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The organisation of dendritic bundles in the prelimbic cortex (area 32) of the rat
BRAIN RESEARCH ELSEVIER
Brain Research 73(1 (1996) 75-86
Research report
The organisation of dendritic bundles in the prelimbic cortex (area 32) of the rat P . L . A . G a b b o t t *, S.J. B a c o n Unicersity Department (~]'Pharmacol.
Accepted 2 April 1996
Abstract Using an antibody against microtubule associated protein 2 (MAP-2: a specific marker fl)r neuronal dendritesL this paper reports the structural organisation of pyramidal cell apical dendrites in the rat prelimbic (PL) cortex. In the coronal plane, MAP-2-immunoreactive apical dendrites of pyramidal neurons in layers 5, 3 and 2 were found bundled together as they ascended radially through the cortex. These bundles of dendrites dispersed in upper layer 2 to form apical dendritic tufts in layer 1. In tangential cross-section, thc immunolabelled bundles were organised into a latticework of discrete clusters of differentially sized profiles. At the boundary between layers 3 and 5. clusters were composed of 26 + 8 dendritic profiles (group mean value + S.D,, five animals), whereas clusters in lavcr 2 contained 55 ± 15 profiles. The number of clusters per unit surface area was not significantly different throughout layers 5, 3 and 2 (760 + 75 per mm 2) with the average centre-to-centre intercluster distance in these layers being 44.2 ± 4.9 gin. The data indicate that apical dendritic bundles are a feature of the radial organisation of PL cortex. These structural subunits may subserve specific integrative functions in the PL area of the rat medial prefrontal cortex. Kevworet~': Medial prefrontal cortex: Cortical module: Pyramidal neuron; Apical dendrilic bundle/cluster; IAmbic/autonomic corlcx
1. Introduction
In the cortex, groups of pyramidal cell apical dendrites aggregate to form distinct vertically aligned bundles [7,23,28,33,37]. The most prominent grouping of dendritic bundles arise from sets of large- and medium-sized pyramidal neurones situated in layer 5 [10,11,13,18,21,23,37]. In the tangential plane, the bundling of apical dendrites is most apparent by the discrete clustering of dendritic profiles at the level of layer 4 - the main thalamorecipient territory of granular areas of the cortex [21,23]. This feature of cortical organisation has been reported in several cortical areas for a variety of mammalian and primate species [6-13.18-33,37-39]. Indeed, it is now thought that a single dendritic bundle, together with the associated neurone populations and synaptic circuitry, represents a fundamental structural subunit underlying specific func-
Cc,rrespondine author. Fax: *-44 (1865) 27-1853: E-mail: paul.gabboil @pharm x~x.ac,u k
tional properties in the cerebral cortex [ 1,7,13,15,23,25,30-33]. The bundling of apical dendrites has not been previously reported in the medial prefrontal cortex (mPFC) of the rat [36]. The aim of this study was to establish whether dendritic bundling was also a structural feature of lhe prelimbic (PL) region (Brodmann area 32) of mPFC in the rat. The approach was to use a specific cytoskeletal marker for dendrites, microtubule associated protein 2 (MAP-2) [4,34], in order to qualitatively and quantitatively investigate the tangential and vertical organisation of dendritic bundles in rat PL cortex. Since PL cortex in the rat is an agranular area (i.e. lacks a cytoarchitecturally distinct layer 4: see Fig. IC), this study concentrated on the distribution of dendritic bundles in upper layer 5 to mid-layer 3 and in layer 2. Of significance is that in PL cortex, the region upper layer 5 / m i d layer 3 is the principal region of innerwltion from the mediodorsal nucleus of the thalamus [17,38], while layer 2 is a region of dense bundling fl)und in other cortical areas
O006-8993/96/%15OO ('op,,right ~, 1996 Elsevier Science B.V. All rights reserved. /W S0006-,";9u ~(,76)0043 7-4
76
P.L.A. Gabbott, S.J. Bacon / Brain Research 730 (1996) 75-8h
[21,23,37,38] and in PL cortex it receives a laminar specific input from the amygdala [3].
B ) PARASAGITTAL t
2. Materials and methods
2.1. Tissue .fixation
Eight adult Sprague-Dawley rats of both sexes (150-250 g) were used in this study. These animals had been deeply anaesthetised and transcardially perfused with a solution of aldehyde fixatives containing 0.05-0.5% glutaraldehyde and 3-4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) [5]. Following perfusion, the brains were removed and serial Vibratome sections (100 Ixm thick) obtained from the parasagittal or coronal planes through the mPFC ([33]; see also Fig. 1A, B). Tissue sections cut parasagittally were kept in order so that their corresponding depth below the pial surface of the medial bank was known (Fig. 1C). Sections were then transferred to Tris-HCl (Tris) buffer (pH 7.4) and processed either for immunocytochemistry using an antiserum against MAP-2, or for conventional post-fixation with OsO 4, dehydration, and resin embedding
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2.2. Anti-MAP-2 immunocvtochemistr 3'
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Sections were initially freeze-thawed or permeabilised by treatment with 0.25% Triton X-100 [5] and incubated as follows: 20% normal goat serum; anti-MAP 2 mouse monoclonal (Sigma Chemicals, UK: clone no. HM-2, product no. M 4403) diluted 1:250-1:5000; anti-mouse ABC kit (Vector Labs, UK). Immunoreactivity was visualised using 0.05% 3,3'-diaminobenzidine (DAB) and 0.01% H202 in Tris buffer (pH 7.4). In some sections, immunoreactivity was revealed using the Vector SG (slate grey) peroxidase substrate kit (Vector). Between the incubation steps, sections were thoroughly washed (3 × 10 min) in Tris buffer (pH 7.4). Sections were then mounted in series on gelatin coated glass slides, air-dried, passed through an ascending series of alcohols, taken through xylene and finally embedded in DPX mountant [5]. Several control sections per animal were either incubated without the primary MAP-2 antiserum or by omitting the biotinylated link antibody of the ABC kit. 2.3. Resin embedded semithin sections
Parasagittal sections (100 ~m thick) from layers 2-5 of PL cortex were postfixed with 1% OsO 4 in 0.1 M phosphate buffer (pH 7.4) for 45 min, dehydrated in a graded series of alcohols (including 1% uranyl acetate in the 70% alcohol for I h in the dark), passed swiftly through propylene oxide, flat-embedded in Durcupan resin, and cured for 2 days at 56°C [5]. Selected regions of PL cortex at
1
2
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bJ 1250 Fig. 1. A, B: line drawings showing the division of the medial prefrontal cortex (mPFC) into cyloarchitectonic areas in the coromd plane (approximately + 3.2 mm anterior to bregma (B)), and in the parasagittal plane (approxmlately 0.4 mm lateral to midline). The prelimbic cortex (PL-area 32) is indicated. Surrounding areas: PrCm. medial precentral cortex: ACd, dorsal anterior cingulate cortex: ACv, ,,entral anterior ciugulate cortex: and IL, infralimbic cortex. Orientation markers: D. dorsal: V, ventral: C, caudal. The corpus callosum is shown shaded. Scale bar = 500 Fro. C: coronal Nissl-stained section of prelimbic cortex (PL). The cytoarchitectural lamination of PL cortex (layers I (~b) is given on the left ol Ihe photomicrograph, and a scale marker indicating depths below the pial surface (Ixm) is given to the right of the photograph. (pitt. pial surfacc:wnl, white matter).
P.L.A. Gabhott. S.J. Bacon/Brain Re.~ear¢'h 730 ~1996~ 75 ~'6
different depths below the pial surface were then carefully excised amt glued onto a blank resin stub [5]. Serial semithin resin sections (I.0 bun in thickness) were subsequently cut using an ultramicrotome and collected in sequence on gelatin coated glass slides. These sections were counterstained with an aqueous solution of 1% toluidine blue and 0.59; sodium borate, washed and dried, and finally mounted in DPX [5]. 2.4. Cvtoarchitecture q f rat m P F C
Additional complete sets of unreacted serial parasagittal and coronal sections were Nissl stained, dehydrated and flat-embedded in DPX. These sections enabled the identification of the cellular lamination and areal cytoarchitecture in mPFC to be defined and helped to delineate the tangential extent of PL cortex (area 32) in parasagittal sections ([32,35]: see Fig. I A - C ) . 2.5. Qualitatirc examination
MAP-2 immunoreacted sections were examined in a photomicroscope equipped with a drawing tube. The appearance of dendritic bundles and clusters in these sections was compared with their appearance in the counterstained semithin resin sections. Structures of interest were recorded through photomicrographs and drawings. The aggregation of apical dendrites in the coronal plane are called 'bundies', whilst in the parasagittal plane they are referred to as "clusters'. (Note that parasagittal sections through the PL area are parallel with the pial surface and reveal the tangential (or horizontal) structure of the cortex.) 2.6. Quontitatize study
The composition and tangential distribution of MAP-2 immunoreacted dendritic clusters at two horizontal sampiing levels m the PL cortex were analysed quantitatively': (i) in layer 2 ( 2 0 0 - 3 0 0 g m below the pial surface: Fi X. IC), and (it) in a region extending from mid-layer 3 to upper layer 5 (400-700 p~m below the pial surface: Fig. IC). Since the latter sampling zone centres on the boundary between layer 3 and layer 5 it is subsequently called l a y e r 3 / 5 ' . (Rat PL area is agranular: see Fig. IC,) Sections could be confidently ascribed to a given cortical lamina (or region within a layer) on the basis of: (i) MAP-2 neuronal staining characteristics, (it) the depth of the section below the pial surface (Fig. IC), and (iii) the cytoarchitecture of the corresponding Nissl stained parasagittal section (Fig. I C). In parasagittal MAP-2 immunoreacted sections, individual dendrites could be clearly recognised and their assembly into discrete clusters was clearly evident (see Section 3). The tangential organisation of dendritic clusters in the t'L cortex most closely resembled a 'hexagonal packing" system (see Fig. 4A, B and Fig. 5) - such a system has
"7
been previously described lor dendritic clusters in the rat visual cortex [21]. Consequently, this packing model was used to derive several quantitative parameters (see below: see also Fig. 5). Sections were viewed in the light microscope ( × 4 0 objective lens and × 10 eyepieces) and a square "lest" counting frame (250 × 250 txm) superimposed on the section through a drawing tube. Each counting frame had an upper and right-side inclusion border and a lower and left-side exclusion border [14]. The centres of dendritic clusters were defined visually ([14], see also [21]). The cluster centres that were inside the "test" area of the counting frame, were marked onto the frame. All the MAP-2-immunoreactive dendritic profiles constituting a 'test' dendritic cluster were drawn onto the countmg" " frame. Boundaries were also drawn by joining the outlying dendritic profiles (using a nearest neighbour procedure: {14]) around each cluster (Fig. 5) 1. In addition, at high magnification (X 100 oil immersion objective lens and × 10 oculars) the number of large-calibre ( > 2.0 ixm) and smallcalibre ( < 2.0 Ixm) dendritic profiles per cluster were calculated for a sample of dendritic bundles (n = 25) al both smnpling levels in each animal. Using a computerised planimeter (Macintosh Quadra operating customised stereology software) the fl~llowing morphometric parameters were recorded or measured l~)r each dendritic clnster (Fig. 5): (1) the total number of dendritic profiles (large- and small-calibre) per bundle (NP), (2) the number of 'test" dendritic clusters under 1 ram: of cortical surface (NC~). (3) centre-to-centre intercluster distance between neighbouring clusters (IC txm) (see Peters and Kara [21] pp. 758-759 lk)r a discussion of "centre-to-centre' versus "nearest neighbour' distances between clusters), (4) the tangential area occupied by a dendritic cluster (CA berne), (5) the tangential area (TA > m -~) of cortex associated with each dendritic cluster. calculated as [TA = (1 m m 2 / N C x ) ] , and (6) the percentage ratio that CA composed of TA [ ( C A / T A ) X 100%]. 2.7. SamplinA, am/statistical analysis
In each of fi~e animals, ten counting frames were applied to parasagitlal sections to examine the clustering of
i In a preliminary sludy, the centre of an irregularly shaped dendritic cluster was visually defined 24 times. Subsequent calculations established that a comparatively small error (coefficient of variation 1.2% in mean r,3 coordinates: [14]) was introduced using lifts subjeclive centring technique. Further, the standardised x,v positions of a sample of visually defined cluster centres (n= 12) were nol statistically different ~o .~,v coordinates of the same clusters generated by a computeriscd planimcter. Additionally, permtffations in drawing the boundar? to a gJ,~cn dendritic cluster produced a coefficient of wtriafion ot 2.6~ in the mean surface area (parameter CA, see main text). These potential sources of error are therefore considered non-significant m d~e derivation of corresponding quantitati,.e data Ik~r Im-ge populations of dendritic clusters ll4].
