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Contacts between medial and lateral perforant pathway fibers and parvalbumin expressing neurons in the subiculum of the rat
Neuroscience 156 (2008) 653– 661
CONTACTS BETWEEN MEDIAL AND LATERAL PERFORANT PATHWAY FIBERS AND PARVALBUMIN EXPRESSING NEURONS IN THE SUBICULUM OF THE RAT F. G. WOUTERLOOD,a* A. J. BOEKEL,a V. ALIANE,a J. A. M. BELIËN,b H. B. M. UYLINGSa,c AND M. P. WITTERa,d
Structures in the medial temporal lobe, in particular the parahippocampal region and the hippocampal formation, are strongly implicated in learning and memory (Eichenbaum, 1999, 2002; Suzuki and Eichenbaum, 2000; Witter and Amaral, 2004). The relevance of this part of the brain is emphasized by changes that occur in cases of mild cognitive impairment, temporal lobe epilepsy and Alzheimer’s disease (Behr and Heinemann, 1996; De Toledo-Morrell et al., 2000; Schwarcz and Witter, 2002; Van Hoesen, 2002). Based on its connectivity the subiculum can be perceived as a crossroad of hippocampal in- and output. The subiculum receives via the perforant pathway a dense and topographically organized projection from layer III neurons in the entorhinal cortex (Naber et al., 2000; Burwell and Witter, 2002). Subicular output projects to forebrain and hypothalamic targets (Shibata, 1989; Wouterlood and Tuinhof, 1992; Naber and Witter, 1998; review in Witter, 2006) and, via a feedback circuit, back to the parahippocampal cortex (Sørensen and Shipley, 1979; Finch et al., 1986; Kloosterman et al., 2003; van Haeften et al., 2003). Yet, in spite of this gross knowledge of connectivity, descriptions of cytological and physiological details (Stewart and Wong, 1993; Harris and Stewart, 2001; Harris et al., 2001; Ishizuka, 2001; Menendez de la Prida et al., 2003; Menendez de la Prida, 2006), and even the discovery of spatially modulated firing of subicular cells (Sharp and Green, 1994; Sharp, 2006), much of the architecture of the neuronal network in this remarkable region is unknown and needs further clarification. In terms of its afferent connectivity, parallels exist between the subiculum and other hippocampal regions. For instance, boutons of perforant pathway fibers form synaptic contacts in the dentate gyrus with dendrites of both principal neurons (granule cells) and interneurons (Freund and Buzsáki, 1996). In field CA1 a similar arrangement has been reported (Freund and Buzsáki, 1996; Kajiwara et al., 2008), and a strong argument in favor of extending this organizational scheme to the subiculum is the finding of feed-forward inhibition here (Finch and Babb, 1980; Finch et al., 1988). As GABA is the most abundant inhibitory neurotransmitter in the hippocampus, subicular GABAergic interneurons are most likely involved. Chemically distinct types of subicular neurons contain either calbindin D-28K (mouse, monkey; Seress et al., 1994; Fujise et al., 1995), nitric oxide synthase (rat; Lin and Totterdell, 1998), or several neuropeptides (rat; Köhler and Chan-Palay, 1982, 1983; Roberts et al., 1984). Recently we observed in the electron microscope that entorhinal input to the subiculum targets to a large extent principal neurons (Baks-te Bulte et al., 2005). However,
a
Graduate School Neurosciences Amsterdam, Research Institute Neurosciences Vrije Universiteit, Department of Anatomy and Neurosciences, Vrije University Medical Center, Room MF-G-136, P.O. Box 7057, 1007 MB Amsterdam, The Netherlands
b
Department of Pathology, Vrije University Medical Center, Amsterdam, The Netherlands
c
Department of Psychiatry and Neuropsychology, Division Brain and Cognition, Maastricht University Medical Center, Maastricht, The Netherlands
d
Kavli Institute for Systems Neuroscience and Center for the Biology of Memory, Norwegian University of Science and Technology, Trondheim, Norway
