<title>1. INTRODUCTION</title> <p>The
entorhinal cortex (Brodman area 28) derives its name from the fact that it is
partially enclosed by the rhinal (olfactory) sulcus. This feature is
particularly striking in nonprimate mammalian species, but also in primates at
least the anterior part of the entorhinal cortex is bordered laterally by a
rhinal sulcus. Interest in the entorhinal cortex arose around the turn of the 20th
century when Ramon y Cajal, in his seminal studies on the anatomy of the
nervous system, described a peculiar part of the posterior temporal cortex
which is strongly connected to the hippocampus with fibers that merge in the
angular bundle and perforate the subiculum. Cajal was so struck by this massive
connection that he suggested that the physiological significance of the
hippocampus had to be related to that of the entorhinal cortex. At that time,
he assumed that the entorhinal cortex was part of the olfactory cortex and so
was, therefore, the hippocampus. He even stated that if this part of the
posterior temporal cortex, which he called the sphenoidal cortex/angular
ganglion, would be visual, so would be the hippocampus [##UREF##0##1##]. How right he was,
in more than one way. Today we conceive the entorhinal cortex as the nodal
point between the hippocampal
formation on the one hand and a variety of multimodal association areas of the
cortex such as parietal, temporal, and prefrontal cortex on the other hand.
So, multimodal sensory and highly processed unimodal inputs converge at the level of
the entorhinal cortex. This input in turn is conveyed to the hippocampal
formation. We also know that the entorhinal cortex harbors different
subdivisions, which specifically mediate the connectivity with functionally
different sets of cortical and subcortical areas in the brain. This has led to
the now quite widely accepted concept of parallel input/output channels as
originally proposed by us and others [##REF##2682783##2##–##UREF##1##5##]. Recent
electrophysiological recordings in lateral and medial entorhinal cortices of the rat have
further elaborated on this point in showing that cells in the medial
subdivision are spatially modulated, whereas in the lateral entorhinal cortex
such modulation is largely absent [##REF##15961670##6##–##REF##15965463##9##]. Cells in the
lateral entorhinal cortex most likely convey olfactory [##UREF##1##5##, ##REF##7371730##10##, ##REF##7350993##11##] and somatosensory
information [##REF##10986344##12##–##REF##17607757##15##].</p> <p>Our current insights into the functional relevance of the hippocampal formation,
and how its anatomy is related to function, are much more detailed than what we
know about the entorhinal cortex. It therefore seems attractive to turn the
argument of Cajal around by stating that in view of the findings that the
hippocampus is crucially involved in conscious, declarative memory processes so
should be the entorhinal cortex. This conjecture is apparently supported by
available functional studies. Although the specific functional contributions of
the entorhinal cortex to memory remain to be established, they are most likely
different from, but complementary to, those of the hippocampus [##UREF##1##5##, ##REF##15664181##16##, ##REF##18215625##17##]. The
finding of the grid cells in medial entorhinal cortex, as well as head
direction and conjunctive cells, and the notion that converging inputs of a
limited number of grid cells onto a single CA1 neuron are sufficient to result in the well-established
place cell properties [##REF##18215625##17##–##REF##17094145##19##] kindled a
renewed interest in the anatomical organization of the entorhinal cortex [##REF##17069897##20##]. A review of the anatomical organization, as part of a special issue on
entorhinal cortex, is therefore appropriate. We aim to provide a comprehensive
description of the entorhinal cortex, with particular emphasis on the intrinsic
organization, based on data from studies in the rat, extensively referring to,
rather than repeating, previously published accounts.</p>
<p>Recommended by Roland S. G. Jones</p> <p>The entorhinal cortex is commonly perceived as a major input and output structure of the hippocampal formation, entertaining the role of the nodal point of cortico-hippocampal circuits. Superficial layers receive convergent cortical information, which is relayed to structures in the hippocampus, and hippocampal output reaches deep layers of entorhinal cortex, that project back to the cortex. The finding of the grid cells in all layers and reports on interactions between deep and superficial layers indicate that this rather simplistic perception may be at fault. Therefore, an integrative approach on the entorhinal cortex, that takes into account recent additions to our knowledge database on entorhinal connectivity, is timely. We argue that layers in entorhinal cortex show different functional characteristics most likely not on the basis of strikingly different inputs or outputs, but much more likely on the basis of differences in intrinsic organization, combined with very specific sets of inputs. Here, we aim to summarize recent anatomical data supporting the notion that the traditional description of the entorhinal cortex as a layered input-output structure for the hippocampal formation does not give the deserved credit to what this structure might be contributing to the overall functions of cortico-hippocampal networks.</p>
<title>2. DEFINITION OF THE ENTORHINAL CORTEX,
SUBDIVISIONS, AND OVERALL ARCHITECTURE</title> <p>A cortical area can be defined in
many different ways, using a variety of different criteria, such as location,
connectivity, cyto- and chemoarchitectonics. For the entorhinal cortex, all these
approaches have been applied, resulting in a confusing variety of borders,
subdivisions, and description of layers. A good lead, since it has withstood
over a century of arguments, is the definition of the entorhinal cortex on the
basis of its connectivity with the hippocampus as originally suggested by Cajal [##UREF##0##1##]. In view of increasing insights into the connectivity of the
hippocampal formation and its subdivisions, quite a few authors have chosen to
take projections to the dentate gyrus as a good defining criterion, in
particular in combination with certain cytoarchitectonic features. In this
paper, such a combined definition will be used and described below.</p> <p>The entorhinal cortex is surrounded by a number of cortical
areas. Anteriorly, it meets with olfactory and amygdaloid cortices, such as the
piriform (olfactory) cortex laterally, and medially it is bordered by the
periamygdaloid cortex and the posterior cortical nucleus of the amygdala. On
its medial side, the entorhinal cortex merges with structures that belong
either to the hippocampal formation or the parahippocampal region, such as the
amygdalo-hippocampal transition, and the parasubiculum. The lateral and
posterior borders are with the other two major constituents of the
parahippocampal region, the perirhinal cortex laterally and the parahippocampal
cortex (in nonprimate species generally referred to as postrhinal cortex)
posteriorly. The lateral and posterior borders are quite easy to establish on
the basis of a variety of cytoarchitectonic and chemoarchitectonic features.
The most prominent features are that the fairly large-sized cells of layer II
in the entorhinal cortex are replaced by much smaller neurons in the perirhinal
and postrhinal cortices, the lamina dissecans disappears, and these changes
coincide with similarly striking changes in the density of parvalbumin-positive
neuropil, high in entorhinal cortex, virtually absent in perirhinal and
parahippocampal areas. The mirror-image pattern appears when staining for heavy
metals (Timm stain) or the calcium binding protein calbindin. All additional
criteria that have been described seem to coincide with these borders. The
anterior and medial borders, in contrast, are somewhat harder to establish.
They apparently coincide with a rather striking change in the ease with which
layers II and III can be differentiated from each other as well as with a loss
of differentiation between the deep layers (medial border) or even complete
disappearance of the deep layers (anterior border). Combined with subtle
changes in chemoarchitectonic features and connectional differences, an overall
consensus has now been reached (for further details see [##REF##7541808##21##–##REF##12625464##25##]).</p> <p>Attempts to subdivide the entorhinal cortex have, likewise, been numerous (see [##UREF##3##26##], for a
detailed review cf. [##REF##2445795##27##]). Whereas Cajal, similar to Lorente de Nó did
not see much merit to subdivide the entorhinal cortex based on
cytoarchitectonic criteria [##UREF##4##28##, ##UREF##5##29##], it was Brodmann [##UREF##6##30##] who parcelled the
entorhinal cortex field 28 into two fields: a lateral area 28a, and a medial
area 28b on the basis of cytoarchitectonic criteria. Lorente de Nó [##UREF##5##29##] instead
argued that the projections to the hippocampal formation support to distinguish
between lateral, intermediate, and medial entorhinal subdivisions. The use of
these two fundamentally different approaches, connectivity versus architecture,
has continued till today, although a merged approach is now becoming accepted.
