonselective
cation currents. These currents could represent an important mechanism through which
ach can regulate neuronal excitability in prefrontal cortex (69).
what would be the physiological impact of these two different calcium-activated non
selective cation currents in neurons? activation of ifadp (fast decay inward after current)
induces a fast transient depolarization probably involved in the initial phasic firing pattern
of layer v pyramidal neurons of prefrontal cortex. in contrast, activation of isadp (slower
inward after current) leads to a long-lasting sadp that can induce sustained spiking activ-
ity. under machr activation these currents may act synergistically to increase neuronal
excitability in response to afferent stimuli (70, 52, 93).
2. Muscarinic modulation of synaptic transmission.
ach has proven to influence synaptic transmission of excitatory and inhibitory cortical
neurons. This topic has been widely treated by several research groups. In vivo experi-
ments conducted in the primary visual cortex tested the effects of iontophoretic application
of ach on responses elicited by visual or electrical stimulation of the lgn. it was found
that the response was facilitated in 74% of the cells while it was depressed in 16%, in both
cases through machr activation (147; see also ). also, in the primary somato-sensory
cortex, iontophoretic application of ach mainly enhanced the response of single cortical
neurons to whisker stimulation (43). response enhancement was observed both in supra-
and infra-granular layers, whereas response suppression was commonly observed in layer
iv. studies conducted in brain slices or in cultured cells showed much more heterogeneous
and not univocal results. for instance, cox et al. (30) using single cell recordings from in
vitro auditory cortex observed an increase of post-synaptic response elicited in neocortical
neurons by puff application of glutamate in the presence of ach; the authors showed that
this enhancement was mediated by machrs. a similar result was obtained by calabresi
et al. (17) in the striatum showing selective nmda enhancement after muscarine appli-
cation. in contrast, vidal and changeux (166) reported that iontophoretic application of
muscarinic agonists (muscarine and acetyl-α-methylcholine) in prefrontal cortex slices,
decreased epsps amplitude through a pre-synaptic action. Kimura and Baughman (90),
using monosynaptically connected cortical cells from cultured neurons, investigated the
modulatory action of ach on both excitatory (epsps) and inhibitory (ipsps) post-synaptic
potentials. Their results showed an ach dose-dependent suppression of transmission in
both types of synapses that was mediated by machrs. The suppression of epsps was
obtained with low concentrations of ach through the activations of m4 machr while the
suppression of ipsps was mediated by m1 machr; in both cases, the action of ach was
pre-synaptic. These data obtained in cell cultures can be coupled with observations made
by several groups in different cortical areas, which underline the importance of ach con-
N. Origlia et al.
centration, the type of cholinergic agonist utilized and the source of connections activating
cholinoceptive areas in determining the sign of cholinergic action on synaptic transmission
(read facilitation versus inhibition). for example, gil and colleagues (62) tested the effects
of cholinergic agonists on synaptic transmission elicited by the electrical stimulation of
two separate pathways: the intracortical and thalamo-cortical pathway in somatosensory
cortex slices. interestingly, they found a selective facilitation of epsps of thalamo-cortical
synapses by nicotine while both pathways were depressed by bath application of muscarine.
remarkably, a low concentration of ach enhanced selectively thalamo-cortical synapses
similarly to nicotine. an input-dependent specificity was also reported in entorhinal cortex
by hasselmo and Bower (73). in particular, bath application of the cholinergic agonist
carbachol strongly reduced the amplitude of field potentials and single cell responses in
layer i neurons elicited by stimulation of intrinsic fibres. in contrast, the responses elic-
ited by stimulation of extracortical afferent fibres were almost unchanged by carbachol.
depression of intracortical synaptic transmission in entorhinal cortex was also observed in
layer ii/iii and layer v after bath application of carbachol (179, 27). similar effects were
induced by bath application of muscarine or ach in the presence of the ache inhibitor in
somatosensory cortex slices (74). recent data on entorhinal cortex using either an in vivo
or an in vitro approach showed that cholinergic activation reduced the excitatory synaptic
strength of inputs from pyriform cortex (71). altogether the reported results permitted to
raise the idea that intracortical connectivity, both at the level of gaBaergic and gluta-
matergic synapses, are suppressed by muscarinic receptors, in contrast with the facilita-
tory action of ach on thalamo-cortical synapses via pre-synaptic nicotinic receptors (89).