Abstract—The entorhinal cortex (EC) projects via the perforant pathway to all subfields in the hippocampal formation. One can distinguish medial and lateral components in the pathway, originating in corresponding medial and lateral subdivisions of EC. We analyzed the innervation by medial and lateral perforant pathway fibers of parvalbumin-expressing neurons in the subiculum. A neuroanatomical tracer (biotinylated dextran amine, BDA) was stereotaxically injected in the medial or lateral entorhinal cortex, thus selectively labeling either perforant pathway component. Transport was allowed for 1 week. Transported BDA was detected with streptavidin-Alexa Fluor™ 488. Parvalbumin neurons were visualized via immunofluorescence histochemistry, using the fluorochrome Alexa Fluor™ 594. Via a random systematic sampling scheme using a two-channel, sequential-mode confocal laser scanning procedure, we obtained image series at high magnification from the molecular layer of the subiculum. Labeled entorhinal fibers and parvalbumin-expressing structures were three dimensionally (3D) reconstructed using computer software. Further computer analysis revealed that approximately 16% of the 3D objects (‘boutons’) of BDA-labeled fibers was engaged in contacts with parvalbumin-immunostained dendrites in the subiculum. Both medial and lateral perforant pathway fibers and their boutons formed such appositions. Contacts are suggestive for synapses. We found no significant differences between the medial and lateral components in the relative numbers of contacts. Thus, the medial and lateral subdivisions of the entorhinal cortex similarly tune the firing of principal neurons in the subiculum by way of parvalbumin positive interneurons in their respective terminal zones. © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: parahippocampal region, hippocampal region, confocal laser scanning, neuroanatomical tracing, inhibition. *Corresponding author. Tel: ⫹31-20-444-8049; fax: ⫹31-20-444-8054. E-mail address: [email protected] (F. G. Wouterlood). Abbreviations: BDA, biotinylated dextran amine; LEA, lateral entorhinal cortex; MEA, medial entorhinal cortex; 3D, three dimensionally. 0306-4522/08 © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2008.08.023
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Fig. 1. Electron micrograph of a BDA-labeled axon terminal (BDA) in the subiculum forming a synapse (arrows) with a thin and nonspinous dendrite. The BDA has been deposited in layer III of MEA (Baks-te Bulte et al., 2005). Nonspinous dendrites in the subiculum may belong to interneurons. Note that the shape of the postsynaptic dendrite is similar to that of thin parvalbumin-immunofluorescent dendrites in the present study. BDA injection in layer III of entorhinal cortex.
approximately 18% of the axon terminals of perforant pathway fibers in the subiculum had synaptic contacts with presumed subicular interneurons (Fig. 1). In the dentate gyrus, entorhinal axons have been shown to target parvalbumin positive neurons (Zipp et al., 1989), and since these cells provide powerful perisomatic inhibitory innervation, this projection has been implicated in the control of granule cell firing. In the subiculum, parvalbumin positive interneurons are likely candidates to receive entorhinal inputs because they have apical dendrites that extend into the perforant path terminal zone (our own unpublished data). The topographical organization of the perforant pathway in the subiculum is striking, i.e. inputs from the medial entorhinal cortex (MEA) innervate a portion of the subiculum not innervated by the lateral entorhinal cortex (LEA) and vice versa (Witter, 2006). In addition, the two components of the perforant pathway likely mediate the transfer of different sensory information and they may show different overall electrophysiological effects as shown in the dentate gyrus (Pöschel and Stanton, 2007). In the present study we therefore explored whether parvalbumin positive interneurons in the subiculum are indeed innervated by entorhinal inputs and if so, whether the lateral and medial components of the pathway differ quantitatively. We combined for this purpose neuroanatomical tracing, immunofluorescence, high-resolution confocal laser scanning, three dimensionally (3D) computer reconstruction and quantification. A preliminary report has been published in abstract form (Boekel et al., 2006).