Cytoarchitectonic parcellation schemes are useful tools to describe
experimental data about connectivity and data on for example the distribution
of receptors [##REF##2682783##2##, ##REF##9136047##22##, ##REF##2445796##31##–##REF##10911886##33##]; they help to navigate through data. Connectionally based subdivision-schemes may better
serve our understanding of the possible functional contributions [##REF##10985279##34##]. In view of the strong implications of the human entorhinal cortex in a variety of brain diseases (see, e.g., [##REF##1336586##35##, ##REF##15312965##36##]), the development of animal models for such
diseases depends strongly on our capabilities to extrapolate the definition of
the entorhinal cortex from rodents to nonhuman and human primates. With this
aim in mind, combinations of the different approaches may lead to the most
detailed and reliable subdivision.</p> <p>A good start to subdivide the
entorhinal cortex is to use the entorhinal-to-dentate projection, which has
been documented in extensive detail in a variety of species. On the basis of the
terminal distribution of this projection in the rat and the mouse, it seems
plausible to divide the entorhinal cortex into two subareas, generally referred
to as the lateral and medial entorhinal cortices (LEC and MEC, resp.). These
areas roughly correspond to the description of Brodmann's areas 28a and b, respectively
[##REF##12625464##25##, ##UREF##6##30##, ##REF##1270625##37##, ##REF##17765711##38##]. In the monkey [##REF##2913203##39##], the terminal distribution of the
entorhinal-to-dentate projection does not provide such a clear criterion to
functionally subdivide the entorhinal cortex. However, a second connection,
which has been proposed to functionally subdivide the entorhinal cortex, is the
input of the presubiculum. In all nonprimate mammalian species studied so far,
including rat, guinea pig, and cat, the innervation of the entorhinal cortex by
presubicular fibers is restricted to a more caudal and dorsal portion, that
coincides with a cyto- and chemoarchitectonically well-defined area, now called
MEC [##REF##4220028##40##–##REF##8429124##44##]. Also in the
monkey, inputs from the presubiculum distribute to only a restricted posterior
portion of the entorhinal cortex ([##REF##6715582##45##, ##REF##2454246##46##]; Witter and Amaral, unpublished
observations), which may thus represent the homologue of MEC as defined in
nonprimates.</p> <p>A note of caution should be added
here: the choice for the terms lateral and medial entorhinal cortex is not
simply related to a particular anatomical position of these areas in relation
to the hippocampal formation and the rhinal fissure. In general, the lateral
area occupies a more rostrolateral position versus a more caudomedial position
for the medial area (see ##FIG##0##Figure 1##(a)).</p> <p>The lamination of the entorhinal
cortex generally is considered the prototype of the transition between the
three-layered allocortex and the six-layered neocortex [##UREF##3##26##]. The superficial
plexiform or molecular layer (layer I) is relatively free of neurons and, in
general, contains a dense band of transversely oriented fibers. The outermost
cell layer (layer II) varies
considerably in appearance among the rostro-to-caudal and lateral-to-medial
extent, but mainly contains so-called “stellate” or “modified pyramidal cells.”
Overall, cells in layer II are fairly large, making them distinctly different
from layer II cells in the adjacent cortical regions with the exception of the
parasubiculum. In the latter area, neurons of layer II are as large as or
somewhat larger than those of the entorhinal cortex, but entorhinal cells stain
darker with a Nissl stain. Layer III is
a wide layer of loosely arranged, large to medium sized cells that are
predominantly of the pyramidal type. The deep border of layer III is the
cell-sparse fiber layer called the lamina dissecans (sometimes referred to as
layer IV). The lamina dissecans is better developed in the medial entorhinal
cortex although species differences are apparent. The next cell layer (layer V)
is clearly stratified and sometimes subdivided into a superficial layer of
large to medium-sized, darkly stained pyramidal cells, which is sometimes
referred to as layer Va. Note that in some lamination schemes, more
particularly so in primates, this layer is referred to as layer IV thus
resulting in some confusion when compared to the present scheme where the lamina
dissecans is referred to layer IV. Subsequent deeper portions of layer V (layer
Vb/Vc) have an overall stratified appearance and mainly consist of rather small
pyramidal cells with a moderately dense packing. In the deepest cell layer VI,
which is delineated by the white matter, multiple layers can be distinguished,
more in particular in primates. However, since the appearance of layer VI is
highly variable at different lateromedial and rostrocaudal levels, generally no
further differentiation between sublayers is made.</p> <title>3. EXTRINSIC CONNECTIVITY</title> <title>3.1. Entorhinal hippocampal connectivity</title> <p>Entorhinal connections with the hippocampal formation in the rat have been comprehensively
described and reviewed in a number of recently published papers and reviews to
which the reader is referred for further details [##REF##2682783##2##, ##REF##16876886##47##–##REF##16344147##52##]. To summarize, all regions of the entorhinal cortex project to all parts of the hippocampal
formation, the dentate gyrus, fields CA3, CA2, CA1, and the subiculum. Overall,
entorhinal fibers synapse most often onto the dendrites of principal cells,
that is, on spines, where they form asymmetrical, excitatory synapses.
Entorhinal fibers also terminate on inhibitory interneurons, forming both
putative excitatory as well as inhibitory synapses with the dendrites of these
interneurons [##REF##17765711##38##, ##REF##16876886##47##, ##REF##16344147##52##–##REF##18000818##54##]. In the
dentate gyrus, entorhinal axons distribute largely to the outer two-thirds of
the molecular layer, although differences between species may exist with
respect to the precise terminal distribution in relation to the origin of these
projections in either LEC or MEC [##REF##17765711##38##, ##REF##1713237##55##]. The projections to the dentate gyrus
arise largely from neurons in layer II. However, projections that arise from
the deep layers have been systematically observed, and it is likely that these
deep originating fibers show a differential terminal distribution, largely
innervating the inner molecular layer of the dentate gyrus ([##REF##8627369##56##]; see also
[##REF##17765711##38##]). The same cells in layer II also form the main origin of the projection
that distributes to the outer portions of stratum lacunosum-moleculare of CA3
and CA2 [##REF##17765711##38##, ##REF##18007015##49##]. In all species studied, the projections to CA1 and the
subiculum originate from cells in layer III of both LEC and MEC. The
terminations of the latter projections exhibit a transverse topography. The
rostral entorhinal cortex in the monkey and LEC in the rat project to the
region around the border between CA1 and subiculum (distal CA1, <italic>furthest away from the dentate gyrus</italic>,
and proximal subiculum, <italic>closest to the
dentate gyrus</italic>) whereas caudal entorhinal cortex in the monkey and MEC in
the rat project to proximal CA1 (<italic>close to
the dentate gyrus</italic>) and distal subiculum (<italic>far from the dentate gyrus</italic>) [##REF##1713237##55##, ##REF##8287110##57##, ##REF##8390320##58##].</p> <p>The CA1-subicular projections are topographically organized along the transverse or
proximodistal axis as well, such that parts of CA1 and subiculum that receive
comparable inputs that are either from LEC or MEC are connected to each other
[##REF##2329188##59##–##REF##11345131##61##]. Finally, the
projections from CA1 and subiculum back to deep layers of LEC or MEC grossly
reciprocate the forward projections [##REF##12454982##51##, ##REF##7538515##62##, ##REF##8395405##63##] (see Figures ##FIG##0##1##(a), ##FIG##0##1##(b)). These data
thus indicate that the entorhinal-CA1-subiculum circuitry exhibits a high
degree of fidelity, which suggests that this circuitry may permit a highly
ordered processing of information. The functional relevance of this strikingly
precise organization needs yet to be established. In this respect, it is of
interest that the CA1 and entorhinal projections targeting the same population
of subicular neurons do not seem to have a high incidence of convergence [##REF##17559098##64##].
In contrast, in CA1, inputs from CA3 and entorhinal cortex do converge on
pyramidal cells as well as onto interneurons with a very high incidence [##REF##18000818##54##]. A final point to make with respect
to entorhinal-hippocampal connectivity has to do with the topographical
organization along the long axis of the hippocampus. Although the orientation
of the hippocampus in various species is quite different [##REF##2682783##2##, ##UREF##7##65##] in all species
the structure has an impressive length, measuring from about 7 mm in mice,
through 9–11 mm in rats, up
to 4.5–5 cm in humans.