78
P.L.A. Gabbott. S.J. Bacon/Brain Rese~rch 730(1996) 75-80
P.L.A. Gabbott, X,I. Bacon/Brain Research 730 f1996) 75-S6
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Fig. 3. A: photomicrograph illustrating MAP-2-immunopositive pyramidal neurones (P) in a coronal section through upper layer 3 and layer 2 t>l' PI_ cortex. The somatic cytoplasm and apical dendrites (arrow) of these neurones are strongly labelled. Scale bar - 2(1 ,urn. 13: langential semithin (I pore thick) resin section through mid-layer 3 of PL cortex stained with toluidine blue. The clustering of processes (an'ows) ascending through the prelimbic cortex is readil~ apparent at this level. The somatic profiles of neighbouring neurones (n) are visible. (c+ capillaries). Scale b a r = 50 ++tm. C: high magnification photomicrograph of a toluidine blue-stained coronal semithin resin section shov, ing a dendritic bundle (thick black arrow) in upper layer 3 ol PL cortex. Large dcridritic profiles comprising a bundle have been indicated by small black dots. The profile of a transversely sectioned process is also indicated
dendrites in layer 2. and ten counting flames were used to examine dendritic clusters in the region layer 3 / 5 (Fig. 1). Mean animal values for each parameter were calculated for clusters in layer 2 and in the region layer 3/5. Mean group values were subsequently derived. Layer 2 and layer 3 / 5 data were subsequently compared statistically using ~NOVAs and post-hoe t-tests [13]. Significant differences were considered to occur when P < 0.05.
3. Results 3. I. Antiserum spec~/icity
The biochemical and cytological specificity of the MAP-2 antiserum used in this study has been reported previously [4,34]. In control sections, incubated either without the primary antiserum or the biotinylated link
t:ig. 2. A: photomicrograph of an unstained coronal Vibratome section through the superficial layers ( I - 3 ) of the PL cortex (area 32) laken rising high contrast optics. The profiles of processes bundled closely together (thick arrows) are clearly visible ascending through the cortex. The bundles disperse on entering layer I. Unstained neuronal somata (n) are indicated, capillaries (c) are also shown. Scale bar 5() p.m. B: rnicrograph of a ',ection through layers 3 and 5 in PI, cortex, reacted immunocytochemically for MAP 2. The somatic cytoplasmata of pyramidal neurones (P) in layer 5 are immunopositive. The ;~pical dendrites of these cells are also strongly immunolabelled (arrows). Indicated is the clustering of processes ascendin~ through the superficial layers of the cortex (boxed region in layer 3). Note the difference in intensity of immunolabelling betv, een the apical dendrite "rod soma ol the pyramidal cell indicated (P'). Also note the emergence of an imrnunolabelled basal dendrite from the cell body of this pyramid (small arrow), c. capillaries. Scale bar 50 gin. C: low magnification photomicrograph of a section reacted immunocytochemically for MAP-2. Bundles of strongly labelled MAP+2+ immunoreactive dendrites (arrows) traverse through layers 1-3 of PL cortex. The somata of unlabelled neurones are indicated (black dots). Scale bar :: 100 [{ Ill.
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P.L.A. Gabbott, S.J. Bacon/ Brain Research 730 (1996) 75 86 sections through the PL cortex, i m m u n o r e a c t i v e dendrites appeared as dark puncta or dark strands aligned along the axis of viewing (Fig. 2D and Fig. 3A). In particular, the apical dendrites of pyramidal neurones, at all levels within the cortex, were strongly immunoreactive, with their perikarya displaying moderate M A P - 2 i m m u n o l a b e l l i n g (Fig. 2B and Fig. 3A). No M A P - 2 - i m m u n o r e a c t i v e dendritic spines were found along i m m u n o l a b e l l e d dendritic processes (Fig. 2B and Fig. 3A, D, E).
antibody, there was no specific i m m u n o l a b e l l i n g of cellular processes nor cell bodies. Control sections possessed a characteristic non-specific light-brown D A B (or light-grey SG) coloration. 3.2. Unstained coronal sections In unstained sections, the b u n d l i n g together of vertically aligned processes can be clearly identified in PL cortex (Fig. 2A). These bundles began at the level of mid-layer 5 and c o n t i n u e d u p w a r d through the cortex until reaching upper layer 2 where they began to diverge and radiate outwards within layer 1 (Fig. 2A). In m a n y sections, the somata of unstained cells in layers upper 5, 3, and 2 were c o m m o n l y located b e t w e e n the unstained ascending b u n dles of processes - these b u n d l e s appeared to be regularly spaced apart (Fig. 2A).
3.4. Qualitati+,e obseruations 3.4.1. Coronal plane In coronal sections, M A P - 2 - i m m u n o r e a c t i v e pyramidal cell apical dendrites could be clearly seen to arise from parent somata that were also i m m u n o l a b e l l e d (Fig. 2B and Fig. 3A). Individual labelled dendrites could be traced from cell bodies in layer 5, through layer 3 and into the upper zone of layer 2, before bifurcating repeatedly into a terminal tuft of apical dendrites in layer I (Fig. 2B, C). The most p r o m i n e n t b u n d l i n g of M A P - 2 - i m m u n o reactive dendrites occurred over the region of cortex extending from upper layer 5 to the layer 2 / 1 border and originated from pyramidal neurones situated in deep- to mid-layer 5 (Fig. 1C; Fig. 2B, C; Fig. 3A). The labelled somata of pyramidal neurones in layer 5 were situated
3.3. MAP-2 immunolabelling in prelimbic cortex In coronal and parasagittal sections through PL cortex, the dendrites of n e u r o n e s in layers 1 - 6 were M A P - 2 - i m m u n o r e a c t i v e (Fig. 2B, C and Fig. 3A). I m m u n o p o s i t i v e dendrites in coronal sections appeared as b r o w n D A B (or grey SG) labelled processes against a comparatively clear b a c k g r o u n d (Fig. 2B; C and Fig. 3A). In parasagittal
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Fig. 4. A: distribution of MAP-2-immunolabelled profiles at the level of mid-layer 3 in PL cortex. Distinct clusters of dendritic profiles are indicated (arrows) and one is shown encircled. (c, capillaries)• Scale bar = 10l) ~xm. B: light microscope drawing showing the tangential distribution of dendritic bundles from the middle of layer 3 in PL cortex. Several dendritic bundles are indicated (arrows). The centres of clusters surrounding a central cluster have been linked together to indicate the predominant hexagonal packing of dendritic clusters• This packing pattern of packing is not perfect since occasional pentagons and heptagons need to be included to complete the lattice (see also Ref. [21]). Additional groups of dendritic profiles occur between the recognised clusters (one example is shown encircled). Scale bar = I00 rxm.
P.L.A. Gabbott, S.Z Bacon / Brain Research Z~'O (•996) 75-86
directly beneath the bundle to which their apical dendrites contributed. The immunolabelled apical dendrites of more superficially located pyramidal neurones in layers upper 5, 3 and 2 joined the periphery of neighbouring bundles (Fig.
IB). The bundling together of immunolabelled apical dendrites from other groups of pyramidal neurones in layers 6a and 3 of PL cortex were present - but their occurrence was not common. Frequently located between immunolabelled dendritic bundles were the cell bodies of moderately immunolabelled or unstained cells (Fig. 2C). In the coronal plane, it was evident that not all labelled dendrites engaged in clustering (Fig. 2B).
3.4.2. Parasagittal plane In the parasagittal plane, the tangential clustering of MAP-2 immunoreacted dendritic profiles are principally identified by the aggregation of the cellular profiles of large apical dendrites (Fig. 3D, E and Fig. 4A). Such a clustering correlates directly with the vertical bundling of profiles seen in the coronal sections, as described above. Dendritic clusters in both layer 2 and the region layer 3 / 5 were defined as being composed of differing numbers of differentially sized MAP-2-immunolabelled profiles that were closely aggregated (see below). Clusters of dendritic profiles were found as discrete structural entities situated between weakly immunolabelled neuronal somata and unstained cell bodies (Fig. 4A). It was uncommon to find a cell body located in the middle of a cluster. The most prominent clustering of labelled protiles occurred in the region of layer 3/5 (Fig. 3El, but
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were also clearly evident at the level of mid-layer 3 (Fig. 4A) and particularly in layer 2 (Fig. 3D). In the region layer 3/5, MAP-2 clusters typically contained large-calibre profiles (about 8), with numerous small-calibre profiles (about 18) commonly distributed towards the centre of each cluster (Fig. 3El. In layer 2, the frequency of the small-calibre profiles within a cluster greatly increased (about 40) (Fig. 3D; Table 1). Dendritic clusters in layer 2 had a full intermixing of dendritic profiles so that the smaller-sized dendritic profiles were found distributed throughout an individual cluster (Fig. 3D). In the parasagittal planes through layers 3 / 5 and 2, nol all of the cross-sectioned MAP-2 profiles formed dendritic clusters (Fig. 3D, E; see also Fig. 4A. B). These profiles occurred either singly or in small groups of 2-6 profiles and did not appear to be specifically, associated with clusters. Note that large-calibre dendritic profiles were sometimes present among these groupings. It was nol ascertained whether such profiles formed independent dendritic clusters (Fig. 4B). The tangential distribution of clusters in the parasagittal plane through the PL cortex was not random, but organised into a lattice (see below and Fig. 4B, Fig. 5). Hexagons were the most frequent shape produced by joining chlster centres (20: Fig. 4B). However, polygons with differing numbers of sides (frequently pentagons and heptagons) had to be incorporated to complete the "honeycombed" latticework [20]. Bundles of basal dendrites from pyramidal cells were not observed at any level of the cortex in parasagittal or coronal sections (Figs. 2-4).
Fable I M o r p h o m e t r i c data c o n c e r n i n g the tangential c o m p o s i t i o n and distribution of M A P - 2 - i m m u n o l a b e l l e d dendritic clusters in parasagiual sections from layer ~ / 5 ~' and layer 2 of the PL cortex in the rat ( m e a n values + S.D.. n = 5 animals: range given in parentheses)
I ) Total n u m b e r of dendritic profilesper bundle (NP) Large-sized profiles per bundle Small-sized profiles per bundle 12) N u m b e r of dendritic bundles under I mm-' of pial surface INdC a) !3) lntercluster distance (IC g i n ) I centre-to-centre distance between n e i g h b o u r i n g clusters) 14) Clnster area ( ( ' A g i n " ) (area bounded by dendritic profiles) (5) Tangential area associated with each cluster (FA i,tm 2) 16) Percentage ralio flint C A occupied of TA I [ C A / T A ] x 100ci )
L a y e r 3 / 5 ~'
Layer 2
26.3 ± 7.8 (10-42) 8.4 ± 2.3 17,9 _+ 5.7 737.8 ± 81,9 (678.9-775.0) 45.1 + 4.9 (15,7-89.2) 619.6 ± 91.9 (242.6-I126.0) 1359.8 ± 79.3 (1072.9 1406.1) 45.6 + 5.9 (22.6-80.1)
55.1 + 14.7 ~15 -70) 15.3 ± 6 5 39.8 + I 1.3 779.2 + t,~l).3 1711.2 932.1) 43.2 + (~7 ( 12. I - 95.3 ) 854.(~ z 151.6 (311.5 1338,4) 1283.5 ~ 121.4 11(190.3-1472,8) 66.6 + 7 I (24.1-90.c,0
:' L a y e r 3 / 5 i~ tile region of cortex between mid-layer 3 and m i d - l a y e r 5. Statistical analysis between layer 3 / 5 and layer 2 data: ( I ) NP: mean value differences significant: two-tailed t-test, t = 3.869. P < 0.005, n = 8. (2) N d C x : mean vahte differences not significant: two-tailed t-test, t = ().759, P = 0.469, n = 8. ( t ) IC: mean value difli~rences not significant: two-tailed t-test, t = 0.512, P = 0.623. n - 8. (~t) CA: mean value ditferences significant: two-tailed t-test, t - 3.436, P < 0.001, n 8. iS) TA: mean value differences not significant: two-tailed t-test, t = 1.177, P ~ 0.273, n - 8. (,~) [ C A / T A ] ~ ; : mean value differences significant: two-tailed t-test, t = 5.086. P < 0.001. n = 8.
