Introduction
Materials and methods
Tissue
Case | Age (years) | Gender | Cause of death | Fresh weight (g) |
---|---|---|---|---|
B01 | 79 | f | Bladder carcinoma | 1350 |
B02 | 56 | m | Rectal carcinoma | 1270 |
B03 | 69 | m | Vascular disease | 1360 |
B04 | 75 | m | Acute glomerulonephritis | 1349 |
B05 | 59 | f | Cardiorespiratory insufficiency | 1142 |
B07 | 37 | m | Cardiac arrest | 1437 |
B08 | 72 | f | Renal arrest | 1216 |
B09 | 79 | f | Cardiorespiratory insufficiency | 1110 |
B13 | 39 | m | Drowning | 1234 |
B14 | 86 | f | Cardiorespiratory insufficiency | 1113 |
AR01 | 78 | m | Multiorgan dysfunction | 1326 |
AR02 | 75 | f | Respiratory insufficiency | 1280 |
AR03 | 79 | m | Cardiac arrest | 1477 |
AR04 | 77 | m | Pulmonary oedema | 1128 |
AR05 | 72 | f | Melanoma | 1326 |
AR06 | 77 | m | Cardiac arrest | 1272 |
Cytoarchitectonic analysis and probabilistic mapping
Receptor autoradiography
Transmitter | Receptor | Ligand | Displacer | Incubation buffer | Preincubation | Main incubation | Final rinsing |
---|---|---|---|---|---|---|---|
Glutamate | AMPA | [3H] AMPA (10 nM) | Quisqualat (10 µM) | 50 mM Tris–acetate (pH 7.2) [+ 100 mM KSCN] | 3 × 10 min, 4 °C | 45 min, 4 °C | (1) 4 × 4 sec, 4 °C (2) Acetone-glutaraldehyde (100 ml + 2.5 ml) 2 × 2 s, 22 °C |
Kainate | [3H] kainite (9.4 nM) | SYM 2081 (100 µM) | 50 mM Tris–acetate (pH 7.1) [+ 10 mM Ca-acetate] | 3 × 10 min, 4 °C | 45 min, 4 °C | (1) 3 × 4 s, 4 °C (2) Acetone-glutaraldehyde (100 ml + 2.5 ml), 2 × 2 s, 22 °C | |
NMDA | [3H] MK-801 (3.3 nM) | ( +)MK-801 (100 µM) | 50 mM Tris–acetate (pH 7.2) + 50 µM glutamate [+ 30 µM glycine + 50 µM spermidine] | 15 min, 4 °C | 60 min, 22 °C | (1) 2 × 5 min, 4 °C (2) Dip in distilled water, 22 °C | |
GABA | GABAA | [3H] Muscimol (7.7 nM) | GABA (10 µM) | 50 mM Tris–citrate (pH 7.0) | 3 × 5 min, 4 °C | 40 min, 4 °C | (1) 3 × 3 s, 4 °C (2) Dip in distilled water, 22 °C |
GABAB | [3H] CGP 54626 (2 nM) | CGP 55845 (100 µM) | 50 mM Tris–HCl (pH 7.2) + 2.5 mM CaCl2 | 3 × 5 min, 4 °C | 60 min, 4 °C | (1) 3 × 2 s, 4 °C (2) Dip in distilled water, 22 °C | |
GABAA/BZ | [3H] Flumazenil (1 nM) | Clonazepam (2 µM) | 170 mM Tris–HCl (pH 7.4) | 15 min, 4 °C | 60 min, 4 °C | (1) 2 × 1 min, 4 °C (2) Dip in distilled water, 22 °C | |
Acetylcholine | M1 | [3H] Pirenzepine (1 nM) | Pirenzepine (2 µM) | Modified Kreb’s buffer (pH 7.4) | 15 min, 4 °C | 60 min, 4 °C | (1) 2 × 1 min, 4 °C (2) Dip in distilled water, 22 °C |
M2 | [3H] Oxotremorine-M (1.7 nM) | Carbachol (10 µM) | 20 mM HEPES-Tris (pH 7.5) + 10 mM MgCl2 + 300 nM pirenzepine | 20 min, 22 °C | 60 min, 22 °C | (1) 2 × 2 min, 4 °C (2) Dip in distilled water, 22 °C | |
M3 | [3H] 4-DAMP (1 nM) | Atropine sulfate (10 µM) | 50 mM Tris–HCl (pH 7.4) + 0.1 mM PSMF + 1 mM EDTA | 15 min, 22 °C | 45 min, 22 °C | (1) 2 × 5 min, 4 °C (2) Dip in distilled water, 22 °C | |
Nic α4/β2 | [3H] Epibatidine (0.5 nM) | Nicotine (100 µM) | 15 mM HEPES (pH 7.5) + 120 mM NaCl + 5.4 mM KCl + 0.8 mM MgCl2 + 1.8 mM CaCl2 | 20 min, 22 °C | 90 min, 22 °C | (1) 5 min, 4 °C (2) Dip in distilled water, 22 °C | |
Serotonin | 5-HT1A | [3H] 8-OH-DPAT (1 nM) | 5-Hydroxy-tryptamine (1 µM) | 170 mM Tris–HCl (pH 7.4) [+ 4 mM CaCl2 + 0.01% ascorbate] | 30 min, 22 °C | 60 min, 22 °C | (1) 5 min, 4 °C (2) Dip in distilled water, 22 °C |