EXPERIMENTAL PROCEDURES Surgery, injection of tracer, kill All experiments were in accordance with local, national and European Community regulations on animal well-being. The smallest number of animals possible was used to substantiate our findings; that is, four female Wistar rats. Four female young Wistar rats (weighing 200 –220 g; Harlan Centraal Proefdierbedrijf, Zeist, The Nether-
lands) were used. Animals were deeply anesthetized with a 4:3 mixture of 10% ketamine (Aesco, Boxtel, The Netherlands) and 2% xylazine (Rompun, Bayer, Mijdrecht, The Netherlands) (total dose of this mixture 1 ml/kg body weight) and mounted in a stereotaxic frame. Biotinylated dextran amine (BDA, MW 10,000, Molecular Probes, Eugene, OR, USA; 5% solution, in 50 mM Tris-buffered saline, TBS, pH 7.4) was injected bilaterally via a vertical (in the sagittal plane) approach into the superficial portion of typical MEA (case 2001215 and 2001216; Bregma ⫺7.80, lateral 4.80 and deep 6.40 from the point of entry of the pipette, dorsal pial surface) or LEA (cases 2001219 and 2002012; Bregma ⫺6.36, lateral 6.80 and deep 5.60 from the dorsal pial surface) of the entorhinal cortex (coordinates derived from the rat brain atlas by Paxinos and Watson, 2005). Injections were made through glass micropipettes (tip diameter 10 – 20 m), using a positive pulsed 6 A DC current (7 s on/off; 20 min). The animals were killed 1 week after the surgery via an overdose of sodium pentobarbital (Nembutal, i.p. 60 mg/kg body weight; Sanofi Sante, Maassluis, The Netherlands). They were subsequently perfused transcardially with saline followed by 500 ml of a mixture of freshly depolymerized 4% paraformaldehyde and 0.1% glutaraldehyde (Merck, Darmstadt, Germany) in 100 mM phosphate buffer, pH 7.4, room temperature. Brains were removed from the skulls and postfixed for an additional 1 h at 4 °C. Sections, 40 –50 m thick, were cut on a vibrating microtome (model VT1000S, Leica Microsystems, Heidelberg, Germany). Brains with an injection of BDA in MEA were sectioned in the horizontal plane whereas the brains with an injection of BDA in LEA were cut in the frontal plane. After cutting, sections were rinsed in 100 mM phosphate buffer, pH 7.4, and further processed.
Immunocytochemistry Sections were rinsed three times with incubation buffer (50 mM Tris/HCl buffer with 150 mM NaCl and 0.5% Triton X-100™ pH 8.0; this buffer was also used for all rinses between incubation steps) (Triton is a trademark of Sigma, St. Louis, MO, USA) and preincubated free-floating (gentle agitation on a rocking plateau) for 1 h at room temp in 5% normal goat serum (Nordic, Tilburg, The Netherlands) and 2% bovine serum albumin (BSA, Sigma). Incubation with the primary antibody (mouse anti-parvalbumin (1:1000, Sigma) took at least 48 h at 4 °C. The next step consisted of a 2-h incubation at room temperature with a cocktail of goat anti-mouse IgG conjugated to Alexa Fluor™ 594 (1:400, Invitrogen–Molecular Probes, Eugene, OR, USA) and streptavidin–Alexa Fluor™ 488 (1:400, Invitrogen–Molecular Probes). Finally, the sections were mounted on glass slides, dried and coverslipped with DPX (Fluka Chemie AG, Buchs, Switzerland). We also incubated sections with a cocktail of streptavidin–Alexa Fluor™ 488 and streptavidin–Alexa Fluor™ 594 for laser scanner calibration purposes. Sections were inspected in a standard fluorescence microscope to chart the sites of BDA injections (Fig. 2A, B).
Confocal laser scanning microscopy: preparation of composite images Sections containing BDA-labeled fibers in the target area (the subiculum) were analyzed with the aid of a Leica TCS-SP2-AOBS confocal laser scanning microscope (CLSM; Leica Microsystems) equipped with an argon/krypton gas laser (488 nm excitation) and a He–Ne laser (594 nm excitation). We selected these excitation wavelengths since fluorescence crosstalk between channels with the fluorochromes Alexa Fluor™ 488 and Alexa Fluor™ 594 can be completely ruled out (Wouterlood, 2006). A channel is a specific configuration of the CLSM designed to detect a particular fluorochrome: laser, emission wavelength, dichroic mirror, range of the emission spectrophotometer detection (‘emission filtering’) and detector. First, we took series of images at low magnification (20⫻ objective) and in ‘simultaneous’ mode, in the 488 and
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Fig. 2. Experimental approach. (A, B) BDA injection site, case MEA 2001215, horizontal section: BDA green, parvalbumin red; injection site centered in the superficial cortical layers (I–III). Fibers run to the subiculum (Sub), fields CA1 and CA3 and the middle one-third of the molecular layer of the dentate gyrus (DG). Very dense terminal fiber labeling is indicated with flat gray in frame B. Note the dense parvalbumin staining in the Sub concentrated in the cell layer. The asterisk indicates layer IV, the lamina dissecans. (C) Sampling scheme. A superimposed grid of 100⫻100 m squares [yellow lines] over the subiculum includes the selection area (molecular layer [ML], outlined with thick white border). Beginning with one randomly selected square, every tenth square was selected for sampling (indicated with numbers). PP⫽perforant pathway; PrSub⫽presubiculum. COM⫽commissural fibers.