82
P.L.A. Gabbott. S.J. B a c o n ~ B r a i n Research 730 (1996) 7 5 - 8 6
P
Fig. 5. Schematic diagram illustrating the 'perfect' tangential organisation of dendritic clusters in PL cortex. The diagram shows the quantitative parameters measured in this study: IC, the centre-to-centre intercluster distance (m); NP, the number of dendritic profiles per cluster (shown as clear profiles of varying sizes within the boundary perimeter of the cluster); CA, the tangential surface area bounded by a dendritic cluster (txm2); NC A, the number of dendritic clusters per unit tangential surface area of tissue (expressed as number under 1 mm: of pial surface); and TA, the tangential area (txm 2) associated with each cluster. The mean values of these parameters were calculated for a sample of 22-48 dendritic clusters per animal (n = 5). Group mean values were subsequently derived. Note that by joining the centres of each cluster in this 'ideal' model produces a perfect bexagonally packed ~honeycomb' lattice. Scale bar = 50 Ixm.
Of significance is that the mean number of immunolabelled profiles per cluster in layer 2 was 110% greater than in layer 3 / 5 - this was related to an increase in the number of both large-calibre ( + 8 2 % ) and small-calibre ( + 122%) profiles. The increased number of profiles per cluster (NP) in layer 2 had consequent effects on the mean area associated with an individual cluster (CA) which was 38% greater than for layer 3 / 5 clusters. Whereas the mean tangential areas associated with each cluster (TA) were not significantly different between the two sampling levels, significant differences were detected between layer 2 and layer 3 / 5 in the ratio that CA composed of TA (Table 1: [(CA/TA) × 100%]). Dendritic clusters in layer 2 occupied a significantly greater proportion of the area associated with each cluster than in layer 3/5. The mean centre-to-centre intercluster distances ti)r clusters in layers 3 / 5 and 2 were directly measured as 45 I~m and 43 Ixm, respectively (Table 1)2 Fig. 6 shows distribution histograms of centre-to-centre intercluster distances for populations of MAP-2 immunolabelled dendritic clusters in parasagittal sections through layer 2 and in layers 3/5.
4. Discussion
The clustering of cellular profiles was also clearly evident in tangential semi-thin sections through layers 2 and 3 / 5 stained with toluidine blue. These sections served to corroborate the observations made from MAP-2 immunoreacted material. In semi-thin sections, profile clusters were composed of a variable number of circular and ovoid profiles, of varying sizes, aggregated together into discrete units (Fig. 3B, C). In layer 3 / 5 , large-calibre profiles were located prominently at the centre of each cluster, with smaller calibre profiles frequently occurring throughout a bundle (Fig. 3B, C). Similar to MAP-2-stained sections, the number of small-calibre profiles associated with bundles in layer 2 was greater than for clusters in the region layer 3/5.
The present study has used a specific antibody against MAP-2 (a recognised marker for neuronal dendrites; Refs. [4,34]), to provide qualitative and quantitative evidence that pyramidal cell apical dendrites in layers 2-5 of the rat prelimbic cortex (PL - area 32) are not randomly distributed, but are organised into discrete radially aligned bundles. In parasagittal sections, these bundles were clearly recognised as distinct clusters of MAP-2 immunolabelled dendritic profiles. The dendritic clusters were readily apparent at both the level of layer 3 / 5 and in layer 2. Quantitative data indicate that although there was no significant difference in the number of clusters per unit area nor in their spatial distribution in these tangential strata~ significant differences did occur in the mean number of profiles composing dendritic clusters between the two levels in PL cortex.
3.6. Quantitative data
4.1. Dendritic bundles in other cortical areas
Quantitative data concerning the composition and spatial distribution of clusters of MAP-2 immunolabelled profiles in parasagittal sections through PL cortex are given in Table 1 (see also Fig. 5). Table 1 provides separate data from layer 2 and from the region layer 3/5. The data indicate that whilst the overall spatial distribution (parameters - NdCA, IC, and TA) of MAP-2 profile clusters were not significantly different between layer 3 / 5 and layer 2, significant differences occurred in the mean composition of each cluster of profiles (parameters - NP and CA) at these two levels in the cortex.
Dendritic bundles have been described previously in the cortices of several mammalian species - the visual cortex
3.5. Resin embedded sections
-" Using the mean TA values and assuming a 'perfect' isohexagonal packing system (Fig. 5), mean centre-to-centre values betwen dendritic clusters can be calculated trigonometrically for layers 3 / 5 (40 tam) and for layer 2 (38 gtm). Alternatively, taking mean TA values, the diameters of the area equivalent circles (D~ir~) associated with dendritic clusters in layers 3 / 5 and layer 2 are 42 g m and 40 ixm, respectively (1),i, ~ = 2. [ T A / v ] ' s ) . These values for 'idealised" packing models are in c(nsc agreement with the directly measured mean centre-to-centre intercluster distances of 45 ixm for layer 3 / 5 and 43 ~ m for layer 2.
P.L.A. Gabbott, S,J. Bacon/Brain Re,search 7,¢0(1996) 75-~'6
of the rat [ 10,11,18,21 ], rabbit [29], cat [ 12,13] and monkey [22], in the rodent posteromedial barrel subfield (PMBSF) of primary somatosensory cortex (SM 1) [6,8,20,28,37], the retrosplenial cortex of the rat [38,39], the frontal cortex of the monkey [26], and in the human motor cortex [19]. The most comprehensive description of cortical dendritic bundling has been provided by Peters and Kara [21] for the primary visual area of the adult rat. In rat visual cortex, the apical dendrites of pyramidal neurones situated in lower layer 5 aggregate to form the central core of a vertically oriented dendritic bundle. The apical dendrites of smaller sized pyramidal cells in upper layer 5 join these bundles and together they ascend in close proximity through the upper layers of the cortex. In the supragranular layers, the bundles are further joined by the apical dendrites of some neighbouring layer 3 and layer 2 pyramidal cells. The apical dendritic bundles begin to disperse in upper layer 2 and divide repeatedly to form apical dendritic tufts in layer 1. Independent dendritic bundles are formed from the apical dendrites of other pyramidal neurones in layers 3 and 2 [21]. Additional bundles are also formed by pyramidal neurones in layer 6a and in layer 4 [21,26]. However, these bundles are not specifically associated with the bundles derived fi'om the neurones in layer 5 nor from those in the supragranular layers. In granular areas of the cortex the bundling of dendrites is most readily apparent within layer 4 [21,23]. The observations of the present study are qualitatively similar to the studies of Peters and Kara [21], White and Peters [37] and to the other studies investigating dendritic bundling in several cortical areas across a range of mammalian species [13,19,22,28]. However, one specific difference between areas concerns the contribution of layer 2 pyramidal cells to apical dendritic bundles. In prelimbic cortex, apical dendritic bundles remain highly clustered in layer 2 (cf. SMI. motor and visual areas of cortex) and closely resemble the dendritic bundling found in granular retrosplenial cortex of the rat [38,39].
4.2. Composition qf" dendritic bundles and intercluster distances The data of Peters and Kara [21] indicate that each cluster, at the level of layer 4 in rat area 17, was composed of 3-15 (mean 8) apical dendrites from large pyramidal cells in lower layer 5 and that dendritic clusters had a mean "centre-to-centre" spacing of 55-60 txm. For con> parison, the mean "centre-to-centre" distance between dendritic clusters in the visual cortex of the monkey was calculated as 31 Fm [26], whereas in the cat visual cortex the mean value is 56 >m [24]. In the mouse PMBSF, White and Peters [37] determined that the mean intercluster distance was about 25 g m in the barrel hollows and approximately 22 b~m in the barrel walls. The size of neurones, differing neuronal densities, and the specific architectures of layer 4 (particularly in mouse SMI) may underlie these species differences ([24] cf. [26] cf. [37]). In the rat visual cortex, Peters and Kara [21] found that not all of the apical dendrites of layer 5 pyramidal neutones engage in bundling. They calculated that al the level of layer 4. 12 large-sized and 38 medium-sized pyramidal neurones were 'associated' with an individual dendritic cluster (see Table 2 in Ref. [21]). Of the large-sized dendrites, approximately two-thirds (J~=8) were ctmtained in clusters (see Table 3 in Ref. [21]) and the somata of these cells were closely situated within layer 5 and lay directly beneath their clustered apical dendrites [21]. By comparison, the apical dendrites of the neurones that did not engage in bundling rose through the cortex either singly or in pairs passing around the bundles of clustered dendrites. Furthermore, Peters and Kara calculated that under I mm e of cortical surface there were approximately 359 dendritic clusters in layer 4 [21]. It should be mentioned that most of the studies cited above used toluidine-blue stained semithin sections to derive information concerning the bundling of apical dendrites in the cortex (cf. the present study using MAP-2 Fig, 3B~ C cf. D, E, see also Refs. [22,37]). Consequently,
Dendritic Bundles - A r e a 32 Layer 2 30
Dendritic Bundles - Area 32 Layer 3/5 3O
ANIMAL 4
25 ~
~2o
S3
Mean: 42.93 SD: 16.81 n: 155
25
~2o E
Z15
r;
<10
<10 5 0
5
~,~ 2O 40 60 80 Intercluster Distance (gm )
0 100
0
20 40 60 80 Intercluster Distance ( ~Jrn )
100
t:ig. 6. Dislribufion histograms for samples of intercluster distances (IC p.m: see Fig. 5) between dendritic bundles in layer 2 and in la~ers 3 / 5 of Pt. cortex in animal 4. Mean values ( + S.D.) and sample size of individual dendritic bundles (n) are gi\,eu in each histogram.