It has now been established that in all species studied, entorhinal-hippocampal
connectivity is present as described above, the striking difference being that
different portions along the long axis of the hippocampal formation are
connected to different bands of the entorhinal cortex. These bands are
differently distanced from the lateral and posterior borders of the entorhinal
cortex with the adjacent perirhinal and postrhinal (rodent) or parahippocampal
(primates) cortices (see Figures ##FIG##0##1##(c), ##FIG##0##1##(d)). In the rat, this longitudinal
topography has been shown to be closely related to the spatial properties of
neurons in both structures. Neurons in the dorsal hippocampus and the related
dorsal portions of entorhinal cortex, more in particular of the medial
entorhinal cortex, exhibit firing patterns that, although qualitatively as well
as quantitatively very different, both represent fairly small areas of the
environment. In contrast, cells that are more ventrally positioned in both
hippocampus and entorhinal cortex
show much larger spatially tuned firing fields [##REF##15333832##8##, ##REF##15965463##9##, ##UREF##8##66##]. Interestingly, the
relationships in this respect between the dorsal to ventral axes both in the
entorhinal cortex and the hippocampus are reflected in a comparable
relationship with respect to the behavioral effects of selective lesions.
Whereas lesions in the dorsal hippocampus and dorsal part of the entorhinal
cortex have comparable detrimental effects on spatial learning and recall,
ventral lesions do not. The latter have a profound effect in fear related
behavior, which is in turn not effected by dorsal lesions [##REF##15664181##16##, ##REF##12149439##67##].</p> <title>3.2. Entorhinal cortical connectivity</title> <p>The
most comprehensive systematic series of studies on entorhinal connectivity in
the rat is from the Burwell lab [##REF##10911865##13##–##REF##17607757##15##, ##REF##9492202##68##–##REF##17604355##71##], with some added studies describing one or a few inputs or outputs in greater detail
[##REF##9136047##22##, ##REF##17598159##72##]. All these studies are in line with earlier influential reports in the
monkey that the perirhinal and parahippocampal cortices form the major cortical
link for the entorhinal cortex [##REF##1156867##73##, ##REF##1156859##74##]. In general, the perirhinal cortex
projects to rostrolateral parts of the entorhinal cortex, whereas the parahippocampal
(primates) or postrhinal (nonprimates) cortex projects preferentially to
caudodorsal portions of the entorhinal cortex [##REF##9261838##3##, ##REF##9492202##68##, ##REF##8126576##75##]. In addition to the
perirhinal/parahippocampal connections, the pre- and parasubiculum [##UREF##2##23##, ##REF##8429124##44##] and
olfactory-related structures provide prominent inputs to the entorhinal cortex
and, in case of olfactory domains, receive a similarly strong output from the
entorhinal cortex. As mentioned above, the spatial distribution of the input
from the presubiculum is currently considered to be one of the defining
features of MEC in different species. Though typically not as strong,
additional cortical afferents and efferents of the entorhinal cortex are
widespread. Cortical afferents are dominated by piriform input, but input also
arises in frontal, cingular, retrosplenial, insular, parietal, and even visual
areas. Similar to what was reported for the monkey, in the rat projections from
the cingulate and retrosplenial cortices preferentially project to the more
caudal portions of the lateral, intermediate, and medial bands of the
entorhinal cortex [##UREF##2##23##, ##REF##17598159##72##, ##REF##14515240##76##–##REF##17436282##78##]. Cortical efferents are widespread, largely reciprocating the cortical afferents. Note
that species differences are apparent indicating that whereas in the rat
cortico-entorhinal reciprocal connectivity is rather limited and confined to
the areas close to the rhinal sulcus [##REF##9136047##22##, ##REF##3020190##79##], such connections in the monkey
are more common and involve a more widespread domain of the entorhinal cortex [##REF##8126576##75##, ##REF##10985281##80##].</p> <p>Although
we will address the layered organization of the entorhinal cortex in more
detail below, it is relevant to point out that entorhinal-cortical projections
largely arise from deep layers, primarily from layer V pyramidal neurons. Possible
exceptions are the entorhinal-infralimbic and entorhinal-olfactory projections,
which appear to arise in layers II and III as well [##REF##9136047##22##, ##REF##7722001##81##]. Regarding entorhinal
afferents, it is clear that most show a distribution largely confined to the
superficial layers I–III with the exception of inputs from infralimbic, and
prelimbic areas together with cingular and retrosplenial inputs that show a
striking preference for deep layers of the entorhinal cortex [##REF##17598159##72##].</p> <title>3.3. Entorhinal subcortical connectivity</title> <p>Studies
conducted in multiple species indicate extensive subcortical connectivity for
the entorhinal cortex. Although differences exist with respect to the detail of
the information, it is safe to conclude that the entorhinal cortex has
connections with the basal forebrain, claustrum, amygdala, basal ganglia,
thalamus, hypothalamus, and brainstem (for review see [##UREF##9##70##]). The entorhinal
cortex sends projections to the nucleus accumbens [##REF##2350704##82##–##REF##7225802##85##] and receives inputs from the ventral tegmental area [##REF##679029##86##]. The entire entorhinal cortex has
strong reciprocal connections with the claustrum [##REF##3680636##32##, ##REF##3362351##87##–##REF##6725648##90##]. Additional
connections exist with basal forebrain structures, in particular the medial
septal nucleus, the nucleus of the diagonal band, and the substantia innominata
[##REF##3680636##32##, ##REF##679029##86##, ##REF##6538106##91##–##REF##3552118##93##].
It is most likely that entorhinal projections to basal forebrain structures
arise in layers II and V.</p> <p>Entorhinal-amygdala connectivity has been
studied in rather detail in both monkey and rat. For recent reviews, the reader
is referred to McDonald [##REF##9643556##94##], Pitkänen et al. [##REF##10911886##33##]; see also Burwell and Witter [##UREF##9##70##]. Although
all parts of the entorhinal cortex are connected with the amygdala, the rostral
subfields are more strongly interconnected with the amygdala than the caudal
subfields. Whereas in monkey the primary connections are with the lateral and
accessory basal nuclei [##REF##10813772##95##, ##REF##12000118##96##], in rat the most prominent inputs arise from
the lateral, basal, and accessory basal nuclei [##REF##9886046##97##]. Amygdala input terminates primarily
in layer III of the entorhinal cortex, and the return projection originates
predominantly from cells in layer V.</p> <p>The
entorhinal cortex is connected with thalamic and hypothalamic structures. Major
thalamic input arises in midline nuclei, particularly the reuniens, paratenial,
and periventricular nuclei [##REF##2445796##31##, ##REF##679029##86##, ##REF##3512627##98##–##REF##12106472##100##]. Additional
but weaker inputs have been described from the anteromedial thalamic nucleus
[##REF##10407123##101##], and the ventromedial nucleus of the hypothalamus [##REF##7814684##102##]. In the rat, it has
been shown that the entorhinal cortex reciprocates the reuniens input [##REF##7514701##103##]. In
the monkey, additional projections have been reported to end in the
magnocellular portion of dorsal medial nucleus, the medial pulvinar, and the
dorsolateral nucleus [##REF##3512627##98##, ##REF##3549796##104##]. The entorhinal cortex also receives input from
midbrain structures such as the dorsal raphe nucleus, the median raphe, and
locus coeruleus [##REF##679029##86##, ##REF##10235645##105##, ##REF##7914799##106##]. Details about entorhinal innervations from
these important modulatory regions of the brain are not available yet.</p> <title>4. INTRINSIC ORGANIZATION OF
THE ENTORHINAL CORTEX</title> <p>Our understanding of the entorhinal cortex is
still rather premature, and to a large extent, influenced by our current
functional concept for MEC. The generally accepted division of the entorhinal
cortex into at least two functionally different domains stresses the need for
an answer to the questions whether or not they differ with respect to their intrinsic
wiring and neuronal makeup, in addition to their gross differences with respect
to cortical and subcortical connectivity summarized above. The entorhinal
network, <italic>grosso modo</italic>, encompasses
three different (groups of) elements, elements receiving inputs, elements that
provide output, and elements that contribute to the intrinsic architecture of
the area. This subdivision into three functionally different elements and roles
to be played by different neurons does not necessarily have an exclusive
character; it is actually quite likely that all three elements might be an
integral part of one and the same neuron; however, specializations may occur.</p> <p>Compared to the details known for the
hippocampal formation and some parts of the neocortex, such as the visual or
barrel cortices in rodents, our understanding of the entorhinal cortex is
rather in its infancy. The first detailed description of the morphology of
entorhinal neurons, based on Golgi impregnated material, was published in 1933
by Lorente de Nó [##UREF##5##29##]. Over the years, this initial description has been
extended, adding details and new cell types, based on a variety of different
techniques. Here, we will summarize the main cell types that are currently
known with a focus on their local connectivity and in particular addressing the
question whether or not the lateral and medial subdivisions differ with respect
to the overall main cell types. We will summarize (see ##FIG##1##Figure 2##) data from
previously published reviews [##UREF##10##107##] supplemented with some recently obtained,
yet unpublished own data.</p> <title>4.1. Cell types in entorhinal cortex</title> <p>
<statement id="head1"><title>Layer I</title><p>In layer I throughout the entorhinal cortex, Lorente de Nó
described two cell types; horizontal cells and short axis cylinder cells,
nowadays known as multipolar neurons (MPNs). This latter category constitutes
the majority of cells in layer I, and generally, they are non- or sparsely spiny.