594 nm channels. From these images we assembled mosaic composite images of the entire subiculum (e.g. Fig. 2C).
Random systematic sampling strategy BDA-labeled perforant pathway fibers were restricted to the outer molecular layer of the subiculum which contains the distal portions
only of dendrites of subicular neurons. The somata of the candidate recipient neurons were invariably present much deeper, in, or close to, the cell layer. Thus for the purpose of determining contacts we needed only samples of the molecular layer. We applied the following procedure which is called ‘random systematic sampling’ (illustrated in Fig. 2C) (cf. Gundersen, 1986). First we placed an overlay sampling grid with 100⫻100 m square units over the composite image and
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Table 1. Sampling and counting Case
MEA 2001215 MEA 2001216 Average MEA LEA 2001219 LEA 2002012 Average LEA Average LEA⫹MEA Totals
Qualifying squares
Samples
Boutons reference density
Boutons mean number⫾S.D.
Contacts normalized number⫾S.D.
Contact ratio: ratio of boutons engaged in contacts/‘free’ labeled boutons
9 6
1006 893
7 10
1406 593
146 119 132.5 147 224 185.5
76 20 48 37 38 37.5 42.75
24.19⫾18.65 6.01⫾3.19 16.03 16.63⫾12.75 22.22⫾16.16 20.01 18
2.80⫾3.05 1.16⫾0.99 2.06 3.25⫾3.15 2.69⫾4.30 2.92 2.56
0.12 0.19 0.15 0.20 0.12 0.16 0.16
Number of sections
3898
636
Two animals (2001215, 2001216) with injection of tracer in MEA and two animals (2001219 and 2002012) with injection of tracer in LEA. All results brought together and averaged if necessary. Note that one qualifying square measures 100⫻100 m. One square corresponds with four samples (all samples having X and Y dimensions of 29.7 m). Boutons and contacts are expressed as the number of 3D objects per 1000 m3 scanned tissue volume.
we outlined the selection area. This area (indicated in Fig. 2C with a thick white line) contained those grid squares over the molecular layer where BDA-labeled fibers were present. Starting from square 1, i.e. a square selected randomly in the first row of squares inside the selection area, we sampled every tenth square. The number of squares per section qualifying for sampling ranged between 95 and 256. From each brain we scanned samples in six to nine separate sections. A survey of the sampling is presented in Table 1.
to calculate from the acquired image the best estimate of the original image (Bertero et al., 1990; Snyder et al., 1992; van der Voort and Strasters, 1995; Hiesinger et al., 2001; Wallace et al., 2001). The images obtained from the double-stained calibration sections were used to check and eliminate sources of possible errors, e.g. pixel ‘shift’ between channels as a result of chromatic aberration or other, instrument-related causes (discussed in Wouterlood, 2006).