5-HT2 | [3H] Ketanserine (1.14 nM) | Mianserin (10 µM) | 170 mM Tris–HCl (pH 7.7) | 30 min, 22 °C | 120 min, 22 °C | (1) 2 × 10 min, 4 °C (2) Dip in distilled water, 22 °C | |
Norepinephrine | α1 | [3H] Prazosin (0.2 nM) | Phentolamine mesylate (10 µM) | 50 mM Na/K-phosphate buffer (pH 7.4) | 15 min, 22 °C | 60 min, 22 °C | (1) 2 × 5 min, 4 °C (2) Dip in distilled water, 22 °C |
α2 | [3H] RX 821002 (1.4 nM) | Phentolamine mesylate (10 µM) | 50 mM Tris–HCl (pH 7.7) + 100 µM MnCl2 | 15 min, 22 °C | 90 min, 22 °C | (1) 5 min, 4 °C (2) Dip in distilled water, 22 °C | |
Dopamine | D1 | [3H] SCH 23390 (1.67 nM) | SKF 83566 (1 µM) | 50 mM Tris–HCl (pH 7.4) + 120 mM NaCl + 5 mM KCl + 2 mM CaCl2 + 1 mM MgCl2 | 20 min, 22 °C | 90 min, 22 °C | (1) 2 × 20 min, 4 °C (2) Dip in distilled water, 22 °C |
Multimodal border definition
Statistical analyses
Results
Multimodal characterization of regions within the hippocampal formation
Total | Left | Right | Male | Female | |
---|---|---|---|---|---|
FD | 806 ± 145 | 800 ± 153 | 811 ± 145 | 760 ± 184 | 851 ± 77 |
CA4 | 137 ± 30 | 136 ± 31 | 138 ± 30 | 131 ± 37 | 142 ± 22 |
CA3 | 248 ± 38 | 247 ± 41 | 249 ± 37 | 225 ± 28 | 271 ± 34 |
CA2 | 183 ± 29 | 180 ± 33 | 187 ± 25 | 167 ± 23 | 200 ± 24 |
CA1 | 1437 ± 239 | 1419 ± 228 | 1456 ± 261 | 1311 ± 252 | 1563 ± 148 |
ProS | 377 ± 61 | 380 ± 65 | 373 ± 60 | 321 ± 22 | 432 ± 25 |
Sub | 531 ± 106 | 529 ± 113 | 533 ± 104 | 475 ± 121 | 587 ± 45 |
PreS | 345 ± 71 | 337 ± 70 | 353 ± 75 | 314 ± 66 | 376 ± 64 |
PaS | 123 ± 39 | 126 ± 41 | 119 ± 39 | 94 ± 11 | 151 ± 36 |
TrS | 115 ± 29 | 112 ± 24 | 118 ± 35 | 120 ± 22 | 110 ± 36 |
Volumetric analysis
Probabilistic maps and maximum probability maps
Discussion
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CA3 has often been merged with CA2, following the scheme proposed by Stephan (1975). However, whereas in rodents and non-human primates the proximal apical dendrites of CA3 display large complex spines, those of CA2 do not have such differentiations (Ramón y Cajal 1911; Lorente de Nó 1934). In the mouse brain, a number of genes, such as those coding for the Purkinje cell protein 4, the Regulator of G-protein signaling 14, and the striatum-enriched protein-tyrosine phosphatase, are selectively expressed in pyramidal neurons of the CA2 region (Cembrowski et al. 2016). Furthermore, in the macaque brain CA3 is targeted by a considerably higher proportion of amygdalohippocampal axons than is CA2 (Wang and Barbas 2018), whereas in the rodent brain the opposite holds true for axonal input from the supramammillary nucleus and the paraventricular nucleus of the hypothalamus (Cui et al. 2013; Zhang and Hernandez 2013). Although the lucidum layer is not easily detectable in cell body stained sections, it can be clearly identified in Timm stained sections due to the high zinc concentrations in mossy fiber synaptic vesicles (Danscher 1981; Becker et al. 2005). Furthermore, the lucidum layer is particularly prominent due to its extremely high kainate and α1 receptor densities. Additionally, CA3 and CA2 also differed in their densities of GABAA, GABAB, M1, M3 and 5-HT1A receptors, as well as of GABAA/BZ binding sites, which were lower in the former area. Additionally, 5-HT2 receptor densities were lower in CA2 than in CA3. Taken together, these data would argue against the merging of CA2 and CA3 into a single region.