MPNs are quite often positive for calretinin (CR) and GABAergic, and two types
have been described. Small CR positive MPNs are more often located just deep to
layer Ia [##REF##10954838##108##], whereas CR positive MPNs with a laterally extending dendritic
tree are mainly located deep in layer I [##REF##10954838##108##].
From the perikaryon of MPNs three to five short, curved smooth dendrites arise
that branch after a short distance and radiate within layer I, sometimes
extending into layer II [##REF##10954838##108##, ##REF##9034897##109##]. The diameter of the dendritic tree is
around 100 <italic>μ</italic>m in small or 150 <italic>μ</italic>m in the other MPNs, respectively. Own recent data
indicate that the axons of layer I neurons travel towards layer II and III [##UREF##11##110##]
where they most likely provide feed-forward inhibition to principal cells [##REF##3902147##111##, ##REF##3249220##112##].
A minority of CR positive layer I neurons can be glutamatergic or contain
calbindin D28K (CB) or neuropeptide-Y (NPY) [##REF##10706219##113##].</p><p>Horizontal cells
are located in the transitional zone between layers I and II [##UREF##5##29##, ##REF##2771045##114##, ##REF##2319007##115##] .
They have a spherical to elongated soma of 13–15 <italic>μ</italic>m. Almost
spine-free dendrites extend laterally and spread horizontally within layer I
and superficial layer II. The horizontal extent can be up to 700 <italic>μ</italic>m (own
unpublished data). The noncollateralizing axon travels towards the deep layers
to the hippocampus [##UREF##11##110##, ##REF##2771045##114##, ##REF##2319007##115##]. Horizontal neurons are GABAergic, in LEC
some are positive for vasoactive intestinal polypeptide (VIP), whereas in MEC,
the dendritic terminals can stain positive for cholecystokinin (CCK) [##REF##2319007##115##–##REF##3514690##117##].</p></statement>
</p> <p>
<statement id="head3"><title>Layer II</title><p>Layer II is mainly made up of densely packed, large and
medium sized pyramidal and stellate cells. The most abundant cell type
throughout layer II in MEC is the stellate cell, with their preferred location
within superficial and middle layer II [##REF##9347353##118##]. The soma of these cells is quite
variable but their spiny dendritic tree is their defining characteristic. The
dendritic arbor comprises multiple, roughly equally sized primary dendrites
that branch widely (average extend of 497 ± 154 <italic>μ</italic>m) and may cover about one half of the
mediolateral extend of the MEC [##REF##9347353##118##–##REF##7689647##121##]. After
reaching the pial surface, the dendrites curve and run parallel to it. The
basal dendritic extent is smaller (average of 231 ± 69 <italic>μ</italic>m) [##REF##9347353##118##]. The relative
thick axon of stellate cells courses straight towards the angular bundle from a
primary dendrite or the base of the soma [##REF##1713171##120##].
Up to 400 <italic>μ</italic>m away from the start, the axon gives off very thin collaterals,
branching repeatedly and reaching the superficial layers, forming a net that
colocalizes with the entire dendritic tree, sometimes extending beyond [##REF##9347353##118##].
Besides, the axon sends occasional collaterals into deep layers III–VI. In the
angular bundle, it gives off one to three collaterals that travel into the
subiculum, continuing to their main targets in the dentate gyrus and CA3 [##REF##8269038##122##].
Most stellate cells are excitatory presumably using glutamate as their main
transmitter [##REF##2884019##123##–##REF##22816##126##] and some
also stain positively for CB [##UREF##10##107##].</p><p>Stellate cells are less common in LEC than in MEC. In LEC, stellate cells are most
likely replaced by a comparable cell type, called fan cells [##REF##1713171##120##, ##REF##16127693##127##]. They
have large polygonal somata with multiple thick sparsely spiny primary
dendrites that fan out from the soma mostly in the horizontal and ascending
direction. This dendritic morphology is thus comparable to that of stellate
cells in MEC. The morphological difference is that fan cells only have small
descending dendrites but there are also physiological differences. The axons
descend and can be followed into the angular bundle, sometimes giving of very
thin ascending collaterals within layers II and III [##REF##16127693##127##].</p><p>Aside from the stellate-like principal neurons, layer II contains a number of
pyramidal-like cells that have medium sized triangular or ovoid shaped soma
with a perpendicular elongation with respect to the pial surface. Most are
located in the deep portion of layer II [##UREF##11##110##, ##REF##8395571##128##, ##REF##9114238##129##]. The majority of these
cells have a prominent spiny thick apical dendrite branching at, or superficial
to the border with layer I. The basal dendrites of all pyramidal types are
spiny, thin, short and straight, with extensive branches within the most
superficial portion of layer III. The maximal mediolateral expanse of the upper
and lower dendritic fields in MEC is around 184 ± 75 <italic>μ</italic>m and is therefore
smaller than that of stellate cells. The smooth and thin axons of the
pyramidal-like cells originate from the soma, some follow a sinusoidal route within
layers II and III, giving off collaterals that distribute in layers I–III [##REF##9114238##129##] with an
extend that can be larger than that of the dendritic tree (own unpublished
data, Alonso et al., 1993). The distribution of the collaterals is comparable to
that of stellate cells, but less profuse [##REF##9347353##118##, ##REF##8395571##128##]. Subtypes of pyramidal-like
cells have been described including neurons with an obliquely oriented soma and
dendrites, called horizontal pyramidal neurons that are mainly located in the
superficial part of layer II [##REF##8269038##122##].</p><p>Another pyramidal
cell type described in LEC has a very thick and sparsely or nonspiny apical
dendrite, which branches in layer II. Thin apical dendritic tufts reach layer
I. The apical dendrite is not as frequently tilted as in MEC pyramidal neurons
[##REF##16127693##127##]. The neurons have thin sparsely spiny basal dendrites and an axon that
has extensive collaterals within layers I–III with many
varicosities. The main axon of these cells cannot always be followed until the
angular bundle but only up to layer III [##REF##16127693##127##, ##REF##7478170##130##].</p><p>Interneurons within layer II are described as MPNs, bipolar, basket, and chandelier cells.
MPNs have polygonal, fusiform, or round cell bodies with multiple, sparsely
spiny dendrites, extending in all directions, reaching layer I and deep into
layer III. It has been described that the axons of MPNs travel to the white
matter but also form local synapses within layer II [##REF##16127693##127##, ##REF##2026788##131##]. Morphologically
they seem to be comparable to stellate cells within the MEC but there are electrophysiological
differences. The family of MPNs contains VIP, substance-P, CCK, SOM, ENK, or
GABA and in the LEC also NPY [##REF##3514690##117##]. The short-axis cylinder cells in layer III
described by Lorento de Nó are comparable to these MPNs [##UREF##5##29##].