however, recent results by our group (97, 98) indicated a more complex scenario where
different concentrations of ach may facilitate or inhibit excitatory synaptic transmission
in the primary visual cortex by acting on different machrs subunits. in particular, a low
concentration of exogenously applied ach induced facilitation of synaptic transmission by
activating m4 and m2 receptors while high ach concentrations inhibited synaptic transmis-
sion acting on multiple machrs. interestingly, the dose of ach inducing facilitation or
inhibition changes as a function of the stimulated intracortical pathway (source) and the
local expression of cholinesterases (ches) [particularly the acetylcholinesterase (ache),
the enzyme determining the breakdown of acetylcholine and the termination of cholinergic
action]. indeed, inhibition of ches or the use of cholinergic agonists, which are insensitive
to che action such as muscarine and carbachol induce a predominant inhibitory effect on
synaptic transmission. we raised the idea that the degree of tonic ach release in cortical
areas should modulate the flow of information by differently influencing the responses to
sensory stimuli in layers primarily responding to extracortical inputs (read layer iv) and
in layers involved in the response refinement (layer ii-iii). additionally, local low/high
ach levels possibly acting through the activation of different machrs sub-types, and the
type of activated cortical connectivity would determine the sign of cholinergic modulation
(facilitation versus inhibition).
Thus, in a more general model ach tonic action does not simply result in facilitation or
depression of synaptic transmission but rather regulates the balance between facilitation and
inhibition of afferent (read thalamic) and intrinsic cortical inputs depending on local con-
centration of ach, the subtypes of activated machrs and the local expression of ches.
the rOle Of chOliNergic system iN NeurONal plasticity
also the activation of nicotinic receptors is capable of modulating cortical responses
mainly by enhancing glutamatergic transmission of thalamic fibers as shown in the soma-
tosensory and prefrontal cortex (126, 100). Thus, the mechanism is similar to that displayed
by muscarinic activation obtained by low ach. however, the difference is that nicotinic
responses are fast, short-lasting (< 100 msec) and rapidly desensitizing, while muscarinic
responses are slow, long lasting (> 100 msec) and more resistant to desensitization. These
characteristics place machrs in a pivotal position to integrate or suppress inputs from dif-
ferent sources (intrinsic versus extrinsic) in different behavioral states and then influencing
visual cortical function and/or high cognitive functions. in agreement with this idea, in
different brain functional studies furey and colleagues (53, 58, 59, 60) showed that cholin-
ergic enhancement induced by inhibition of ache increased the responses in visual cortical
areas during working memory tests in young and older healthy adults.
what is the holistic reason why ach modulates visual responses and what would be its
influence on perception, attention and/or memory? one possibility is that ach would par-
ticipate directly to the discrimination of the visual stimulus by increasing the selectivity of
cortical visual neurons depending on the characteristics of sensory stimuli (for example, ach
increases the signal-to-noise ratio and the selectivity for stimulus orientation in the visual
cortex; [155, 148, 183]). following this hypothesis ach may contribute to sculpt the visual
stimuli respect to the background and in this way facilitating attentive and learning process.
a corollary hypothesis is that ach would increase the attention for novel stimuli versus
attended stimuli by modulating the encoding versus retrieval processes in visual corti-
cal areas. in this context, a cholinergic enhancement should affect the encoding or the
retrieval process, depending on how strong and, possibly, how long the activation of local
cholinergic projections would be capable of modulating glutamatergic and/or gaBaergic
transmission in different cortical circuitries. facilitation of extrinsic input coupled to sup-
pression of intrinsic inputs by ach in visual cortex as well as in other cortical areas such
as the prefrontal, perirhinal and entorhinal cortex would enhance encoding of new visual
information. This possibility implicitly assumes that modulation of sensory perception by
ach in sensory cortical areas represents the first step in the process of redistributing neural
activity within visual cortex and other cortical areas with the aim of enhancing attention,
learning and memory.