High-resolution confocal laser scanning
Automated contact recognition and quantification. Automated 3D object and contact recognition was performed with SCIL_Image software (TNO, Delft, The Netherlands). The basic premises and the procedures necessary to run this software in automated contact recognition are discussed elsewhere (Wouterlood et al., 2007, 2008). Briefly, the software first outlined 3D objects in the Z-series of images in each channel by applying isodensity envelopes: ‘boutons’ in the 488 nm channel and ‘dendrites’ in the 594 nm channel. Isodensity envelope parameters (thresholds) were acquired as follows. For the 488 nm channel we used an objective, automated procedure based on the plugin ‘3D object counter’ (Fabrice Cordelières, Institut Curie, Orsay, France; e-mail [email protected]) of the public-domain program ImageJ (Rasband, 1997–2006; automated procedure described in detail in Wouterlood et al., 2008). For the 594 nm channel we determined the isodensity envelope threshold manually with a ‘best-match’ procedure since big, single objects like dendrites with inhomogeneous intense fluorescence contents provide unsatisfactory results in the automated procedure (Wouterlood et al., 2008). SCIL_Image was instructed via macros to first identify 3D objects in images in both channels and to analyze subsequently which of the 488 nm channel 3D objects showed partial voxel overlap with identified 3D objects in the 594 nm channel (explained in Wouterlood et al., 2008). After running the SCIL_Image based software we verified all ‘large’ contacts manually since the software showed a tendency to report one big contact where, in manual 3D reconstructions, a BDA-labeled fiber appears to make series of two or more small contacts with a parvalbumin dendrite. For this purpose we 3D reconstructed boutons and dendrites by means of Amira™ visualization/modeling software (Mercury Computer Systems, Inc., website at www.tgs.com), using the same thresholds implemented in the 3D object recognition in SCIL_Image.
Proper visualization of the involved structures required a high final magnification: 63⫻ immersion objective lens, NA 1.3, electronic zoom setting of 8⫻. At this setting one 100⫻100 m sampling square in the sampling grid was too large to scan completely in one pass. Since at the final magnification the dimensions of individual samples were 29.7 m in the X- and 29.7 m in the Y direction, we scanned in each 100⫻100 m square four immediately adjacent Z-series of images. It should be mentioned at this point that the drying of the sections on the slide and the subsequent embedding in mounting medium had resulted in a shrinkage of the section in the Z-direction of approximately 70%. Thus, tissue deformation in the Z-direction is most likely to be present (see Gardella et al., 2003). Final scanning parameters were: pinhole 1.00 arbitrary units [Airy disk], Z-increment fixed at 80 nm, image frame 512⫻512 pixels at eight-bit sampling, Z-series covering the entire section thickness, 70 –90 successive image frames. We applied the option ‘sequential scanning, alternating between frames’ through which the instrument acquires one image frame in the 488 nm channel followed by one image frame in the 594 nm channel and then moves one increment along the Z-axis to repeat the scanning. For calibration purposes we took during each acquisition session several images of the sections in which BDA-labeled fibers had been double-stained on purpose with both fluorochromes (see above). The scanning parameters for the calibration series were the same as for the experimental series. Since the perforant pathway does not include parvalbumin expressing elements (Wouterlood et al., 1995), colocalization of both markers was ruled out.
Post acquisition image processing Image deconvolution. All images were processed by means of an iterative maximum likelihood estimation algorithm (Huygens II deconvolution software, Scientific Volume Imaging, Hilversum, the Netherlands, website at www.svi.nl) running on an Origin 300 computer (SGI, Mountain View, CA, USA). Deconvolution is a statistical procedure, using the instrument’s point spread function,
Normalization of fiber densities. Deposition of a small amount of a tracer in regions of the CNS (e.g. our injections in MEA and LEA), leads most often to labeling of only a fraction of the fibers projecting from that region. A typical termination zone
F. G. Wouterlood et al. / Neuroscience 156 (2008) 653– 661 (e.g. the molecular layer of the subiculum) then contains a very dense focus of labeled fibers that rapidly thins out toward the edges. As a consequence, the proportion of labeled versus unlabeled fibers varies between sections as well as topographically within individual sections. In Fig. 2A and B these phenomena are clearly visible. A detail that may look trivial while it definitely affects our results is that we notice in the microscope only labeled fibers, remaining literally in the dark with respect to unlabeled fibers. Variations in the proportion of labeled versus unlabeled fibers, e.g. at the edges of the selection area, have profound effect on the absolute number of observed contacts. To correct for this bias we adopted a normalization procedure based on two assumptions, first that the integral fiber density of the perforant pathway in the molecular layer, or the sum of labeled and unlabeled fibers, was constant. The second assumption was that in the center of the selection area, where labeled fiber density is highest, all perforant pathway fibers were labeled. The highest encountered actual density of labeled fibers was therefore considered to be our ‘reference density’ for that particular experimental case. Normalization included three steps. First we determined the actual and reference densities of BDA labeled boutons. Then we calculated for each sample the proportion of the actual density to the reference density, and finally we multiplied the number of actually found contacts with the inverse proportion factor. Actual and reference densities of BDA-labeled boutons had been obtained during the isodensity threshold analysis with the plugin ‘3D object counter’ of ImageJ (see above).