84
P.L.A. Gabbott, S.J. Bacon/Brain Research Z~0 (1996) 75-86
in defining the number of profiles constituting a 'dendritic' bundle, these studies may have included large-calibre glial cell processes and unmyelinated axonal profiles or excluded small-calibre apical dendritic profiles - the latter exclusion error being most pronounced in the more superficial layers of cortex. 4.3. Dendritic bundles in prelimbic L~ersus cisual cortex
The number of central large-calibre dendritic profiles composing each cluster is similar between the PL and visual cortices (about 8 dendritic profiles - Table 3, p. 578, Ref. [21]; and Table 1, this study). However, differences between these two areas do occur in the mean 'centre-to-centre' intercluster distance (visual, 56 ~m; prelimbic, 45 ~m) and in the number of clusters under 1 mm 2 of cortical surface (visual, 360/ram 2 ; prelimbic, 7 5 9 / m m 2 ). These data indicate the much higher packing density of dendritic bundles in the PL area (proisocortex; Refs. [35,36]) compared with the primary visual area (neocortex) and that large-calibre dendrites form a quantitatively similar component of dendritic clusters in both cortical areas. The high number of dendritic profiles in the discrete clusters found in layer 2 compared with layer 3 / 5 is due to the addition of the apical dendrites of pyramidal neurones in upper layer 3 and lower layer 2. A similar finding is reported for other cortical areas including the visual cortex [10,11,13,18,21,29,37]. However, unlike other cortical areas, layer 2 in PL cortex contains a very high density of many small-sized pyramidal neurones - some of which possess two apical dendrites [35,38,39]. Additionally, the independent groups of apical dendritic bundles formed by pyramidal cells in layers 6, 4, and 3, that are found in visual and other cortical areas [21,37] were not conspicuous in prelimbic cortex. 4.4. Pyramidal cell modules in prelimbic cortex
Neurones in many cortical areas of the mammalian brain are arranged into functional columns on the basis of their specific physiological response properties [7,25,27,33,36]. This functional organisation is considered to be produced by anatomical modules that have similar neuronal compositions and internal connections but with different afferent and efferent pathways [7,25,31,33]. The fundamental structural subunits within a cortical module are thought to be the neurones and synaptic circuitry associated with a single bundle of apical dendrites originating f r o m pyramidal neurones in l a y e r 5 [6,7,13,22,23,25,27,31,33]. The structural subunit of which a single dendritic bundle forms the central component (see Fig. 5) can be conveniently modelled by two concentric cylinders extending from white matter to pia [21,22,26]. The data from the present study indicate that in the PL cortex of the rat the outer cylinder is approximately 41 Ixm in diameter with
the internal central cylinder, containing the bundle of radially aligned apical dendrites, being about 31 p~m in diameter. The region between the two cylinders would contain other neuronal (somata, dendrites and axonal arbors) and glial elements as well as cerebral vasculature [23,26]. Although electrophysiological and anatomical columns have not, as yet, been demonstrated in the rat PL cortex [36], a dendritic bundle together with its associated surrounding cylinder of neural tissue could therefore represent the common structural subunit of both functional and structural columns in the prelimbic cortex (see below). 4.5. Dendritic bundles in prelimbic cortex: q[lerents, ~ e r ents and Jimctional considerations
Speculations about the functional capacity of dendritic bundles have centred on their ability to correlate or synchronise the activity between vertically aligned sets of pyramidal cells in the superficial and deep layers of the cortex in response to afferent input [I,15,27,30] - particularly from the thalamus [17,36]. This being so, then the clustering of profiles associated with dendritic bundles in lower layer 3 of PL cortex may serve to enhance the electrotonic coupling between neighbouring apical dendrites in response to direct innervation from the mediodof sal thalamus [1.15,17,30,36]. In addition, facilitatory synchronisation within a dendritic bundle may involve specific "local circuit" GABAergic neurones that are also innervated monosynaptically by the same cortical afferents [35,36]. Indeed. little is known about the specificity and quantitative distribution of 'excitatory" and "inhibitory' connections within and between dendritic bundles [23,36]. The bundling of apical dendrites in the PL cortex may also be related to the distribution of other afferent pathways to the PL cortex [36], for example fi'om the amygdala [3] or hippocampus [16], and serve as their principle foci of termination [31]. Afferents from the basolateral nucleus of the amygdala to the PL cortex specifically innervate spine-bearing processes in layers 5 and 2 [3]. Importantly, individual amygdalocortical axons were found Io course vertically from layer 5 into layer 2 and give rise to a highly clustered axon field of varicose processes - these terminal arbors were approximately 100 tzm × 100 lain (see Fig. 4 in Ref. [3]. Individual axons could therefore provide selective innervation to approximately five bundles of apical dendrites in layer 2. Given the high degree of overlap between individual fibres in layer 2 and that the amygdalocortical projection is topographically organised, then entirely different sets of pyramidal cell modules would be innervated at different tangential distances within this layer [6,23]. Similar considerations may apply to the selective innervation of the prelimbic cortex by CA1 hippocampal afferents [16] and other afferent fibre systems [36]. Since "functional columns' in the cortex overlap, then the underlying neuronal machinery of the anatomical
P.L.A. Gabbott. S.J. B a c o n / B r a i n Re.~ea/'ch 7.¢() f19961 75 A'6
columns and consequently of the dendritic bundles must perform dilterent physiological tasks in parallel [7,33,36]. Such parallel and distributive processing is particularly suited to the imegrative function of the mammalian medial prefrontal cortex [36] and may underlie the integration of information from diverse afferent sources in the prelimbic cortex of the rat [ 16,17]. With regard to the output from PL cortex, each dendritic bundle in area 32 may contain the apical dendrites from a defined set of projection pyramids in layers 2 / 3 and 5 / 6 that project to a specific spectrum of other cortical fields and several subcortical targets - for exampie, to the insular, infralimbic, or cingulate cortices, the striatum, amygdala, thalamus, medulla or to the spinal cord
[2.36]. In conclusion, this study has demonstrated that dendritic bundles are a principal structural component of the rat PL cortex. Moreover, dendritic bundles may provide the elementary physiological units whose integration underlies specific aspects of the functional organisation of the PL cortex in the rat [ 16.17,31,36].
Acknowledgements The technical help of Paul Jays, Angela Robinson and Tracy-Ann Warner is acknowledged with gratitude. This research was supported by the MRC and the Belt Memorial Fund. P.G. is a Belt Memorial Research Fellow.
References [I] Amitai Y., Friedman A., Connors B.W. and Gutnick M.J., Regenerative activity in apical dendrites of pyramidal cells in neocortex, Cerebral ('orte~, 3 (1993) 26-38. [2] Bacon, S.J, and Smith. A.D., A monosynaptic pathway from an identified vasomotor centre m the medial prefrontal cortex to an autonomic area in the thoracic spinal cord, Neuros~ience, 54 (1993) 719 72~. [3] Bacon, S.J,, Headlam, J.N.. Gabbott, P.L.A. and Smith, A.D.. Amygdala mpul to medial prefrontal cortex (mPFC) in the rat. A light and electr, m microscope study, Brain Res., 720 (1996) 211 219. [4] Bernhardt, R. and Matus, A., Light and electron microscopic studies of the distribution of microtubule-associated protein 2 in rat brain: A difference between dendritic and uxonal cytoskeletons, ,I. Conq~. Neurol., 226 ( 1~841 2113 221. 15] Bolam. ,[,P. tEd.), £]rperime~ttal Neuroanatomv. A Practical Ap proach, IRL Pwss, OUP Oxford, 1992, !6] l)et>'er. K.. Course and distribution of apical dendrites of layer 5 pyramids in the barrel field and area purietalis of the mouse, Anat ['2mhrvol l Ber/in ~. 149 (1976) 251-258. 7] Eccles, J.C,, The cerebral neocortex. A theory of its operation. In E.G. Jones and A. Peters (Eds.), Ceret~ral Cortex. Vol. 2. Funclional Propertie~ ol C,,rti~'al Cell~. Plenum Press, New York, 1984, pp. 1-38. g ] Escobar, M.I, Pimienta, H., Caviness, V.S., Jacobson, M., Crandall, J.|~. and Ko,,ik, K.S.. Architecture of apical dendrites in the murinc
S5
neocortex: Dual apical dendrilic systems. Neuro~cieHcc, 17 11981~) 975-989. [9] Feldman. M.L., Morphology of the ncocorUcal pyramidal neuron. In A. Peters and E.G. Jones (Eds.). ('~'r~'hrol ('ortc.~. ~4d. I. Cellular Compeme*zts ¢~/' the Cerehral C~,rle~. Plenunl Press, New York, 1984, pp. 123-200. [10] Feldman, M.L. and Peters, A.. A stud 3 ol barrels and pyramidal dendritic clusters in the cerebral cortex, Brain Re.v, 77 (1974) 55-76 [I 1] Fleischauer. K., Petsche, H. and ~ ittko,,v,,ki, '~., Vertical bundles of dendrites in the neocortex, Z Anat [:)tmickl. 1~6 (1972) 213 223. [12] Fleischauer, K.. On different patterns of dendritic bundling in the cerebral cortex of the cat. Z Anal. Etttwick].. 143 (1974) 115 12~'~. [13] Fleischauer, K. and l)etzer, K.. Dendritic bundling in the cerebral cortex. In G.W. Kreutzberg ted.), Adl'allcc~ iH N~,u~wlogy. Vol. 12, Plly,~iolog 3 apul Patholovy c!t l)eJldritc~. Raven Pa-e',s. Nc~ York, 1975. pp. 71 78. [ 14] Fry, ,I.(7. ted.), Biolo~,i~'at Dora Amffvsi~. I I'ra~'li~'a/ ,ll~l,'r,a~'h. 1RE Press. OUP. Oxfl~rd, 1993, [15] Horowitz, B., Neuronal plasticfly: ho,a change,, m dendritic urchilcclure curt affect the spread of posls}llaplic f,oterltials, Brain Rev. 224 (1981} 412 -418. [16] Jay. T.M., GIo,ainski, J. and Thicrr 3. A.-M., Selcctivity of the hippocampal pr~jection to the Pl. area of the prefrontal cortex in the rat, Brain Res., 5(15 (I989)337-340. [17] Kumda M,, Murakami, K., Kishi, K. and Price..I.L., Thalamocorli cal synapses between axons fi-om the mediodorsal thalamic nucleus and pyramidal cells in the prelimbic torte,, of lhe rat. ,/. ('omp. Neurol., 356 (1995) 143-151. [18] l,ohmann, H, und KiSppcn, H.d., Postnatal development o1" pyramidal cell dendritic and axonal bundles in Ihe ~isual corte'~ of the rat, J. Brain R~,~., 36 (1995) 10[ 111. [19] Meyer, G.. Forms and spatial arrangement ~1 neurones in the primary motor cortex of man../. ('omp Nt, m-ol., 262 11987) 402 428. [20] Peters, A. and W"alsh, T.M.. A study of lhe organisation of apical dendrites in the somatic sensory cortex of the rat. I ('ontp, Neurol.. 14-4 11972) 253 268. [21] Peters, A. and Kara. I).A., The neuronal o,)nlposilioll ol urea 17 ol the rat \isual cortex. IV. The organisalion ~f pyramidal cells, ,/ Uomp. Neurol., 2(~0 (1987) 573-590, [22] Peters, A. and Sethares, C., Organisation of pyramidal neurones m area 17 of monke5 visual cortex, ,I. C~,np. Nemol., 31)611991 )1-23 [23] Peters, A., Pyramidal cell modules in rat xisual cortex. Their s t r u t lure and development. In S.C. Sharma and J.W. [:a,acetl (J(ds.). kormalion and Re generaliotl ~{/' Ver/c ('oHJ~cctirm,s. l:;irkhaser, Boston, 1993, pp. 102 120. [24] Peters. A. and Yihna/, E., Neuronal organisalion in arca 17 ~1 call xisual cortex, ('erehral Cort~cv, 3 (1993~ 4 ¢~S [25] Roney, KJ., Scheihel, A.B. and Shay,, (;.I,., l)endritic btmdles: Survey of anatomical experiments and physioh~gical theories, Brain Res. Re~., 1 11979) 225-271. [26] Sukui. M,, Dendritic bundles fl~rmed b 5 laver VI pyramidal cells m the monkey frontal association cortex, EIV~. ICraht Rc~., 5S (I<)N5) 609 612. [27] Schiebcl. M.E. and Schiebel. A.B.. Dendrites as neuronal couplers: The dendritic bundle. In M. Santini tEd. t. (;o&,i ('cttlemlio] A~Vml~Osi,m Proceedinq,s. Raven Press, New York. 1975. pp. 347 354. [28] Schierhorn. H., Postnatal development of lamina V pyramidal cells in sensorimotor c~rtex of albino rats, (~'k'¢'llll¢lttt~ Mo~7~hol .lahr/~., 1241197S) I 23. [29] Schmolkc, C., The ontogeny of dendrite bundles m rabbit visual cortex, Anat. Emt,Tol., 180 (1989) 37I 3Sl. [30] Shaw, G.L., Hath. E. and Scheibel. A.R., ('ooperativity in brain function: Assemblies of approximately t0 neurones. K~?~. Nettrol., 77 (1982) 324-358. [31] Shepherd, G.M., Apical dendritic spines of cortical pyramidal cells: Remarks on their possible roles in the higher brain functiolls,
86
[32] [33] [34]
[35]
[36]
P.L.A. Gabbott, S.J. Bacon/Brain Research 730(1996) 75-86 including memory. In G. Lynch (Ed.), Synapses. Circuits. and the Beginnings (~fMemory, MIT Press, London, UK, 1986. Swanson, L.W., Brain Maps: Structure c~["the Rat Brain, Elsevier, Amsterdam, 1992. Szentagothai, J., The 'module concept" in cerebral architecture, Brain Res., 95 (1975) 475-496. Tucker, R.P., Binder, L.I. and Matus, A.I., Neuronal microtubule-associated proteins in the embryonic avian spinal cord, J. Comp. Neurol.. 271 (1988) 44-55. Vogt, B.A. and Peters, A., Form and distribution of neurones in rat cingulate cortex: Areas 32, 24 and 29, J. Comp. Neurol., 262 (1981) 256-270. Vogt, B.A. and Gabriel, M. (Eds.) Neurobiology ~( Cingulate
Cortex and Limbic Thalamus. A ComprehensiL,e Handbook. Birkhiiuser, Basel, 1993. [37] White, E.L. and Peters A., Cortical modules in the posteromedial barrel subfield (SMI) of the mouse, J. Comp. Neurol., 334 (1993) 86-96, [38] Wyss, J.M., Van Groen, T. and Sripanidkulchai, K., Dendritic bundling in layer 1 o1" granular retrospleuial cortex: intracellular labelling and selectivity of innervatiom J. Comp. Neurol., 295 (1990) 33-42. [39] Wyss, J.M. and Van Groen, T., Early breakdown of dendritic bundles in the retrosplenial granular cortex of hypertensive rats: Prevention by antihypertensive therapy. Cerebral Corte~, 2 (1992) 468-476.