3. Cholinergic system and synaptic plasticity.
different approaches have been used to show the involvement of the cholinergic system
on neuronal plasticity in several brain regions. in the neocortex, homogeneous results were
reported in assigning a relevant role to cholinergic innervations in modulating plastic phe-
nomena. depletion of cholinergic afferents to the somatosensory cortex of cats prevented
the expansion of topographic maps that normally occurs after removal of one digit (85).
moreover, selective damage of the cholinergic cells of the BfB induced by 192 igg-saporin
leads to reduced neuronal activity-dependent plasticity (9, 182) and retards the development
of the barrel cortex (181). similarly, BfB cholinergic neurons modulate the reorganization
of the sensory map in auditory cortex (88).
The primary visual cortex represents the most studied area for investigating mechanisms
underlying neuronal activity-dependent synaptic plasticity. manipulation of binocular
N. Origlia et al.
vision during a restricted time-window of early postnatal development (critical period),
causes a dramatic change in visual cortex connectivity (80, 81). These pioneer studies
conducted in kittens demonstrated that closing one of the eyelids for several days or weeks
(monocular deprivation, md) during the critical period shifts the ocular dominance distri-
bution of neurons in visual cortex towards the undeprived eye. in other words, most of the
neurons are driven by the stimulation of the open eye. This is particularly evident in animals
that present a clear anatomical organization in ocular dominance columns such as monkeys,
cats and ferrets. md has maximal effects when performed during the critical period, which
corresponds to first postnatal period but differs in length and starting point according to the
mammalian species. The degree of irreversibility of md effects varies in different species
being maximal in monkeys, cats, ferrets and rats and low in mice. sensory experience is
a strong determinant for the duration of critical periods and the lack of visual experience
(dark rearing) prolongs critical periods for monocular deprivation.
The combined destruction of the cortical noradrenergic and cholinergic innervations
reduces the shift in ocular dominance induced by monocular deprivation during the critical
period, although the alternate lesion of either system is ineffective (11). however, pharma-
cological blockade of cholinergic transmission by muscarinic antagonists infused into the
visual cortex is by itself sufficient to suppress ocular dominance changes (68).
in order to study the cellular and biochemical mechanisms underlying brain plasticity
several investigators addressed their efforts to settle experimental protocols inducing stable,
reliable and consistent neuronal changes both in vivo and in vitro. The efficacy of synaptic
transmission can be regulated over a wide range of temporal scales, ranging from millisec-
onds, to minutes and hours (short-term plasticity, long-term plasticity). concerning long
lasting modifications, two forms of synaptic plasticity can be experimentally induced: long-
term potentiation (lTp) and long-term depression (lTd). lTp and lTd can be considered
part of experience-dependent synaptic plasticity that endows the brain with an ongoing
ability to accommodate to a dynamically changing environment.
The expression of long term synaptic plasticity (lTp and lTd) has been extensively
studied in the rat visual cortex showing that the critical period for the induction of lTp
evoked by stimulation of thalamo-cortical connections almost overlaps the duration of
the critical period for ocular dominance (91). This feature is much less evident in the
mouse visual cortex. in addition to the thalamo-cortical connections lTp and lTd can be
elicited by stimulation of intrinsic connections both during postnatal development and in
adulthood. The different forms of lTp and lTd expressed in different cortical layers and
elicited by the stimulation of different cortical pathways are only in part nmda-depend-
ent (142, 19).
Both, lTp and lTd in the visual cortex appear to be modulated by the cholinergic sys-
tem. Brocher et al. (14) showed that lTp in layer ii/iii cells was facilitated by concomitant
application of muscarinic and noradrenergic agonists. however, no effects were observed
when each of them was administrated individually. on the other hand, pesavento et al.