</p><p>Sparsely
spiny horizontal bipolar cells although considered to be local/interneurons
project to the hippocampus [##REF##2319007##115##, ##REF##7286122##119##, ##REF##2026788##131##]. The soma is located in layer II at
the border to layer I. The dendrites are oriented horizontally along the border
between layers I and II [##REF##2026788##131##]. Vertically orientated bipolar cells have a
spindle shaped perikaryon continuing into one smooth thin ascending and one descending
primary dendrite that branch into thinner dendrites more distally [##REF##10954838##108##]. CR,
VIP, and the corticotrophin releasing factor (CRF) have been found in
subpopulations of bipolar cells. In the LEC also ENK, CCK and NPY might be present
in this class of neurons [##REF##2319007##115##, ##REF##3514690##117##, ##REF##7286122##119##, ##REF##2026788##131##].</p><p>Fast
spiking basket-like cells have small spherical cell bodies with sparsely spiny
dendrites that often ramify into layer I. The extensive axonal arbor is mainly
confined to layer II. They form basket-like complexes mainly around the soma of
other cells, preferably forming symmetric, inhibitory synapses with stellate or pyramidal cells [##REF##3514690##117##, ##REF##8469376##132##]. Basket
cells are known to contain GAD and maybe CCK. Throughout the EC, PV positive
axons have been found that form symmetric synapses with principal neurons in
layers II and III. These terminals have a basket-like axosomatic configuration.
Therefore, it is suggested that basket cells in the whole EC contain PV [##REF##3514690##117##, ##REF##8469376##132##, ##REF##7745443##133##].</p><p>Chandelier
or axo-axonic cells are characterized by vertical
aggregations of axonal boutons, called candles, which preferably are located
superficial to the cell body. The somata of chandelier cells are medium sized
with different shapes. The almost nonspiny, poorly ramifying dendrites
originate from the basal and apical poles of the somata, displaying a bipolar
or bitufted arbor that often stays within layer II/III. Vertical chandelier
cells that are restricted to MEC issue a vertically oriented axonal tree that
is around 200–300 <italic>μ</italic>m wide and
300–450 <italic>μ</italic>m high with the main axonal branch dividing into several
collaterals that form the characteristic vertical aggregations within the upper
portion of layer II/III [##REF##8276990##134##]. Horizontally organized chandelier cells are
located in the MEC and LEC, and their axonal plexi are smaller (250–350 <italic>μ</italic>m wide and
100–200 <italic>μ</italic>m high) than
that of vertically oriented chandelier cells. Chandelier cells are GABAergic,
often PV-positive and form symmetric contacts with
initial axon segments of principal cells [##REF##2470472##135##–##REF##922488##138##].</p></statement>
</p> <p>
<statement id="head2"><title>Layer III</title><p>MEC and LEC layer III
pyramidal neurons have comparable morphological as well as electrophysiological
characteristics [##UREF##4##28##, ##UREF##5##29##, ##REF##16127693##127##]. According to some authors, in MEC an anatomical
distinction can be made between spiny and nonspiny pyramidal cells [##REF##2771045##114##]. In
the LEC, only sparsely spinous pyramidal cells exist that belong to the spiny
pyramidal group [##REF##16127693##127##]. The somata of spiny pyramidal cells (SPCs), which are
located throughout layer III, give rise to a prominent apical dendrite that
bifurcates, become spiny afterwards, and branch extensively. The spiny basal
dendrites spread further in the horizontal direction 389 ± 36 <italic>μ</italic>m compared to the
vertical direction 203 ± 31 <italic>μ</italic>m, allowing for widespread local connectivity [##REF##9070741##139##].
Apical and basal dendrites together lead to a mean vertical extent of 410 ± 23 <italic>μ</italic>m and a horizontal extent of 312 ± 37 <italic>μ</italic>m. The main axon projects via the angular
bundle to the subiculum [##REF##9070741##139##]. Some axonal collaterals spread within layers III
and II but also in the lamina dissecans and layer V, occasionally with a broader
horizontal extent than the dendritic tree [##UREF##11##110##, ##REF##9070741##139##, ##REF##9749802##140##].</p><p>Nonspiny pyramidal
cells (NSPCs), also called type 2 cells [##REF##9070741##139##], have triangular to spherically shaped
somata of different sizes. The nonspiny apical dendrite that, compared to SPC,
branches significantly closer to the soma also branches frequently in the
superficial layers, finally reaching the pia [##REF##9070741##139##, ##REF##9330357##141##]. The vertical dendritic
extent of these neurons is comparable to that of SPCs, whereas the horizontal
extent, specifically of the basal dendrites, is less [##REF##9070741##139##]. NSPCs thus have a
more circular basal dendritic tree around the soma than SPC. The axons of NSPCs
travel towards the angular bundle. Collaterals leave the main axon close to the
soma and may remain within the corresponding cell layer and/or distribute over
all other layers of the entorhinal cortex [##REF##9070741##139##]. The collaterals, which travel
towards the superficial layers, sometimes form a net over the entire own dendritic
extent and occasionally extending over an even larger domain [##UREF##11##110##, ##REF##9070741##139##].</p><p>Layer III also
contains stellate cells, in particular in the upper part of the layer. The somata
of these neurons are elongated, polygonal, or spherical. Cells belonging to the
latter subgroup sometimes have evenly distributed spiny dendrites around the
somata, whereas others have one or two spiny basal dendrites and a variety of
ascending dendrites that branch in layer I. The axons reach the white matter,
and collaterals are formed in layer III and the lamina dissecans [##REF##2771045##114##].</p><p>Also located
within layer III are principal MPN somata. These MPNs are either small and spherical,
with laterally extending dendrites, or they are large. The largest MPNs are
located in the outer half of layer III of the LEC with a conspicuous spatial
lateral separation (500 <italic>μ</italic>m) between each cell body. The cell body of large MPNs
is 15–18 <italic>μ</italic>m in diameter
with multiple sparsely spiny dendrites that
elongate in all directions showing moderate branching. The thickest dendrites
face towards the superficial layers whereas the thinner ones radiate laterally
towards the deep layers. The axons of MPNs reach the hippocampus via the white
matter with collaterals distributed in the vicinity of the parent cell soma [##REF##2771045##114##].</p><p>Multipolar local
circuit neurons, mainly described in MEC, are characterized by wide-ranging
apical dendrites that reach the cortical surface, multiple compact basal
dendrites, and a prominent axonal arborization. The axon reaches layers I to
III but rarely extends into the lamina dissecans or superficial layer V [##REF##9070741##139##].
At least subgroups of MPNs contain GABA, CCK, SOM, substance-P and very rarely
SRIF, VIP or ENK [##REF##10706219##113##, ##REF##6137169##116##, ##REF##4073532##142##]. Another subgroup of inhibitory MPNs has
sparsely spiny dendrites that extend with their multipolar dendritic arbor
towards deep layers instead of superficial layers. In addition, these neurons have
an axon extending locally with some collaterals projecting to and reaching
layer I [##REF##16641241##143##].</p><p>Interneurons
resembling pyramidal cells, the so-called pyramidal looking interneurons (PLIs)
have also been described as Type 3-(Gloveli) or Type 1-(Kumar) cells [##REF##9070741##139##, ##REF##16641241##143##].