RESULTS BDA: injection sites, projection patterns and termination areas Each BDA injection resulted in the labeling of a cluster of neurons in a distinct site in the entorhinal cortex (e.g. case 2001215L shown in Fig. 2A–B). In all cases, irrespective of the injection in either LEA or MEA, several contingents of labeled fibers were visible coursing to the subiculum and the hippocampal formation. First, a robust projection consisting of relatively thick, labeled fibers with few varicosities (‘passing fibers’) descended radially through the adjacent deep layers of the entorhinal cortex, and either passed through the white matter into the subiculum or entered the angular bundle. Collectively these fibers form the perforant pathway. Second, a dense contingent of projection fibers was present coursing at the surface of the brain in the medial direction, through the molecular layers of the parasubiculum and presubiculum into the molecular layer of the subiculum, where the fibers blended with the fibers arriving via the perforant pathway. A third contingent occurred deep in the white matter of the temporal cortex, contributing to commissural and rostrocaudal intracortical projections. Finally, a few scattered fibers from the angular bundle entered the deepest layer of the subiculum and could be traced through the alveus of field CA1, curving perpendicularly at their final destination into CA1, traversing the stratum pyramidale and radiatum on their way to stratum lacunosum-moleculare of CA1 (the alvear or temporo-ammonic tract; cf. Deller et al., 1996). The molecular layer of the subiculum contained the terminal plexus: densely woven patterns of labeled fibers studded with numerous boutons. The position of the labeled terminal plexus, along the transverse or so-called proximo-distal axis of the subiculum, varied with respect to the origin of the entorhinal
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fibers. In case of an injection in MEA, BDA-labeled terminal fibers were most prominent in the distal subiculum (immediately adjacent to the presubiculum, cf. Witter and Groenewegen, 1990; Witter, 2006). In cases 2001219 and 2002012 (tracer injection in LEA), the position of the labeled terminal fibers was shifted toward a position close to the border with field CA1, i.e. the proximal part of the subiculum. In line with previously published accounts on the organization of the perforant pathway, we observed in cases 2001215 and 2001216 (injections in MEA) anterogradely labeled fibers in the middle one-third of the molecular layer of the dentate gyrus, in the inner half of the stratum lacunosum-moleculare of field CA3 and in stratum lacunosum-moleculare of the proximal part of field CA1. In the cases with LEA injections, labeling in the dentate gyrus was confined to the outer one-third of the molecular layer and, in field CA3, in the outer half of stratum lacunosummoleculare. In field CA1, labeling was present in stratum lacunosum-moleculare overlying the distal CA1, i.e. adjacent to the subiculum. In the subiculum, individual BDA-labeled fibers in the terminal plexus were very thin. Boutons on these thin fibers were very prominent in the confocal microscope at the high magnification applied for the sampling. By far most labeled terminal boutons occurred as spindle-like swellings in en passant positions on the fibers (Fig. 3). The dimensions of these boutons varied considerably. The smallest boutons had a diameter of approximately 0.5 m whereas the diameters of the largest boutons reached approximately 1.0 m. Parvalbumin neurons, dendrites and fibers in the subiculum The terms ‘parvalbumin neurons,’ ‘-cell bodies,’ ‘-dendrites’ and ‘-fibers’ are used as shortcuts for parvalbumin immunopositive neurons, and similarly, for parvalbumin immunopositive cell bodies, dendrites and fibers. Cell bodies of parvalbumin neurons occurred in the deep (polymorph) layer and in the cell layer of the subiculum. Rarely a parvalbumin cell body was present in the molecular layer. The latter