Brain Research 73(1 (1996) 75-86
Research report
The organisation of dendritic bundles in the prelimbic cortex (area 32) of the rat P . L . A . G a b b o t t *, S.J. B a c o n Unicersity Department (~]'Pharmacol.
Accepted 2 April 1996
Abstract Using an antibody against microtubule associated protein 2 (MAP-2: a specific marker fl)r neuronal dendritesL this paper reports the structural organisation of pyramidal cell apical dendrites in the rat prelimbic (PL) cortex. In the coronal plane, MAP-2-immunoreactive apical dendrites of pyramidal neurons in layers 5, 3 and 2 were found bundled together as they ascended radially through the cortex. These bundles of dendrites dispersed in upper layer 2 to form apical dendritic tufts in layer 1. In tangential cross-section, thc immunolabelled bundles were organised into a latticework of discrete clusters of differentially sized profiles. At the boundary between layers 3 and 5. clusters were composed of 26 + 8 dendritic profiles (group mean value + S.D,, five animals), whereas clusters in lavcr 2 contained 55 ± 15 profiles. The number of clusters per unit surface area was not significantly different throughout layers 5, 3 and 2 (760 + 75 per mm 2) with the average centre-to-centre intercluster distance in these layers being 44.2 ± 4.9 gin. The data indicate that apical dendritic bundles are a feature of the radial organisation of PL cortex. These structural subunits may subserve specific integrative functions in the PL area of the rat medial prefrontal cortex. Kevworet~': Medial prefrontal cortex: Cortical module: Pyramidal neuron; Apical dendrilic bundle/cluster; IAmbic/autonomic corlcx
1. Introduction
In the cortex, groups of pyramidal cell apical dendrites aggregate to form distinct vertically aligned bundles [7,23,28,33,37]. The most prominent grouping of dendritic bundles arise from sets of large- and medium-sized pyramidal neurones situated in layer 5 [10,11,13,18,21,23,37]. In the tangential plane, the bundling of apical dendrites is most apparent by the discrete clustering of dendritic profiles at the level of layer 4 - the main thalamorecipient territory of granular areas of the cortex [21,23]. This feature of cortical organisation has been reported in several cortical areas for a variety of mammalian and primate species [6-13.18-33,37-39]. Indeed, it is now thought that a single dendritic bundle, together with the associated neurone populations and synaptic circuitry, represents a fundamental structural subunit underlying specific func-
Cc,rrespondine author. Fax: *-44 (1865) 27-1853: E-mail: paul.gabboil @pharm x~x.ac,u k
tional properties in the cerebral cortex [ 1,7,13,15,23,25,30-33]. The bundling of apical dendrites has not been previously reported in the medial prefrontal cortex (mPFC) of the rat [36]. The aim of this study was to establish whether dendritic bundling was also a structural feature of lhe prelimbic (PL) region (Brodmann area 32) of mPFC in the rat. The approach was to use a specific cytoskeletal marker for dendrites, microtubule associated protein 2 (MAP-2) [4,34], in order to qualitatively and quantitatively investigate the tangential and vertical organisation of dendritic bundles in rat PL cortex. Since PL cortex in the rat is an agranular area (i.e. lacks a cytoarchitecturally distinct layer 4: see Fig. IC), this study concentrated on the distribution of dendritic bundles in upper layer 5 to mid-layer 3 and in layer 2. Of significance is that in PL cortex, the region upper layer 5 / m i d layer 3 is the principal region of innerwltion from the mediodorsal nucleus of the thalamus [17,38], while layer 2 is a region of dense bundling fl)und in other cortical areas
O006-8993/96/%15OO ('op,,right ~, 1996 Elsevier Science B.V. All rights reserved. /W S0006-,";9u ~(,76)0043 7-4
76
P.L.A. Gabbott, S.J. Bacon / Brain Research 730 (1996) 75-8h
[21,23,37,38] and in PL cortex it receives a laminar specific input from the amygdala [3].
B ) PARASAGITTAL t
2. Materials and methods
2.1. Tissue .fixation
Eight adult Sprague-Dawley rats of both sexes (150-250 g) were used in this study. These animals had been deeply anaesthetised and transcardially perfused with a solution of aldehyde fixatives containing 0.05-0.5% glutaraldehyde and 3-4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) [5]. Following perfusion, the brains were removed and serial Vibratome sections (100 Ixm thick) obtained from the parasagittal or coronal planes through the mPFC ([33]; see also Fig. 1A, B). Tissue sections cut parasagittally were kept in order so that their corresponding depth below the pial surface of the medial bank was known (Fig. 1C). Sections were then transferred to Tris-HCl (Tris) buffer (pH 7.4) and processed either for immunocytochemistry using an antiserum against MAP-2, or for conventional post-fixation with OsO 4, dehydration, and resin embedding
~],
ANTERIORB÷3"2mm
C
AOd
LATERAL
0'4mrn
CORONAL
[51.
Pl
2.2. Anti-MAP-2 immunocvtochemistr 3'
Depth o pm
Sections were initially freeze-thawed or permeabilised by treatment with 0.25% Triton X-100 [5] and incubated as follows: 20% normal goat serum; anti-MAP 2 mouse monoclonal (Sigma Chemicals, UK: clone no. HM-2, product no. M 4403) diluted 1:250-1:5000; anti-mouse ABC kit (Vector Labs, UK). Immunoreactivity was visualised using 0.05% 3,3'-diaminobenzidine (DAB) and 0.01% H202 in Tris buffer (pH 7.4). In some sections, immunoreactivity was revealed using the Vector SG (slate grey) peroxidase substrate kit (Vector). Between the incubation steps, sections were thoroughly washed (3 × 10 min) in Tris buffer (pH 7.4). Sections were then mounted in series on gelatin coated glass slides, air-dried, passed through an ascending series of alcohols, taken through xylene and finally embedded in DPX mountant [5]. Several control sections per animal were either incubated without the primary MAP-2 antiserum or by omitting the biotinylated link antibody of the ABC kit. 2.3. Resin embedded semithin sections
Parasagittal sections (100 ~m thick) from layers 2-5 of PL cortex were postfixed with 1% OsO 4 in 0.1 M phosphate buffer (pH 7.4) for 45 min, dehydrated in a graded series of alcohols (including 1% uranyl acetate in the 70% alcohol for I h in the dark), passed swiftly through propylene oxide, flat-embedded in Durcupan resin, and cured for 2 days at 56°C [5]. Selected regions of PL cortex at
1
2
25o
3 5OO
750
6a
moo
bJ 1250 Fig. 1. A, B: line drawings showing the division of the medial prefrontal cortex (mPFC) into cyloarchitectonic areas in the coromd plane (approximately + 3.2 mm anterior to bregma (B)), and in the parasagittal plane (approxmlately 0.4 mm lateral to midline). The prelimbic cortex (PL-area 32) is indicated. Surrounding areas: PrCm. medial precentral cortex: ACd, dorsal anterior cingulate cortex: ACv, ,,entral anterior ciugulate cortex: and IL, infralimbic cortex. Orientation markers: D. dorsal: V, ventral: C, caudal. The corpus callosum is shown shaded. Scale bar = 500 Fro. C: coronal Nissl-stained section of prelimbic cortex (PL). The cytoarchitectural lamination of PL cortex (layers I (~b) is given on the left ol Ihe photomicrograph, and a scale marker indicating depths below the pial surface (Ixm) is given to the right of the photograph. (pitt. pial surfacc:wnl, white matter).
P.L.A. Gabhott. S.J. Bacon/Brain Re.~ear¢'h 730 ~1996~ 75 ~'6
different depths below the pial surface were then carefully excised amt glued onto a blank resin stub [5]. Serial semithin resin sections (I.0 bun in thickness) were subsequently cut using an ultramicrotome and collected in sequence on gelatin coated glass slides. These sections were counterstained with an aqueous solution of 1% toluidine blue and 0.59; sodium borate, washed and dried, and finally mounted in DPX [5]. 2.4. Cvtoarchitecture q f rat m P F C
Additional complete sets of unreacted serial parasagittal and coronal sections were Nissl stained, dehydrated and flat-embedded in DPX. These sections enabled the identification of the cellular lamination and areal cytoarchitecture in mPFC to be defined and helped to delineate the tangential extent of PL cortex (area 32) in parasagittal sections ([32,35]: see Fig. I A - C ) . 2.5. Qualitatirc examination
MAP-2 immunoreacted sections were examined in a photomicroscope equipped with a drawing tube. The appearance of dendritic bundles and clusters in these sections was compared with their appearance in the counterstained semithin resin sections. Structures of interest were recorded through photomicrographs and drawings. The aggregation of apical dendrites in the coronal plane are called 'bundies', whilst in the parasagittal plane they are referred to as "clusters'. (Note that parasagittal sections through the PL area are parallel with the pial surface and reveal the tangential (or horizontal) structure of the cortex.) 2.6. Quontitatize study
The composition and tangential distribution of MAP-2 immunoreacted dendritic clusters at two horizontal sampiing levels m the PL cortex were analysed quantitatively': (i) in layer 2 ( 2 0 0 - 3 0 0 g m below the pial surface: Fi X. IC), and (it) in a region extending from mid-layer 3 to upper layer 5 (400-700 p~m below the pial surface: Fig. IC). Since the latter sampling zone centres on the boundary between layer 3 and layer 5 it is subsequently called l a y e r 3 / 5 ' . (Rat PL area is agranular: see Fig. IC,) Sections could be confidently ascribed to a given cortical lamina (or region within a layer) on the basis of: (i) MAP-2 neuronal staining characteristics, (it) the depth of the section below the pial surface (Fig. IC), and (iii) the cytoarchitecture of the corresponding Nissl stained parasagittal section (Fig. I C). In parasagittal MAP-2 immunoreacted sections, individual dendrites could be clearly recognised and their assembly into discrete clusters was clearly evident (see Section 3). The tangential organisation of dendritic clusters in the t'L cortex most closely resembled a 'hexagonal packing" system (see Fig. 4A, B and Fig. 5) - such a system has
"7
been previously described lor dendritic clusters in the rat visual cortex [21]. Consequently, this packing model was used to derive several quantitative parameters (see below: see also Fig. 5). Sections were viewed in the light microscope ( × 4 0 objective lens and × 10 eyepieces) and a square "lest" counting frame (250 × 250 txm) superimposed on the section through a drawing tube. Each counting frame had an upper and right-side inclusion border and a lower and left-side exclusion border [14]. The centres of dendritic clusters were defined visually ([14], see also [21]). The cluster centres that were inside the "test" area of the counting frame, were marked onto the frame. All the MAP-2-immunoreactive dendritic profiles constituting a 'test' dendritic cluster were drawn onto the countmg" " frame. Boundaries were also drawn by joining the outlying dendritic profiles (using a nearest neighbour procedure: {14]) around each cluster (Fig. 5) 1. In addition, at high magnification (X 100 oil immersion objective lens and × 10 oculars) the number of large-calibre ( > 2.0 ixm) and smallcalibre ( < 2.0 Ixm) dendritic profiles per cluster were calculated for a sample of dendritic bundles (n = 25) al both smnpling levels in each animal. Using a computerised planimeter (Macintosh Quadra operating customised stereology software) the fl~llowing morphometric parameters were recorded or measured l~)r each dendritic clnster (Fig. 5): (1) the total number of dendritic profiles (large- and small-calibre) per bundle (NP), (2) the number of 'test" dendritic clusters under 1 ram: of cortical surface (NC~). (3) centre-to-centre intercluster distance between neighbouring clusters (IC txm) (see Peters and Kara [21] pp. 758-759 lk)r a discussion of "centre-to-centre' versus "nearest neighbour' distances between clusters), (4) the tangential area occupied by a dendritic cluster (CA berne), (5) the tangential area (TA > m -~) of cortex associated with each dendritic cluster. calculated as [TA = (1 m m 2 / N C x ) ] , and (6) the percentage ratio that CA composed of TA [ ( C A / T A ) X 100%]. 2.7. SamplinA, am/statistical analysis
In each of fi~e animals, ten counting frames were applied to parasagitlal sections to examine the clustering of
i In a preliminary sludy, the centre of an irregularly shaped dendritic cluster was visually defined 24 times. Subsequent calculations established that a comparatively small error (coefficient of variation 1.2% in mean r,3 coordinates: [14]) was introduced using lifts subjeclive centring technique. Further, the standardised x,v positions of a sample of visually defined cluster centres (n= 12) were nol statistically different ~o .~,v coordinates of the same clusters generated by a computeriscd planimcter. Additionally, permtffations in drawing the boundar? to a gJ,~cn dendritic cluster produced a coefficient of wtriafion ot 2.6~ in the mean surface area (parameter CA, see main text). These potential sources of error are therefore considered non-significant m d~e derivation of corresponding quantitati,.e data Ik~r Im-ge populations of dendritic clusters ll4].