PLIs have a pyramidal shaped cell body and non-spiny basal and apical dendrites
branch extensively, forming a dense local network in superficial layers with a
circular appearance [##REF##9070741##139##]. The apical dendrites often do not reach the pia and
have a vertical dendritic extent of 347 ± 73 <italic>μ</italic>m and a horizontal extend of 269 ± 98 <italic>μ</italic>m. The basal dendrites extend horizontally comparable to that of the
apical dendrites. PLIs have a dense axonal plexus in the local vicinity
surrounding the cell body, and extending superficially into layer II [##REF##16641241##143##].</p><p>Bipolar
cells have been described in layer III of MEC and LEC. They have a spindle-like
perikaryon with one ascending and one descending smooth, thin and sometimes
long dendrite. The ascending dendritic collaterals traverse throughout layer
II, reaching layer I. The extent of the descending dendrites has not been
described yet. The axon arises from the primary descending dendrite and extends
into layer III and the lamina dissecans, deep to the parent cell body [##REF##10954838##108##]. At
least a subpopulation of bipolar cells is known to contain VIP or CR. The
latter are more common in LEC than in MEC [##REF##10954838##108##, ##REF##9034897##109##].</p></statement>
</p> <p>
<statement id="head4"><title>Lamina dissecans (layer IV)</title><p>Occasionally, pyramidal-shaped neurons are located in
the lamina dissecans, at the borders to layers III and V. These neurons have
the morphological and physiological properties of either layer III or layer V
pyramidal neurons, respectively (own unpublished data).</p><p>Furthermore, bipolar
cells, whose dendrites grow horizontally instead of vertically to the pial
surface, with axonal collaterals that can travel towards superficial layer III
and deep layers, have been found in the lamina dissecans (unpublished data). It
has been shown that bipolar cells might contain VIP, CCK, and CRF [##REF##3514690##117##].</p></statement>
</p> <p>
<statement id="head5"><title>Layer V</title><p>
There is no difference between layer V principal neurons
in LEC and MEC [##REF##12209840##144##, ##REF##10713573##145##]. The apical
dendrites as well as axon collaterals often travel towards superficial layers,
sometimes even reaching the pial surface. The basal dendritic tree spreads
mainly within deep layers. The main axon travels towards the angular bundle and
the subiculum [##REF##12209840##144##, ##REF##10713573##145##]. In general, layer V consists of large pyramidal cells
located immediately below the lamina dissecans, while the deeper part of layer
V contains smaller cells. The somata of larger pyramidal cells can have
different forms. Usually pyramidal formed somata are observed but sometimes
also star-shaped cell bodies can be seen. All large pyramidal cells have one
distinct large and spiny apical dendrite that often branches close to the soma
with the main dendrite reaching the pial surface after branching into a tuft in
superficial layers II and I. In case of large pyramidal neurons, spines occur
on the dendrites after the first or second bifurcation [##REF##1713171##120##]. The basal
dendrites are thinner compared to the apical dendrites and can extent profusely
in all directions within layers V and VI [##REF##11168548##146##]. Compared to large pyramidal
cells, small neurons have more basal dendrites that are also more densely
occupied with spines. These basal dendrites of these smaller cells also extent
further in the deep layers. The main axon of the pyramidal cells travels
towards the angular bundle, eventually reaching the dentate gyrus via the
subiculum [##REF##11168548##146##]. Collaterals of these axons also split within layer V, forming
collaterals which travel toward the lamina dissecans, reaching the vicinity of
the soma [##REF##12209840##144##, ##REF##10713573##145##]. Some collaterals also travel towards superficial layers
[##UREF##11##110##]. Some pyramidal cells in entorhinal cortex layer V contain SOM [##REF##3514690##117##].</p><p>A second
principal cell type described in layer V is generally referred to as a type of horizontal
cell [##UREF##5##29##, ##REF##1713171##120##, ##REF##12209840##144##, ##REF##10713573##145##]. Somata of these cells are polygonal rather than
pyramidal in shape. A distinct, sparsely spiny, apical dendrite extends to the
pial surface, branching extensively in layer I up to the lamina dissecans. In
MEC, in contrast to LEC, the primary apical dendrite is not thicker than the
other dendrites but is spinier.
The characteristic, slightly spiny basal dendritic plexus extents horizontally
sometimes up to 1mm from the soma within layers V and VI. Axons of horizontal
cells travel to the angular bundle, giving off branches into layers V and VI [##UREF##11##110##, ##REF##12209840##144##, ##REF##10713573##145##].</p><p>A third type of
principal neurons is polymorphic MPNs [##REF##12209840##144##–##REF##11168548##146##]. The somata
of these cells are spherical to slightly pyramidal with average diameters of 13–24 <italic>μ</italic>m. Instead of having
a prominent apical dendrite, these neurons have a multipolar spiny dendritic
arborization that extents for long distances in all directions some even into
the subiculum ([##REF##12209840##144##–##REF##11168548##146##]; own
unpublished data). The axon branches within layer V but reaches the angular
bundle and travels through the subiculum, finally reaching the dentate gyrus [##REF##11168548##146##].
Members of the family of MPNs can express PV, SOM, NPY, and substance-P [##REF##6137169##116##, ##REF##3514690##117##].</p><p>Fusiform cells
that project to the hippocampus were found in superficial layer V [##REF##1713171##120##]. They
also have a single ascending dendritic tree that sometimes even reaches the pia
and one descending dendritic tree. The axon spreads locally but the main axon
projects towards the hippocampus. Fusiform neurons can contain CR [##REF##10954838##108##, ##REF##1713171##120##].</p><p>Superficial
layer V further harbours bipolar cells with a spindle-like soma having an
average diameter along the short axis of around 12 <italic>μ</italic>m [##REF##10954838##108##, ##REF##1713171##120##]. Dendrites
originate from the apical and basal poles of the spindle shaped cell body. Except close to the soma,
the dendrites are spiny and extend from the soma to the subiculum in one
direction and to layer I in the other direction, extensively branching in
layers II and I [##REF##1713171##120##]. However, most dendrites are found within the deep layers.
The main axon travels towards deep layers and perforates the subiculum,
reaching the dentate gyrus [##REF##11168548##146##]. Globular cells have very spiny and highly branched
dendrites, originating radially from the soma [##REF##1713171##120##]. Somata have a size of
19.5 <italic>μ</italic>m and up to 12 dendrites that branch within layers III–V. The axon
projects towards the angular bundle and within the layer V ([##REF##1713171##120##] own unpublished
data). It has been described that multipolar but not explicitly globular cells
in the deep layers of the entorhinal cortex might contain SOM, substance-P, NPY,
and GABA [##REF##3514690##117##].</p></statement>
</p> <p>
<statement id="head6"><title>Layer VI</title><p>The multilaminated layer VI borders the white matter.
MPNs are located throughout layer VI. They have a spherical soma with a
diameter of approximately 14 <italic>μ</italic>m. The spiny dendrites have multiple swellings
and extend mainly within layer VI, parallel to the layering. The dendrites also
extend towards the angular bundle and rarely to layer III [##REF##1713171##120##]. We found MPNs
with basal dendrites with no apical dendrite that surround the soma facing all
directions. The axons and collaterals reach the subiculum, whereas other
collaterals sometimes reach the superficial layers (own unpublished data).</p><p>The somata of
classical pyramidal cells in the MEC are medium sized. Pyramidal cells in the
LEC have not been described yet. The difference compared to layer V or III
pyramidal cells is that the dominant dendrite does not always travel radially
towards superficial layers but also either horizontally within layers VI and V
or descends towards the angular bundle and the subiculum (own unpublished
data). The basal dendrites and the widely spreading collaterals spread within
layers VI and V. The axons of pyramidal cells travel towards the angular bundle
and subiculum as well as towards superficial layers. Their axon collaterals are
located within layers V, VI, the angular bundle, and the subiculum [##UREF##11##110##].</p><p>In conclusion, there are
differences between cell types and the distribution of cell types in LEC and
MEC (see ##FIG##1##Figure 2##). In layers I and II, the differences between cell morphology
and electrophysiology in the LEC compared to the MEC are more prominent than in
layers III and V. We know for example that different subtypes of layer I MPNs
neurons show a different distribution within layer I. The same holds for
chandelier and basket cells in layer II. In addition, there are major
differences in the distribution of for example PV- and CR-positive neurons and
neuropil. This suggests that LEC and MEC are different with respect to the
types of interneurons present. Furthermore, layer II principal neurons in LEC
and MEC do not only have a somewhat different morphology but differ also
electrophysiologically. Taken together these findings might be an indication
that the microcircuits within layers I and II in the LEC and MEC are different.