layer contained numerous parvalbumin dendrites, many of which physically emerged from the parvalbumin cell bodies in the cell layer. Parvalbumin dendrites often had a beaded appearance (thickenings and constrictions along the dendrite in a pseudocyclical way) (Fig. 3A, B). Parvalbumin dendrites did not bear spines. Thick parvalbumin dendrites could be distinguished with ease because of their diameter, cylindrical shape and attachment to parvalbumin cell bodies. Located in between these traceable dendrites and thick dendritic fragments were numerous thin truncated dendrites, and parvalbumin fibers. Because of the relatively strong weight of the criterion ‘diameter’ on the decision to classify a parvalbumin structure as a ‘dendrite,’ correspondingly the thinner parvalbumin dendrites were increasingly harder classifiable with certainty as dendrites. An elongated aspect of a thin parvalbumin structure together with a relatively stable diameter compared with a typical bouton was decisive to classify such a structure as a dendrite. Conse-
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Fig. 3. (A) Z-projection overlay image of a deconvoluted image series. Arrows indicate boutons of BDA-labeled fibers (green) close to parvalbumin dendrites (red). Frame size was in all samples 29.7⫻29.7 m. The inset shows one of the BDA-labeled fibers with its boutons at high magnification. (B) 3D Reconstruction of these image series. (C) Detail of squared area of B. Several contacts are visible (arrows). (D) Graphical output of SCIL_image analysis. Z-projection image. Yellow indicates overlap of voxels belonging to BDA-labeled boutons (color coded green) and parvalbumin dendrites (color coded red).
quently, at the lower range of the diameter scale it was impossible to distinguish the thinnest parvalbumin dendrites from thick parvalbumin axons. Such small profiles were eliminated from computer analysis by introducing an offset of 100 voxels. Contacts between BDA-labeled boutons and parvalbumin containing dendrites Typically, BDA-labeled fibers showed along their course continuous variation of their diameters (Fig. 3A). Close apposi-
tions (i.e. contacts) occurred between boutons (swellings) of these fibers and the shafts of dendrites of parvalbumin immunopositive neurons. In all sections studied, we found in the sampled areas in the molecular layer numerous varicosities of BDA-labeled fibers in contact with the parvalbumin dendrites (Fig. 3). In total our software analyzed 265 3D reconstructions in the subiculum after injections of BDA in MEA, and 371 3D reconstructions in the subiculum after injections of BDA in LEA. The results are shown in Table 1. Examples of appositions complying with the nu-
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merical criteria for a ‘contact’ are shown in Fig. 3B–D. The reconstructed BDA-labeled fiber in Fig. 3C has three en passant boutons in contact with the parvalbumin dendrite. The density references between cases differed considerably. The upper and lower extremes were both found in the MEA group of cases: case MEA 200215 showing the highest density reference of 76 3D objects per 1000 m3 and case MEA 200216 expressing the lowest density reference with 20 3D objects per 1000 m3. These differences in density references reflect individual variability in the density of fiber labeling. The average number of contacts in case of the medial perforant pathway, as measured in the sections taken from MEA injection cases 2001215 and 2001216, was 2.06/ 1000 m3; in case of the lateral perforant pathway this number, based on the analyses of sections taken from cases LEA 2001219 and 2002012 brains, was 50% higher: 2.92/1000 m3. In all cases the ‘contact ratio,’ which is the ratio of identified contacts to labeled boutons was remarkably similar, on average 0.15– 0.16, which implies that on average 15% to 16% of the observed boutons in each sample was in contact with a parvalbumin dendrite, irrespective whether the labeled fibers originated from MEA or LEA.