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Amaral and Inausti (1990) described CA3 and CA4 as a single region, although they are not only identifiable based on the presence/absence of specific cytoarchitectonic layers, but also differed in their connectivity patterns and densities of multiple receptor types. Thus, whereas CA4 only has a pyramidal layer, CA3 is composed of the oriens, pyramidal, lucidum, radiatum and lacunosum-molecular layers. The CA4 region and the multiform layer of FD are heavily innervated by the locus coeruleus, whereas CA3 displays only a moderately dense plexus of dopamine-ß-hydroxylase fibres (Swanson and Hartman 1975). We found the lucidum layer to be also clearly distinguishable by its conspicuously high kainate and α1 receptor densities, which is in accordance with previously described observations in both human and rodent brains (Tremblay et al. 1985; Represa et al. 1987; Zilles et al. 2002; Palomero-Gallagher et al. 2003; Zeineh et al. 2017). The border between these two regions can also be identified based on NMDA, M1 and α2 receptor densities, which were lower in the pyramidal layer of CA3 than in CA4.
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In some studies the CA4 region was merged with the multiform layer of FD to build a single region, namely the hilus (Amaral 1978; West and Gundersen 1990; Frahm and Zilles 1994). However, CA4 and the multiform layer differ in their connectivity patterns, since the raphe nuclei project heavily to the multiform layer of FD, but only moderately to the CA4 region (Moore and Halaris 1975). Our results provide further support for the concept of CA4 as being a separate region, since it contained higher AMPA, NMDA, GABAB, M1 and M3 receptor densities and higher GABAA/BZ binding site concentrations, but lower α1 receptor densities than did the multiform layer of FD.
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The existence of ProS has been a subject of particular debate. Whereas some authors define it as a distinct subfield of the subicular complex (Rose 1927; Rosene and Van Hoesen 1987; Ding 2013), others (von Economo and Koskinas 1925; Stephan 1975; Braak 1980; Insausti and Amaral 2012) include the superficial layers of ProS in the CA1 region, and the deeper ones in the Sub, since they consider ProS to be a mere transition zone resulting from the overlap of elements from the hippocampus proper and the subicular complex. However, the gradual disappearance of CA-like pyramids and concomitant appearance of subicular-like pyramids in the pyramidal layer of ProS is not the only criterion by which the existence and extent of this region can be defined. Rather, the borders of ProS can also be identified by abrupt changes in lamination pattern, in the expression levels of diverse molecular and chemical markers, as well as by specific connectivity patterns. Cytoarchitectonically, the border between ProS and CA1 can be characterized by the abrupt disappearance of the radiatum layer in the former region. SMI-32 immunoreactivity in ProS is lower than in Sub, but higher than in CA1, and acetylcholinesterase staining is considerably higher in ProS than in either of the two neighbouring regions (Ding 2013). Furthermore, ProS presents higher expression levels of the neurotensin and tyrosine hydroxylase genes than do CA1 or Sub (Ding 2013). Region and layer-specific expression levels of the htr2a gene also enable the delineation of ProS (Ding 2013), and were comparable to those we found for the 5-HT2 receptor as labelled with [3H]ketanserin, an antagonist which primarily targets the 5-HT2A subtype (Yadav et al. 2011). Our results concerning the remaining examined receptor types would also argue against the definition of ProS as a transition region, since it differed considerably in its receptor expression levels from both adjoining regions. Finally, the CA1, ProS and Sub regions also differ in their amygdalohippocampal connectivity patterns, with the highest density of terminal projections found in ProS and the lowest in Sub (Ding 2013; Wang and Barbas 2018). Whereas ProS projects to the perirhinal cortex, CA1 and Sub do so to the entorhinal cortex (Blatt and Rosene 1998).
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PaS and TrS were initially described as being transition regions between PreS and the entorhinal cortex, and between PreS and area BA35, respectively (Braak 1978, 1980; Braak and Braak 1993). However, PreS, PaS and TrS show different patterns of Alzheimer-related extracellular amyloid and intraneuronal neurofibrillary changes (Kalus et al. 1989), and we here found marked differences in receptor expression levels in PaS and TrS with respect to PreS. Therefore, we here classify them as distinct architectonic entities and not mere transition regions.