Layer III and V principal neurons of both LEC and MEC are more comparable regarding
morphology as well as the electrophysiological properties. Having said this, we
need to be aware of the fact that our understanding of the different cell types
in the entorhinal cortex and how they are wired together is still rather
fragmentary.</p></statement>
</p> <title>4.2. Intrinsic organization</title> <p>The entorhinal cortex contains a substantial
system of associational connections that are best described at two different
levels. The first is that in all species studied, intraentorhinal fibers are
organized in a limited number (generally three) of rostrocaudally oriented
bands. Connections that link different transverse (or mediolateral) regions of
the entorhinal cortex, thus providing connectivity between these bands, are
rather sparse [##REF##9703027##147##–##REF##17154267##151##]. The associational
connections within these bands originate in both superficial and deep layers. Results
of anatomical tracing experiments have provided convincing evidence that projections
originating from layers II and III tend to terminate mainly in the superficial
layers, whereas projections originating from deep layers terminate both in the
deep and superficial layers. The finding of rather extensive superficial to
superficial connectivity seems at odds with results suggesting that there is
only sparse collateral innervation among layer II principal cells but see Kumar
and Buckmaster [##REF##16641241##143##], who showed layer II to layer II
excitatory connectivity with an up to 500 <italic>μ</italic>m distance, and inhibitory
connectivity (see also ##FIG##1##Figure 2##). Among layer III principal cells, collateral
innervation is more common [##REF##11029534##152##]. One naturally has to take the nature of these
local connections into account, and the anatomical results [##REF##9703027##147##–##REF##3379162##149##] do not
indicate whether we are dealing with excitatory connections among principal
cells or connections of principal cells with putative inhibitory interneurons
or even with excitatory local neurons [##REF##10954838##108##]. For example, the pyramidal-like
interneurons in layer III or the multipolar interneuron at the border between
layers II and III (see ##FIG##1##Figure 2##) is likely candidates to contribute to these
intrinsic associative networks, but this remains to be established.</p> <p>The overall organization of the longitudinal
intrinsic connections is best considered in relation to the organization of the
reciprocal entorhinal connections with the hippocampal formation. Interconnected
portions of the LEC and MEC close to the rhinal fissure, in rats referred to as
the dorsolateral band of entorhinal cortex, are connected to the dorsal
(nonprimate) or posterior (primate) part of the hippocampal formation (see
Figures ##FIG##0##1##(c), ##FIG##0##1##(d)). Interconnected cells in the intermediate band, encompassing
again parts of both LEC and MEC, connect to the intermediate hippocampal
formation, whereas the most medially interconnected band of entorhinal cortex
is mainly connected to the ventral (nonprimate) or anterior (primate)
hippocampal formation. Cells located in each of these entorhinal bands thus
give rise to associational connections to other cells in the same region, but
not in any substantial way to portions of the entorhinal cortex that are
connected with other levels of the hippocampal formation. Thus, the
associational connections seem to be organized to integrate all of the
information that targets a particular portion of the entorhinal cortex, and
that portion of entorhinal cortex interacts selectively with a particular
longitudinal level of the hippocampal formation [##REF##2682783##2##, ##REF##9703027##147##, ##UREF##12##153##, ##REF##9703026##154##]. This
implies that at the level of the entorhinal cortex integration across input
modalities may occur and this is in line with reports that in the monkey
entorhinal cortex, single neurons apparently respond to different types of
sensory inputs [##REF##9307135##155##]. It is
still an open question whether these longitudinally organized associative
intrinsic networks really support association between the two sets of inputs
that reach MEC and LEC, respectively. It is also not known whether this network
originates partially or completely from the same neurons that contribute to the
more focal intrinsic connectivity that will be described subsequently.</p> <p>The second organizational level deals with
the local connectivity within and among layers of more restricted portions of
the entorhinal cortex. As we know from the studies summarized above, neurons in
different layers have very different inter- and intralaminar connectional
patterns that include axon collaterals confined to the parent cell layer or
spanning several layers. But not only the axonal distribution is of importance,
the dendritic trees may also play an essential role in that they either span
several layers or are more restricted to the parent cell layer. Although
detailed information for quite a few of neuronal types in the entorhinal cortex
is still lacking, it is safe to say that the entorhinal network, on the basis
of its neuronal composition alone, cannot be properly described in terms of
superficial and deep layers as more or less independent layers. All this may
not come as a surprise since comparable concepts have been described with
respect to the organization of the neocortex [##REF##17964242##156##]. This second level of
intrinsic organization has not yet been seriously incorporated into our working
concept about entorhinal cortex. This is essential however in order to properly
understand how inputs to the entorhinal cortex will be processed by the
entorhinal network and what the eventual information is that will be conveyed
to the hippocampal formation on the one hand and to other cortical and
subcortical areas on the other hand. The following paragraphs will provide a
description of recent most salient findings that may be related to this second
level of the entorhinal intrinsic organization (see ##FIG##2##Figure 3##).</p> <p>One important anatomical observation already
reported by Cajal [##UREF##0##1##, ##UREF##4##28##] is that neurons in the deep layers are connected to
superficial layers by way of axonal projections ([##REF##12209840##144##, ##REF##10713573##145##, ##REF##9703027##147##–##REF##3379162##149##, ##REF##14750656##157##]; see
also ##FIG##1##Figure 2##: the small and large pyramidal cells in layer V of both LEC and
MEC). These anatomical findings have been corroborated in a number of
functional studies [##REF##8638163##158##–##REF##9405558##162##]. Recently,
these connections have been studied in more detail in the rat with respect to
projections from the subiculum, using both anatomical and electrophysiological
techniques [##REF##15390170##50##, ##REF##12454982##51##, ##REF##14750656##157##, ##REF##14656299##163##]. The majority of the axons from deep to
superficial layers are likely excitatory and target both interneurons as well
as principal neurons in almost equal percentages [##REF##14750656##157##]. This thus constitutes
the substrate for powerful excitation as well as feedforward inhibition to
neurons in the superficial layers. Stimulation of the subiculum in vivo, not
only resulted in population activity in layers II and III of the entorhinal
cortex, but subsequently activated the dentate gyrus and CA1 [##REF##15390170##50##, ##REF##14656299##163##].
Moreover, the transfer of activity from layer II to DG and from layer III to
CA1 depended on the anesthetic used, suggesting that two functionally distinct
parallel reentrant routes exist in the entorhinal-hippocampal system. Although
it has not yet been established in freely moving animals whether these two
parallel pathways function as separately controlled inputs to DG/CA3 on the one
hand and CA1/subiculum on the other hand, the findings are of interest in
relation to recently published ideas that DG, together with CA3, is
preferentially involved in pattern separation whereas CA1 might be more
relevant for pattern completion processes [##REF##15272123##164##–##UREF##13##167##]. It should
be mentioned here that there is also convincing anatomical as well as
electrophysiological evidence supporting the existence of connections between
cells in layers III and II [##REF##2771045##114##, ##REF##3958250##148##, ##REF##3379162##149##, ##REF##3419592##168##–##REF##16177045##171##] suggesting
that they may function in concert as well.</p> <p>As illustrated in ##FIG##1##Figure 2##, all layers of
both LEC and MEC, with the possible exception of layer VI where details are
still lacking, contain neurons, mainly of the pyramidal type, with an apical
dendrite that extends all the way up to layer I, quite often forming a
dendritic tuft in layers II and I. This is a feature that is strikingly similar
to what has been reported for the neocortex. Although the functional
significance of this general pattern is still poorly understood, the
commonality of it, even in evolutionary older parts of the cortex, must be an
indication for its significance. In the neocortex, layer I is a main recipient
of feedback projections and inputs from subcortical structures [##REF##17964242##156##]. In
contrast, in the entorhinal cortex, like in the hippocampus, layer I
constitutes a major input layer; for example, the densest innervations from
olfactory portions of the cortex, including the olfactory bulb, terminate in
layer I [##REF##7371730##10##, ##REF##3902147##111##, ##REF##6866324##172##, ##REF##6499975##173##]. Likewise, in quite a few instances, inputs to the
entorhinal cortex that densely terminate in layers II, III, or V have a
component to the deep portion of layer I as well [##REF##2682783##2##, ##REF##17598159##72##]. In addition, the
apical dendrites of entorhinal neurons not only receive synaptic inputs at
their tufts in layer I, but in case of neurons in layer V of MEC, we have shown
that they are among the postsynaptic targets of projections from the
presubiculum [##REF##15196963##174##]. Note that presubicular inputs to MEC, like those from the
perirhinal cortex and some nuclei of the amygdaloid complex, densely terminate
in layers III and deep I, almost avoiding layers II and V. Presubicular fibers
contact neurons in layer III, and do so with a high density [##REF##7843307##175##, ##REF##8987807##176##]. Recent
electrophysiological in vitro and in vivo data have corroborated that
presubicular fibers synapse onto neurons in layers III and V, but also onto
neurons in layer II [##REF##15987257##170##, ##REF##16177045##171##]. No data are available with respect to
perirhinal and amygdale inputs to LEC, that show a similar laminar
distribution, but in view of the overall similarity of the networks and cell
types in both entorhinal areas, it is likely that for example inputs from
perirhinal cortex and amygdale target neurons in layers II, III, and V. Data on
inputs that specifically distribute to layer II of MEC, and to a lesser extent
of LEC, such as fibers that originate in the parasubiculum, are not available.