78
P.L.A. Gabbott. S.J. Bacon/Brain Rese~rch 730(1996) 75-80
P.L.A. Gabbott, X,I. Bacon/Brain Research 730 f1996) 75-S6
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Fig. 3. A: photomicrograph illustrating MAP-2-immunopositive pyramidal neurones (P) in a coronal section through upper layer 3 and layer 2 t>l' PI_ cortex. The somatic cytoplasm and apical dendrites (arrow) of these neurones are strongly labelled. Scale bar - 2(1 ,urn. 13: langential semithin (I pore thick) resin section through mid-layer 3 of PL cortex stained with toluidine blue. The clustering of processes (an'ows) ascending through the prelimbic cortex is readil~ apparent at this level. The somatic profiles of neighbouring neurones (n) are visible. (c+ capillaries). Scale b a r = 50 ++tm. C: high magnification photomicrograph of a toluidine blue-stained coronal semithin resin section shov, ing a dendritic bundle (thick black arrow) in upper layer 3 ol PL cortex. Large dcridritic profiles comprising a bundle have been indicated by small black dots. The profile of a transversely sectioned process is also indicated
dendrites in layer 2. and ten counting flames were used to examine dendritic clusters in the region layer 3 / 5 (Fig. 1). Mean animal values for each parameter were calculated for clusters in layer 2 and in the region layer 3/5. Mean group values were subsequently derived. Layer 2 and layer 3 / 5 data were subsequently compared statistically using ~NOVAs and post-hoe t-tests [13]. Significant differences were considered to occur when P < 0.05.
3. Results 3. I. Antiserum spec~/icity
The biochemical and cytological specificity of the MAP-2 antiserum used in this study has been reported previously [4,34]. In control sections, incubated either without the primary antiserum or the biotinylated link
t:ig. 2. A: photomicrograph of an unstained coronal Vibratome section through the superficial layers ( I - 3 ) of the PL cortex (area 32) laken rising high contrast optics. The profiles of processes bundled closely together (thick arrows) are clearly visible ascending through the cortex. The bundles disperse on entering layer I. Unstained neuronal somata (n) are indicated, capillaries (c) are also shown. Scale bar 5() p.m. B: rnicrograph of a ',ection through layers 3 and 5 in PI, cortex, reacted immunocytochemically for MAP 2. The somatic cytoplasmata of pyramidal neurones (P) in layer 5 are immunopositive. The ;~pical dendrites of these cells are also strongly immunolabelled (arrows). Indicated is the clustering of processes ascendin~ through the superficial layers of the cortex (boxed region in layer 3). Note the difference in intensity of immunolabelling betv, een the apical dendrite "rod soma ol the pyramidal cell indicated (P'). Also note the emergence of an imrnunolabelled basal dendrite from the cell body of this pyramid (small arrow), c. capillaries. Scale bar 50 gin. C: low magnification photomicrograph of a section reacted immunocytochemically for MAP-2. Bundles of strongly labelled MAP+2+ immunoreactive dendrites (arrows) traverse through layers 1-3 of PL cortex. The somata of unlabelled neurones are indicated (black dots). Scale bar :: 100 [{ Ill.
8[)
P.L.A. Gabbott, S.J. Bacon/ Brain Research 730 (1996) 75 86 sections through the PL cortex, i m m u n o r e a c t i v e dendrites appeared as dark puncta or dark strands aligned along the axis of viewing (Fig. 2D and Fig. 3A). In particular, the apical dendrites of pyramidal neurones, at all levels within the cortex, were strongly immunoreactive, with their perikarya displaying moderate M A P - 2 i m m u n o l a b e l l i n g (Fig. 2B and Fig. 3A). No M A P - 2 - i m m u n o r e a c t i v e dendritic spines were found along i m m u n o l a b e l l e d dendritic processes (Fig. 2B and Fig. 3A, D, E).
antibody, there was no specific i m m u n o l a b e l l i n g of cellular processes nor cell bodies. Control sections possessed a characteristic non-specific light-brown D A B (or light-grey SG) coloration. 3.2. Unstained coronal sections In unstained sections, the b u n d l i n g together of vertically aligned processes can be clearly identified in PL cortex (Fig. 2A). These bundles began at the level of mid-layer 5 and c o n t i n u e d u p w a r d through the cortex until reaching upper layer 2 where they began to diverge and radiate outwards within layer 1 (Fig. 2A). In m a n y sections, the somata of unstained cells in layers upper 5, 3, and 2 were c o m m o n l y located b e t w e e n the unstained ascending b u n dles of processes - these b u n d l e s appeared to be regularly spaced apart (Fig. 2A).
3.4. Qualitati+,e obseruations 3.4.1. Coronal plane In coronal sections, M A P - 2 - i m m u n o r e a c t i v e pyramidal cell apical dendrites could be clearly seen to arise from parent somata that were also i m m u n o l a b e l l e d (Fig. 2B and Fig. 3A). Individual labelled dendrites could be traced from cell bodies in layer 5, through layer 3 and into the upper zone of layer 2, before bifurcating repeatedly into a terminal tuft of apical dendrites in layer I (Fig. 2B, C). The most p r o m i n e n t b u n d l i n g of M A P - 2 - i m m u n o reactive dendrites occurred over the region of cortex extending from upper layer 5 to the layer 2 / 1 border and originated from pyramidal neurones situated in deep- to mid-layer 5 (Fig. 1C; Fig. 2B, C; Fig. 3A). The labelled somata of pyramidal neurones in layer 5 were situated
3.3. MAP-2 immunolabelling in prelimbic cortex In coronal and parasagittal sections through PL cortex, the dendrites of n e u r o n e s in layers 1 - 6 were M A P - 2 - i m m u n o r e a c t i v e (Fig. 2B, C and Fig. 3A). I m m u n o p o s i t i v e dendrites in coronal sections appeared as b r o w n D A B (or grey SG) labelled processes against a comparatively clear b a c k g r o u n d (Fig. 2B; C and Fig. 3A). In parasagittal
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Fig. 4. A: distribution of MAP-2-immunolabelled profiles at the level of mid-layer 3 in PL cortex. Distinct clusters of dendritic profiles are indicated (arrows) and one is shown encircled. (c, capillaries)• Scale bar = 10l) ~xm. B: light microscope drawing showing the tangential distribution of dendritic bundles from the middle of layer 3 in PL cortex. Several dendritic bundles are indicated (arrows). The centres of clusters surrounding a central cluster have been linked together to indicate the predominant hexagonal packing of dendritic clusters• This packing pattern of packing is not perfect since occasional pentagons and heptagons need to be included to complete the lattice (see also Ref. [21]). Additional groups of dendritic profiles occur between the recognised clusters (one example is shown encircled). Scale bar = I00 rxm.
P.L.A. Gabbott, S.Z Bacon / Brain Research Z~'O (•996) 75-86
directly beneath the bundle to which their apical dendrites contributed. The immunolabelled apical dendrites of more superficially located pyramidal neurones in layers upper 5, 3 and 2 joined the periphery of neighbouring bundles (Fig.
IB). The bundling together of immunolabelled apical dendrites from other groups of pyramidal neurones in layers 6a and 3 of PL cortex were present - but their occurrence was not common. Frequently located between immunolabelled dendritic bundles were the cell bodies of moderately immunolabelled or unstained cells (Fig. 2C). In the coronal plane, it was evident that not all labelled dendrites engaged in clustering (Fig. 2B).
3.4.2. Parasagittal plane In the parasagittal plane, the tangential clustering of MAP-2 immunoreacted dendritic profiles are principally identified by the aggregation of the cellular profiles of large apical dendrites (Fig. 3D, E and Fig. 4A). Such a clustering correlates directly with the vertical bundling of profiles seen in the coronal sections, as described above. Dendritic clusters in both layer 2 and the region layer 3 / 5 were defined as being composed of differing numbers of differentially sized MAP-2-immunolabelled profiles that were closely aggregated (see below). Clusters of dendritic profiles were found as discrete structural entities situated between weakly immunolabelled neuronal somata and unstained cell bodies (Fig. 4A). It was uncommon to find a cell body located in the middle of a cluster. The most prominent clustering of labelled protiles occurred in the region of layer 3/5 (Fig. 3El, but
~I
were also clearly evident at the level of mid-layer 3 (Fig. 4A) and particularly in layer 2 (Fig. 3D). In the region layer 3/5, MAP-2 clusters typically contained large-calibre profiles (about 8), with numerous small-calibre profiles (about 18) commonly distributed towards the centre of each cluster (Fig. 3El. In layer 2, the frequency of the small-calibre profiles within a cluster greatly increased (about 40) (Fig. 3D; Table 1). Dendritic clusters in layer 2 had a full intermixing of dendritic profiles so that the smaller-sized dendritic profiles were found distributed throughout an individual cluster (Fig. 3D). In the parasagittal planes through layers 3 / 5 and 2, nol all of the cross-sectioned MAP-2 profiles formed dendritic clusters (Fig. 3D, E; see also Fig. 4A. B). These profiles occurred either singly or in small groups of 2-6 profiles and did not appear to be specifically, associated with clusters. Note that large-calibre dendritic profiles were sometimes present among these groupings. It was nol ascertained whether such profiles formed independent dendritic clusters (Fig. 4B). The tangential distribution of clusters in the parasagittal plane through the PL cortex was not random, but organised into a lattice (see below and Fig. 4B, Fig. 5). Hexagons were the most frequent shape produced by joining chlster centres (20: Fig. 4B). However, polygons with differing numbers of sides (frequently pentagons and heptagons) had to be incorporated to complete the "honeycombed" latticework [20]. Bundles of basal dendrites from pyramidal cells were not observed at any level of the cortex in parasagittal or coronal sections (Figs. 2-4).
Fable I M o r p h o m e t r i c data c o n c e r n i n g the tangential c o m p o s i t i o n and distribution of M A P - 2 - i m m u n o l a b e l l e d dendritic clusters in parasagiual sections from layer ~ / 5 ~' and layer 2 of the PL cortex in the rat ( m e a n values + S.D.. n = 5 animals: range given in parentheses)
I ) Total n u m b e r of dendritic profilesper bundle (NP) Large-sized profiles per bundle Small-sized profiles per bundle 12) N u m b e r of dendritic bundles under I mm-' of pial surface INdC a) !3) lntercluster distance (IC g i n ) I centre-to-centre distance between n e i g h b o u r i n g clusters) 14) Clnster area ( ( ' A g i n " ) (area bounded by dendritic profiles) (5) Tangential area associated with each cluster (FA i,tm 2) 16) Percentage ralio flint C A occupied of TA I [ C A / T A ] x 100ci )
L a y e r 3 / 5 ~'
Layer 2
26.3 ± 7.8 (10-42) 8.4 ± 2.3 17,9 _+ 5.7 737.8 ± 81,9 (678.9-775.0) 45.1 + 4.9 (15,7-89.2) 619.6 ± 91.9 (242.6-I126.0) 1359.8 ± 79.3 (1072.9 1406.1) 45.6 + 5.9 (22.6-80.1)
55.1 + 14.7 ~15 -70) 15.3 ± 6 5 39.8 + I 1.3 779.2 + t,~l).3 1711.2 932.1) 43.2 + (~7 ( 12. I - 95.3 ) 854.(~ z 151.6 (311.5 1338,4) 1283.5 ~ 121.4 11(190.3-1472,8) 66.6 + 7 I (24.1-90.c,0
:' L a y e r 3 / 5 i~ tile region of cortex between mid-layer 3 and m i d - l a y e r 5. Statistical analysis between layer 3 / 5 and layer 2 data: ( I ) NP: mean value differences significant: two-tailed t-test, t = 3.869. P < 0.005, n = 8. (2) N d C x : mean vahte differences not significant: two-tailed t-test, t = ().759, P = 0.469, n = 8. ( t ) IC: mean value difli~rences not significant: two-tailed t-test, t = 0.512, P = 0.623. n - 8. (~t) CA: mean value ditferences significant: two-tailed t-test, t - 3.436, P < 0.001, n 8. iS) TA: mean value differences not significant: two-tailed t-test, t = 1.177, P ~ 0.273, n - 8. (,~) [ C A / T A ] ~ ; : mean value differences significant: two-tailed t-test, t = 5.086. P < 0.001. n = 8.