(140) showed that a simple application of ach in slices containing the visual cortex was
sufficient to induce lTp through stimulation of machrs after the end of the critical period.
successively, pesavento et al. (139) and origlia et al. (127) using a transgenic mouse that
produces antibodies against ngf and displays reduced cortical cholinergic innervation,
the rOle Of chOliNergic system iN NeurONal plasticity
found an impaired lTp in visual cortex slices. exogenous application of ach, however,
rescued lTp suggesting an essential role of this neurotransmitter in cortical synaptic plastic-
ity. in agreement with these results Kuczewski et al. (97, 98) showed that the immunolesion
of BfB cholinergic neurons with igg-192 saporin was able to impair lTp in visual cortex.
a biphasic action of the cholinergic system on cortical synaptic plasticity was suggested by
Kirkwood et al. (92) who reported that, in visual cortex, also lTd is favored by cholinergic
agonists such as carbachol. origlia et al. (128) showed that nmda-dependent lTp and
lTd rely on the activation of different machrs in the primary visual cortex. indeed, using
single and double muscarinic receptor knock-out mice, they demonstrated that a normal
lTp was expressed when m2 and m4 can be co-activated while lTd relays more on m1 and
m3 receptor in agreement with previous results obtained by choi et al. (28).
altogether these results in visual cortex slices suggest that the direction of synaptic plas-
ticity depends on the combined activity of different machrs (see figure 1 for a drawing
of machrs and their intracellular pathways of signal transduction involved in nmdar-
dependent forms of long term synaptic plasticity). an intriguing possibility is that ach,
by acting on different machr subtypes, would regulate the Bienenstock-cooper-munro
(Bmc) threshold for synaptic modification. according to the Bmc model, a given synaptic
stimulus may produce either lTp or lTd depending on whether or not it is able to over-
come a certain modification threshold (θm; reviewed by ).
fig. 1. - drawing of machrs and their intracellular pathways of signal transduction involved in
nmdar-dependent forms of long term synaptic plasticity.
N. Origlia et al.
These results suggest that the cholinergic system through different machrs expressed in
visual cortex would be able to modulate different forms of experience-dependent synaptic
plasticity that are primarily driven by glutamate and nmdar. The reported results indicate
a clear relationship between ach modulation of visual cortex plasticity during the critical
period and the cholinergic influence on lTp and/or lTd.
recent work by Tsanov and manahan-vaughan (162) showed that lTp and lTd in the
visual cortex of freely moving adult rats, evoked by stimulation of lgn are influenced by
changes in luminance levels, as it occurs during diurnal cycle. in particular, acute 12 hour
light exposure leads to synaptic potentiation while acute dark rearing enhances synaptic
depression. in addition, short period of monocular deprivation was sufficient to induce lTp
following the stimulation of undeprived eye in adult mice (149). These data suggest that also
in the adulthood the visual cortex is in a dynamic state driven by visual experience. in agree-
ment with this idea, it is known that practicing certain visual tasks (perceptual learning) in
adulthood results in high visual performance suggesting plasticity of neuronal circuitries at
the cortical level (87, 57). remarkably, perceptual training using basic visual stimuli features
was able to improve visual functions in adult amblyopic patients that suffered from binocular
abnormalities during the critical period (141). however, it remains to be elucidated the role of
the cholinergic system and different cholinergic receptors in adult visual cortex plasticity.
c o n c l u s i o n s
changes in the activity of basal forebrain cholinergic nuclei coupled with activation of
different cholinergic receptors appear to be responsible for the fine modulation of cortical
responsiveness to visual inputs. in particular, acetylcholine local level acting through specific
muscarinic receptors subtypes is able to modulate the cortical flow of visual information. This
mechanism may play a key role in switching the cortex through different cognitive states
associated with high or low activation of the cholinergic system. moreover, the direction
of long term synaptic plasticity (lTp/lTd) involved in the formation and/or maintenance
of cortical visual maps depends on the combined activity of different muscarinic receptors.
Thus, using the visual system as a model we integrated information on pattern of cholinergic
projections, including their development and maturation, distribution of muscarinic receptors
and cholinesterases, local release of acetylcholine to define the cholinergic signature and the
functional role of acetylcholine on neuronal plasticity and cognitive functions.
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