The potential for functionally relevant interaction between neurons in the deep
layers of the entorhinal cortex and superficially terminating inputs has yet
another dimension. It has been argued that hippocampal output that leads to
firing of cells in layer V of the entorhinal cortex may result in back
propagation into the superficial layers, along the dendrites of layer V cells.
Back propagation has been documented in the neocortex and in CA1 and may occur
in the entorhinal cortex as well. Back-propagating action potentials may
increase the influence exerted by inputs to distal portions of the dendrite
[##REF##14656299##163##]. This combination of distally terminating inputs from local axon
collaterals in layers II and III and back propagation along dendrites of layer
V cells provides the most likely substrate for observations that activation of
superficial entorhinal layers may lead to subsequent activation of deep layers
of entorhinal cortex [##REF##8638163##158##, ##REF##12611981##159##]. Although axons of layers II and III
occasionally send a collateral into deep layers of entorhinal cortex ([##REF##16127693##127##];
own observations, illustrated in ##FIG##1##Figure 2##), the overall direct connectivity
from superficial to deep layers is rather sparse and therefore may not be
sufficient to mediate this rather strong superficial to deep activation.</p> <p>What
then is the functional relevance of inputs from for example the medial
prefrontal, cingular, and retrosplenial areas? Afferents from these areas
preferentially, and in some instances even exclusively, terminate in the deep
layers of the entorhinal cortex. Note that in the monkey, however, it has
recently been reported that projections from the retrosplenial cortex densely
innervate entorhinal layer I [##REF##17436282##78##]. Do inputs to the deep layers of the
entorhinal cortex modulate the transfer of hippocampal output to the cortex,
interact with the integrative capacities of the entorhinal network, or both?
These questions are relevant not only for our understanding of the functional
relevance of the entorhinal cortex in relation to functions of the hippocampus
but also since these cortical areas form part of the default mode network,
implicated in higher-order cognitive functions [##UREF##14##177##–##REF##18003904##179##].</p> <title>5. PERSPECTIVES</title> <p>The functional relevance of the
organization of networks in the brain is often interpreted on the basis of a surprisingly
restricted point of view. Debates on the functional organization of the
hippocampal formation have been strongly influenced by the idea that the
prevailing hippocampal circuitry is unidirectional. With regards to the entorhinal
cortex, the breakthrough discovery, that deep entorhinal layers receive
hippocampal output from CA1 and the subiculum on the one hand, and that these
same layers are the origin of strong cortical projections, has biased our view towards
the rather simple concept that the deep layers mediate hippocampal-to-cortical
connectivity, similar to superficial layers providing the way in for cortical
inputs to the hippocampal formation. If the entorhinal cortex is such an
important hub, similar to the central station of a large city, and that is what
all data seem to converge on, it is quite likely that it serves yet another
role. In addition to serving simply to get into the city or leave the city, the
station also provides the powerful potential for new interactions between and
among incoming and outgoing people. This potential for “new” interactions has been
grossly neglected in case of the entorhinal cortex. The potential of the
entorhinal cortex to act as an interactive hub, contributing essentially to the
functions of the cortico-hippocampal system instead of just transferring
information, has been underscored not only by the recent finding of the unique
spatial firing properties of grid cells in the entorhinal cortex [##REF##16675704##7##–##REF##15965463##9##], but also by
reports that the spatial firing properties of CA1 cells likely depend on inputs
from the entorhinal cortex [##REF##18215625##17##–##REF##17094145##19##]. More in
particular, the findings that spatially tuned neurons are present in all layers
of MEC, and that a clear relationship is apparent between closely associated
portions of MEC across layers underscore the concept of the entorhinal cortex
as an important higher-order association cortex where understanding the
interactions between the layers will provide us the key into its functional
relevance [##REF##16675704##7##, ##REF##17069897##20##].</p> <p>Similar to the yet unresolved
mystery of the relevance of cortical inputs to deep layers of the entorhinal
cortex, it remains to be established what the functional relevance is of LEC.
The data summarized above indicate that with the exception of neurons in layer
II, it is likely that both LEC and MEC are largely similar with respect to
their intrinsic wiring, both in terms of neuronal elements that comprise the nodal
points of the network as well as with respect to how these are wired together
(see Figures ##FIG##1##2##, ##FIG##2##3##, [##REF##17069897##20##]). What then accounts for the strikingly different
features of LEC and MEC when spatially modulated neuronal firing is concerned?
Most likely, differences in input and output characteristics will set the scene
as eloquently summarized recently [##UREF##1##5##]. However, how convincing this may look,
it may not be the complete story. Additional differences in modulatory
connectivity from not only the septal complex, but also from the raphe nuclei,
the ventral tegmental area, and locus coeruleus may turn out to be most relevant.
Unfortunately, with the partial exception of inputs from the medial septum,
very little detailed information is available regarding these inputs in terms
of their overall distribution and topography in relation to both extrinsic and
intrinsic wiring of the entorhinal cortex. Furthermore, detailed information of
the postsynaptic targets of these modulatory inputs is largely missing. One
final approach to further our understanding of the entorhinal cortex may be to
make use of the striking involvement of the entorhinal cortex in an impressive
list of brain diseases [##REF##1336586##35##] and to focus on alterations in the circuitry that
likely occur during development, ageing, and disease and their effect on
entorhinal functioning.</p>
<title>ACKNOWLEDGMENTS</title> <p>The preparation of this paper and
the original research on entorhinal neurons are supported by the Kavli
Foundation and a Centre of Excellence grant from the Norwegian Research
Council. Cathrin B. Canto thanks the Department of Anatomy and Neurosciences, VU
University medical center for the generous hospitality and warm atmosphere.</p>
<fig id="fig1" position="float"><label>Figure 1</label><caption><p>Schematic representation of the overall organization of the
entorhinal cortex and its connectivity. (a) Position of the
entorhinal cortex and surrounding cortices and hippocampus in the
rat left hemisphere. Indicated are the dorsoventral extent of the
hippocampus, positions of LEC and MEC, and the approximate position
of a representative horizontal section, illustrated in (b). (b)
Horizontal section illustrating entorhinal-hippocampal
connectivity (see text for more details). (c) and (d) Representation of the
topographical arrangement of entorhinal-hippocampal reciprocal
connections. A dorsolateral band of entorhinal cortex (magenta) is
preferentially connected to the dorsal hippocampus. Increasingly,
more ventral and medial bands of entorhinal cortex (purple to
blue) are connected to increasingly more ventral levels of the
hippocampus. Yellow line in (c) indicates the border between LEC
and MEC. (e) Enlarged entorhinal cortex, taken from (c), indicating
the main connectivity of different portions of entorhinal cortex.
Brain areas preferentially connected to LEC are printed in green,
those connected to MEC are in magenta. The color of the arrows
indicates preferential connectivity to the
dorsolateral-toventromedial bands of entorhinal cortex (magenta or
blue, resp.) or that no preferential gradient is present
(green).
</p></caption></fig> <fig id="fig2" position="float"><label>Figure 2</label><caption><p>Summary diagram of the morphology of main cell types in LEC and MEC. (a) Cells in superficial layers I-III. (b) Cells in deep layers IV-VI. See text for more details.</p></caption></fig> <fig id="fig3" position="float"><label>Figure 3</label><caption><p>Schematic representation of laminar distribution and synaptic
interactions between inputs and principle cells of the entorhinal
cortex. Different inputs are represented by color-coded arrows;
position of the arrows indicates the main laminar distribution.
Circles indicate putative synaptic contacts between inputs and
principle cells. Main output connectivity of principle cells is
indicated as well. The figure emphasizes the integrative capacity
of layer V cells.</p></caption></fig>
<graphic xlink:href="NP2008-381243.001"/> <graphic xlink:href="NP2008-381243.002"/> <graphic xlink:href="NP2008-381243.003"/>
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Neural Plast. 2008 Aug 28; 2008:381243