82
P.L.A. Gabbott. S.J. B a c o n ~ B r a i n Research 730 (1996) 7 5 - 8 6
P
Fig. 5. Schematic diagram illustrating the 'perfect' tangential organisation of dendritic clusters in PL cortex. The diagram shows the quantitative parameters measured in this study: IC, the centre-to-centre intercluster distance (m); NP, the number of dendritic profiles per cluster (shown as clear profiles of varying sizes within the boundary perimeter of the cluster); CA, the tangential surface area bounded by a dendritic cluster (txm2); NC A, the number of dendritic clusters per unit tangential surface area of tissue (expressed as number under 1 mm: of pial surface); and TA, the tangential area (txm 2) associated with each cluster. The mean values of these parameters were calculated for a sample of 22-48 dendritic clusters per animal (n = 5). Group mean values were subsequently derived. Note that by joining the centres of each cluster in this 'ideal' model produces a perfect bexagonally packed ~honeycomb' lattice. Scale bar = 50 Ixm.
Of significance is that the mean number of immunolabelled profiles per cluster in layer 2 was 110% greater than in layer 3 / 5 - this was related to an increase in the number of both large-calibre ( + 8 2 % ) and small-calibre ( + 122%) profiles. The increased number of profiles per cluster (NP) in layer 2 had consequent effects on the mean area associated with an individual cluster (CA) which was 38% greater than for layer 3 / 5 clusters. Whereas the mean tangential areas associated with each cluster (TA) were not significantly different between the two sampling levels, significant differences were detected between layer 2 and layer 3 / 5 in the ratio that CA composed of TA (Table 1: [(CA/TA) × 100%]). Dendritic clusters in layer 2 occupied a significantly greater proportion of the area associated with each cluster than in layer 3/5. The mean centre-to-centre intercluster distances ti)r clusters in layers 3 / 5 and 2 were directly measured as 45 I~m and 43 Ixm, respectively (Table 1)2 Fig. 6 shows distribution histograms of centre-to-centre intercluster distances for populations of MAP-2 immunolabelled dendritic clusters in parasagittal sections through layer 2 and in layers 3/5.
4. Discussion
The clustering of cellular profiles was also clearly evident in tangential semi-thin sections through layers 2 and 3 / 5 stained with toluidine blue. These sections served to corroborate the observations made from MAP-2 immunoreacted material. In semi-thin sections, profile clusters were composed of a variable number of circular and ovoid profiles, of varying sizes, aggregated together into discrete units (Fig. 3B, C). In layer 3 / 5 , large-calibre profiles were located prominently at the centre of each cluster, with smaller calibre profiles frequently occurring throughout a bundle (Fig. 3B, C). Similar to MAP-2-stained sections, the number of small-calibre profiles associated with bundles in layer 2 was greater than for clusters in the region layer 3/5.
The present study has used a specific antibody against MAP-2 (a recognised marker for neuronal dendrites; Refs. [4,34]), to provide qualitative and quantitative evidence that pyramidal cell apical dendrites in layers 2-5 of the rat prelimbic cortex (PL - area 32) are not randomly distributed, but are organised into discrete radially aligned bundles. In parasagittal sections, these bundles were clearly recognised as distinct clusters of MAP-2 immunolabelled dendritic profiles. The dendritic clusters were readily apparent at both the level of layer 3 / 5 and in layer 2. Quantitative data indicate that although there was no significant difference in the number of clusters per unit area nor in their spatial distribution in these tangential strata~ significant differences did occur in the mean number of profiles composing dendritic clusters between the two levels in PL cortex.
3.6. Quantitative data
4.1. Dendritic bundles in other cortical areas
Quantitative data concerning the composition and spatial distribution of clusters of MAP-2 immunolabelled profiles in parasagittal sections through PL cortex are given in Table 1 (see also Fig. 5). Table 1 provides separate data from layer 2 and from the region layer 3/5. The data indicate that whilst the overall spatial distribution (parameters - NdCA, IC, and TA) of MAP-2 profile clusters were not significantly different between layer 3 / 5 and layer 2, significant differences occurred in the mean composition of each cluster of profiles (parameters - NP and CA) at these two levels in the cortex.
Dendritic bundles have been described previously in the cortices of several mammalian species - the visual cortex
3.5. Resin embedded sections
-" Using the mean TA values and assuming a 'perfect' isohexagonal packing system (Fig. 5), mean centre-to-centre values betwen dendritic clusters can be calculated trigonometrically for layers 3 / 5 (40 tam) and for layer 2 (38 gtm). Alternatively, taking mean TA values, the diameters of the area equivalent circles (D~ir~) associated with dendritic clusters in layers 3 / 5 and layer 2 are 42 g m and 40 ixm, respectively (1),i, ~ = 2. [ T A / v ] ' s ) . These values for 'idealised" packing models are in c(nsc agreement with the directly measured mean centre-to-centre intercluster distances of 45 ixm for layer 3 / 5 and 43 ~ m for layer 2.
P.L.A. Gabbott, S,J. Bacon/Brain Re,search 7,¢0(1996) 75-~'6
of the rat [ 10,11,18,21 ], rabbit [29], cat [ 12,13] and monkey [22], in the rodent posteromedial barrel subfield (PMBSF) of primary somatosensory cortex (SM 1) [6,8,20,28,37], the retrosplenial cortex of the rat [38,39], the frontal cortex of the monkey [26], and in the human motor cortex [19]. The most comprehensive description of cortical dendritic bundling has been provided by Peters and Kara [21] for the primary visual area of the adult rat. In rat visual cortex, the apical dendrites of pyramidal neurones situated in lower layer 5 aggregate to form the central core of a vertically oriented dendritic bundle. The apical dendrites of smaller sized pyramidal cells in upper layer 5 join these bundles and together they ascend in close proximity through the upper layers of the cortex. In the supragranular layers, the bundles are further joined by the apical dendrites of some neighbouring layer 3 and layer 2 pyramidal cells. The apical dendritic bundles begin to disperse in upper layer 2 and divide repeatedly to form apical dendritic tufts in layer 1. Independent dendritic bundles are formed from the apical dendrites of other pyramidal neurones in layers 3 and 2 [21]. Additional bundles are also formed by pyramidal neurones in layer 6a and in layer 4 [21,26]. However, these bundles are not specifically associated with the bundles derived fi'om the neurones in layer 5 nor from those in the supragranular layers. In granular areas of the cortex the bundling of dendrites is most readily apparent within layer 4 [21,23]. The observations of the present study are qualitatively similar to the studies of Peters and Kara [21], White and Peters [37] and to the other studies investigating dendritic bundling in several cortical areas across a range of mammalian species [13,19,22,28]. However, one specific difference between areas concerns the contribution of layer 2 pyramidal cells to apical dendritic bundles. In prelimbic cortex, apical dendritic bundles remain highly clustered in layer 2 (cf. SMI. motor and visual areas of cortex) and closely resemble the dendritic bundling found in granular retrosplenial cortex of the rat [38,39].
4.2. Composition qf" dendritic bundles and intercluster distances The data of Peters and Kara [21] indicate that each cluster, at the level of layer 4 in rat area 17, was composed of 3-15 (mean 8) apical dendrites from large pyramidal cells in lower layer 5 and that dendritic clusters had a mean "centre-to-centre" spacing of 55-60 txm. For con> parison, the mean "centre-to-centre" distance between dendritic clusters in the visual cortex of the monkey was calculated as 31 Fm [26], whereas in the cat visual cortex the mean value is 56 >m [24]. In the mouse PMBSF, White and Peters [37] determined that the mean intercluster distance was about 25 g m in the barrel hollows and approximately 22 b~m in the barrel walls. The size of neurones, differing neuronal densities, and the specific architectures of layer 4 (particularly in mouse SMI) may underlie these species differences ([24] cf. [26] cf. [37]). In the rat visual cortex, Peters and Kara [21] found that not all of the apical dendrites of layer 5 pyramidal neutones engage in bundling. They calculated that al the level of layer 4. 12 large-sized and 38 medium-sized pyramidal neurones were 'associated' with an individual dendritic cluster (see Table 2 in Ref. [21]). Of the large-sized dendrites, approximately two-thirds (J~=8) were ctmtained in clusters (see Table 3 in Ref. [21]) and the somata of these cells were closely situated within layer 5 and lay directly beneath their clustered apical dendrites [21]. By comparison, the apical dendrites of the neurones that did not engage in bundling rose through the cortex either singly or in pairs passing around the bundles of clustered dendrites. Furthermore, Peters and Kara calculated that under I mm e of cortical surface there were approximately 359 dendritic clusters in layer 4 [21]. It should be mentioned that most of the studies cited above used toluidine-blue stained semithin sections to derive information concerning the bundling of apical dendrites in the cortex (cf. the present study using MAP-2 Fig, 3B~ C cf. D, E, see also Refs. [22,37]). Consequently,
Dendritic Bundles - A r e a 32 Layer 2 30
Dendritic Bundles - Area 32 Layer 3/5 3O
ANIMAL 4
25 ~
~2o
S3
Mean: 42.93 SD: 16.81 n: 155
25
~2o E
Z15
r;
<10
<10 5 0
5
~,~ 2O 40 60 80 Intercluster Distance (gm )
0 100
0
20 40 60 80 Intercluster Distance ( ~Jrn )
100
t:ig. 6. Dislribufion histograms for samples of intercluster distances (IC p.m: see Fig. 5) between dendritic bundles in layer 2 and in la~ers 3 / 5 of Pt. cortex in animal 4. Mean values ( + S.D.) and sample size of individual dendritic bundles (n) are gi\,eu in each histogram.
84
P.L.A. Gabbott, S.J. Bacon/Brain Research Z~0 (1996) 75-86
in defining the number of profiles constituting a 'dendritic' bundle, these studies may have included large-calibre glial cell processes and unmyelinated axonal profiles or excluded small-calibre apical dendritic profiles - the latter exclusion error being most pronounced in the more superficial layers of cortex. 4.3. Dendritic bundles in prelimbic L~ersus cisual cortex
The number of central large-calibre dendritic profiles composing each cluster is similar between the PL and visual cortices (about 8 dendritic profiles - Table 3, p. 578, Ref. [21]; and Table 1, this study). However, differences between these two areas do occur in the mean 'centre-to-centre' intercluster distance (visual, 56 ~m; prelimbic, 45 ~m) and in the number of clusters under 1 mm 2 of cortical surface (visual, 360/ram 2 ; prelimbic, 7 5 9 / m m 2 ). These data indicate the much higher packing density of dendritic bundles in the PL area (proisocortex; Refs. [35,36]) compared with the primary visual area (neocortex) and that large-calibre dendrites form a quantitatively similar component of dendritic clusters in both cortical areas. The high number of dendritic profiles in the discrete clusters found in layer 2 compared with layer 3 / 5 is due to the addition of the apical dendrites of pyramidal neurones in upper layer 3 and lower layer 2. A similar finding is reported for other cortical areas including the visual cortex [10,11,13,18,21,29,37]. However, unlike other cortical areas, layer 2 in PL cortex contains a very high density of many small-sized pyramidal neurones - some of which possess two apical dendrites [35,38,39]. Additionally, the independent groups of apical dendritic bundles formed by pyramidal cells in layers 6, 4, and 3, that are found in visual and other cortical areas [21,37] were not conspicuous in prelimbic cortex. 4.4. Pyramidal cell modules in prelimbic cortex
Neurones in many cortical areas of the mammalian brain are arranged into functional columns on the basis of their specific physiological response properties [7,25,27,33,36]. This functional organisation is considered to be produced by anatomical modules that have similar neuronal compositions and internal connections but with different afferent and efferent pathways [7,25,31,33]. The fundamental structural subunits within a cortical module are thought to be the neurones and synaptic circuitry associated with a single bundle of apical dendrites originating f r o m pyramidal neurones in l a y e r 5 [6,7,13,22,23,25,27,31,33]. The structural subunit of which a single dendritic bundle forms the central component (see Fig. 5) can be conveniently modelled by two concentric cylinders extending from white matter to pia [21,22,26]. The data from the present study indicate that in the PL cortex of the rat the outer cylinder is approximately 41 Ixm in diameter with
the internal central cylinder, containing the bundle of radially aligned apical dendrites, being about 31 p~m in diameter. The region between the two cylinders would contain other neuronal (somata, dendrites and axonal arbors) and glial elements as well as cerebral vasculature [23,26]. Although electrophysiological and anatomical columns have not, as yet, been demonstrated in the rat PL cortex [36], a dendritic bundle together with its associated surrounding cylinder of neural tissue could therefore represent the common structural subunit of both functional and structural columns in the prelimbic cortex (see below). 4.5. Dendritic bundles in prelimbic cortex: q[lerents, ~ e r ents and Jimctional considerations
Speculations about the functional capacity of dendritic bundles have centred on their ability to correlate or synchronise the activity between vertically aligned sets of pyramidal cells in the superficial and deep layers of the cortex in response to afferent input [I,15,27,30] - particularly from the thalamus [17,36]. This being so, then the clustering of profiles associated with dendritic bundles in lower layer 3 of PL cortex may serve to enhance the electrotonic coupling between neighbouring apical dendrites in response to direct innervation from the mediodof sal thalamus [1.15,17,30,36]. In addition, facilitatory synchronisation within a dendritic bundle may involve specific "local circuit" GABAergic neurones that are also innervated monosynaptically by the same cortical afferents [35,36]. Indeed. little is known about the specificity and quantitative distribution of 'excitatory" and "inhibitory' connections within and between dendritic bundles [23,36]. The bundling of apical dendrites in the PL cortex may also be related to the distribution of other afferent pathways to the PL cortex [36], for example fi'om the amygdala [3] or hippocampus [16], and serve as their principle foci of termination [31]. Afferents from the basolateral nucleus of the amygdala to the PL cortex specifically innervate spine-bearing processes in layers 5 and 2 [3]. Importantly, individual amygdalocortical axons were found Io course vertically from layer 5 into layer 2 and give rise to a highly clustered axon field of varicose processes - these terminal arbors were approximately 100 tzm × 100 lain (see Fig. 4 in Ref. [3]. Individual axons could therefore provide selective innervation to approximately five bundles of apical dendrites in layer 2. Given the high degree of overlap between individual fibres in layer 2 and that the amygdalocortical projection is topographically organised, then entirely different sets of pyramidal cell modules would be innervated at different tangential distances within this layer [6,23]. Similar considerations may apply to the selective innervation of the prelimbic cortex by CA1 hippocampal afferents [16] and other afferent fibre systems [36]. Since "functional columns' in the cortex overlap, then the underlying neuronal machinery of the anatomical
P.L.A. Gabbott. S.J. B a c o n / B r a i n Re.~ea/'ch 7.¢() f19961 75 A'6
columns and consequently of the dendritic bundles must perform dilterent physiological tasks in parallel [7,33,36]. Such parallel and distributive processing is particularly suited to the imegrative function of the mammalian medial prefrontal cortex [36] and may underlie the integration of information from diverse afferent sources in the prelimbic cortex of the rat [ 16,17]. With regard to the output from PL cortex, each dendritic bundle in area 32 may contain the apical dendrites from a defined set of projection pyramids in layers 2 / 3 and 5 / 6 that project to a specific spectrum of other cortical fields and several subcortical targets - for exampie, to the insular, infralimbic, or cingulate cortices, the striatum, amygdala, thalamus, medulla or to the spinal cord
[2.36]. In conclusion, this study has demonstrated that dendritic bundles are a principal structural component of the rat PL cortex. Moreover, dendritic bundles may provide the elementary physiological units whose integration underlies specific aspects of the functional organisation of the PL cortex in the rat [ 16.17,31,36].
Acknowledgements The technical help of Paul Jays, Angela Robinson and Tracy-Ann Warner is acknowledged with gratitude. This research was supported by the MRC and the Belt Memorial Fund. P.G. is a Belt Memorial Research Fellow.
References [I] Amitai Y., Friedman A., Connors B.W. and Gutnick M.J., Regenerative activity in apical dendrites of pyramidal cells in neocortex, Cerebral ('orte~, 3 (1993) 26-38. [2] Bacon, S.J, and Smith. A.D., A monosynaptic pathway from an identified vasomotor centre m the medial prefrontal cortex to an autonomic area in the thoracic spinal cord, Neuros~ience, 54 (1993) 719 72~. [3] Bacon, S.J,, Headlam, J.N.. Gabbott, P.L.A. and Smith, A.D.. Amygdala mpul to medial prefrontal cortex (mPFC) in the rat. A light and electr, m microscope study, Brain Res., 720 (1996) 211 219. [4] Bernhardt, R. and Matus, A., Light and electron microscopic studies of the distribution of microtubule-associated protein 2 in rat brain: A difference between dendritic and uxonal cytoskeletons, ,I. Conq~. Neurol., 226 ( 1~841 2113 221. 15] Bolam. ,[,P. tEd.), £]rperime~ttal Neuroanatomv. A Practical Ap proach, IRL Pwss, OUP Oxford, 1992, !6] l)et>'er. K.. Course and distribution of apical dendrites of layer 5 pyramids in the barrel field and area purietalis of the mouse, Anat ['2mhrvol l Ber/in ~. 149 (1976) 251-258. 7] Eccles, J.C,, The cerebral neocortex. A theory of its operation. In E.G. Jones and A. Peters (Eds.), Ceret~ral Cortex. Vol. 2. Funclional Propertie~ ol C,,rti~'al Cell~. Plenum Press, New York, 1984, pp. 1-38. g ] Escobar, M.I, Pimienta, H., Caviness, V.S., Jacobson, M., Crandall, J.|~. and Ko,,ik, K.S.. Architecture of apical dendrites in the murinc
S5
neocortex: Dual apical dendrilic systems. Neuro~cieHcc, 17 11981~) 975-989. [9] Feldman. M.L., Morphology of the ncocorUcal pyramidal neuron. In A. Peters and E.G. Jones (Eds.). ('~'r~'hrol ('ortc.~. ~4d. I. Cellular Compeme*zts ¢~/' the Cerehral C~,rle~. Plenunl Press, New York, 1984, pp. 123-200. [10] Feldman, M.L. and Peters, A.. A stud 3 ol barrels and pyramidal dendritic clusters in the cerebral cortex, Brain Re.v, 77 (1974) 55-76 [I 1] Fleischauer. K., Petsche, H. and ~ ittko,,v,,ki, '~., Vertical bundles of dendrites in the neocortex, Z Anat [:)tmickl. 1~6 (1972) 213 223. [12] Fleischauer, K.. On different patterns of dendritic bundling in the cerebral cortex of the cat. Z Anal. Etttwick].. 143 (1974) 115 12~'~. [13] Fleischauer, K. and l)etzer, K.. Dendritic bundling in the cerebral cortex. In G.W. Kreutzberg ted.), Adl'allcc~ iH N~,u~wlogy. Vol. 12, Plly,~iolog 3 apul Patholovy c!t l)eJldritc~. Raven Pa-e',s. Nc~ York, 1975. pp. 71 78. [ 14] Fry, ,I.(7. ted.), Biolo~,i~'at Dora Amffvsi~. I I'ra~'li~'a/ ,ll~l,'r,a~'h. 1RE Press. OUP. Oxfl~rd, 1993, [15] Horowitz, B., Neuronal plasticfly: ho,a change,, m dendritic urchilcclure curt affect the spread of posls}llaplic f,oterltials, Brain Rev. 224 (1981} 412 -418. [16] Jay. T.M., GIo,ainski, J. and Thicrr 3. A.-M., Selcctivity of the hippocampal pr~jection to the Pl. area of the prefrontal cortex in the rat, Brain Res., 5(15 (I989)337-340. [17] Kumda M,, Murakami, K., Kishi, K. and Price..I.L., Thalamocorli cal synapses between axons fi-om the mediodorsal thalamic nucleus and pyramidal cells in the prelimbic torte,, of lhe rat. ,/. ('omp. Neurol., 356 (1995) 143-151. [18] l,ohmann, H, und KiSppcn, H.d., Postnatal development o1" pyramidal cell dendritic and axonal bundles in Ihe ~isual corte'~ of the rat, J. Brain R~,~., 36 (1995) 10[ 111. [19] Meyer, G.. Forms and spatial arrangement ~1 neurones in the primary motor cortex of man../. ('omp Nt, m-ol., 262 11987) 402 428. [20] Peters, A. and W"alsh, T.M.. A study of lhe organisation of apical dendrites in the somatic sensory cortex of the rat. I ('ontp, Neurol.. 14-4 11972) 253 268. [21] Peters, A. and Kara. I).A., The neuronal o,)nlposilioll ol urea 17 ol the rat \isual cortex. IV. The organisalion ~f pyramidal cells, ,/ Uomp. Neurol., 2(~0 (1987) 573-590, [22] Peters, A. and Sethares, C., Organisation of pyramidal neurones m area 17 of monke5 visual cortex, ,I. C~,np. Nemol., 31)611991 )1-23 [23] Peters, A., Pyramidal cell modules in rat xisual cortex. Their s t r u t lure and development. In S.C. Sharma and J.W. [:a,acetl (J(ds.). kormalion and Re generaliotl ~{/' Ver/c ('oHJ~cctirm,s. l:;irkhaser, Boston, 1993, pp. 102 120. [24] Peters. A. and Yihna/, E., Neuronal organisalion in arca 17 ~1 call xisual cortex, ('erehral Cort~cv, 3 (1993~ 4 ¢~S [25] Roney, KJ., Scheihel, A.B. and Shay,, (;.I,., l)endritic btmdles: Survey of anatomical experiments and physioh~gical theories, Brain Res. Re~., 1 11979) 225-271. [26] Sukui. M,, Dendritic bundles fl~rmed b 5 laver VI pyramidal cells m the monkey frontal association cortex, EIV~. ICraht Rc~., 5S (I<)N5) 609 612. [27] Schiebcl. M.E. and Schiebel. A.B.. Dendrites as neuronal couplers: The dendritic bundle. In M. Santini tEd. t. (;o&,i ('cttlemlio] A~Vml~Osi,m Proceedinq,s. Raven Press, New York. 1975. pp. 347 354. [28] Schierhorn. H., Postnatal development of lamina V pyramidal cells in sensorimotor c~rtex of albino rats, (~'k'¢'llll¢lttt~ Mo~7~hol .lahr/~., 1241197S) I 23. [29] Schmolkc, C., The ontogeny of dendrite bundles m rabbit visual cortex, Anat. Emt,Tol., 180 (1989) 37I 3Sl. [30] Shaw, G.L., Hath. E. and Scheibel. A.R., ('ooperativity in brain function: Assemblies of approximately t0 neurones. K~?~. Nettrol., 77 (1982) 324-358. [31] Shepherd, G.M., Apical dendritic spines of cortical pyramidal cells: Remarks on their possible roles in the higher brain functiolls,
86
[32] [33] [34]
[35]
[36]
P.L.A. Gabbott, S.J. Bacon/Brain Research 730(1996) 75-86 including memory. In G. Lynch (Ed.), Synapses. Circuits. and the Beginnings (~fMemory, MIT Press, London, UK, 1986. Swanson, L.W., Brain Maps: Structure c~["the Rat Brain, Elsevier, Amsterdam, 1992. Szentagothai, J., The 'module concept" in cerebral architecture, Brain Res., 95 (1975) 475-496. Tucker, R.P., Binder, L.I. and Matus, A.I., Neuronal microtubule-associated proteins in the embryonic avian spinal cord, J. Comp. Neurol.. 271 (1988) 44-55. Vogt, B.A. and Peters, A., Form and distribution of neurones in rat cingulate cortex: Areas 32, 24 and 29, J. Comp. Neurol., 262 (1981) 256-270. Vogt, B.A. and Gabriel, M. (Eds.) Neurobiology ~( Cingulate
Cortex and Limbic Thalamus. A ComprehensiL,e Handbook. Birkhiiuser, Basel, 1993. [37] White, E.L. and Peters A., Cortical modules in the posteromedial barrel subfield (SMI) of the mouse, J. Comp. Neurol., 334 (1993) 86-96, [38] Wyss, J.M., Van Groen, T. and Sripanidkulchai, K., Dendritic bundling in layer 1 o1" granular retrospleuial cortex: intracellular labelling and selectivity of innervatiom J. Comp. Neurol., 295 (1990) 33-42. [39] Wyss, J.M. and Van Groen, T., Early breakdown of dendritic bundles in the retrosplenial granular cortex of hypertensive rats: Prevention by antihypertensive therapy. Cerebral Corte~, 2 (1992) 468-476.