DISCUSSION The present results reveal close spatial interactions in the subiculum between boutons of fibers originating in entorhinal cortex and dendrites of neurons expressing parvalbumin immunoreactivity. That the projections from the MEA and LEA ‘behave’ the same in this respect is expressed by the ‘contact ratio’: the number of boutons (‘3D objects’) in contact with parvalbumin dendrites divided by the number of contacts plus ‘free’ BDA-labeled boutons. This contact ratio was in the order of magnitude of 16% and it appeared to be independent of the reference density. Although in cases MEA 2001215 and 2001216 the respective reference densities differed considerably, the average reference density in the MEA projections to the subiculum (48/ 1000 m3) corresponded reasonably well with the average reference density of the entorhino-subicular projection seen in LEA cases (37.5/1000 m3). In spite of the differences in absolute numbers of labeled fibers and boutons, the contact ratios were remarkably stable across all cases. We regard the differences between the density references in the MEA cases therefore as extremes of individual variability associated with subtle differences in injection circumstances, injection parameters, sizes and positions of the injection sites, and final uptake of the neuroanatomical tracer by projection neurons. Also the differences between MEA and LEA projections in absolute numbers of fibers labeled, which are in the order of magnitude of 30%, are not unfamiliar in tracing experiments. At the level of resolution offered by our instrument, the reported interactions consist of boutons of the labeled perforant pathway fibers abutting the shafts of parvalbumin dendrites. From electron microscopic observations (e.g. Fig. 1) we know that boutons on fibers are swellings that
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contain synaptic vesicles and mitochondria, and that they are engaged in synaptic contacts (see also Wouterlood and Groenewegen, 1985; Wouterlood and Jorritsma-Byham, 1993). The observation under light microscopic conditions of two structures in close apposition does not necessarily imply a synaptic contact between those two labeled elements. However, previously we have observed numerous synaptic contacts of entorhinal efferents in the subiculum with dendritic spines and with shafts of nonspinous dendrites (Baks-te Bulte et al., 2005; cf. Fig. 1), while, from the point of view of light microscopy, the physical apposition of a fiber and a dendrite is a prerequisite for a synaptic contact. Furthermore, even if only a fraction of the observed appositions would in reality be engaged in synaptic contact, then still a considerable number of contacts would be present. What might happen, yet indistinguishable with the present method is that different ratios of synapses with parvalbumin dendrites might exist in MEA with LEA. The present data however do not suggest such a difference. The strength of showing appositions between cellular elements of different identity is at the same time the shortcoming of our method: we can only estimate the total which is the sum of casual contacts plus functional synapses. However, in a recent electron microscopical analysis we noted that entorhinal fibers originating from either MEA or LEA, in addition to terminating onto spiny dendrites, are also engaged in synaptic contacts with dendritic shafts most likely belonging to non-spiny, interneurons (Baks-te Bulte et al., 2005). The present results at the confocal level of resolution are in line with these findings. It is noteworthy that Baks-te Bulte et al. (2005) reported approximately 18% of the perforant pathway synapses on dendritic shafts of subicular neurons (14% asymmetrical and 4.2% symmetrical axodendritic synapses), a figure which is quite similar to the contact ratios seen in the present tracing study between boutons of entorhinal fibers and parvalbumin positive dendritic elements. It should be noted here that shrinkage of the tissue (cf. Gardella et al., 2003) which we have not further taken into account, does not affect the contact ratio. Because this ratio was calculated from the actual image series without taking the reference density into account, it is not affected by differences in reference density either. Interestingly, our data indicate that the connectivity of the medial and lateral perforant pathway with parvalbumin positive elements in the molecular layer of the subiculum is quantitatively reasonably similar. This quantitative similarity implies that notwithstanding the reported differences in types of information transferred by way of these two routes (Eichenbaum et al., 2007) the overall connectional schemes may be rather similar. Role of parvalbumin neurons in the subiculum Comparison of the present findings of perforant pathway contacts on parvalbumin positive dendrites (16%) with our previously published, more general EM findings in which the dendritic targets (18%) were not further characterized (Baks te Bulte et al., 2005), indicates that the parvalbuminpositive interneuron population most likely represents the
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main recipient of entorhinal input. Thus, although a variety of interneurons have been differentiated electrophysiologically (Menendez de la Prida et al., 2003; Menendez de la Prida, 2006) as well as anatomically (Köhler and ChanPalay, 1982, 1983; Roberts et al.,1984; Seress et al., 1994; Fujise et al., 1995; Lin and Totterdell, 1998), the parvalbumin-positive interneurons should be considered as the most likely mediators for the reported weak inhibitory response of principal neurons in the subiculum upon entorhinal stimulation (dorsal subiculum; Gigg et al., 2000). The latter authors suggested that the inhibition they found was inflicted via a monosynaptic excitatory influence combined with a multisynaptic inhibitory route via CA1 neurons. The contacts between entorhinal afferents and subicular parvalbumin neurons presently reported suggest a direct polysynaptic inhibitory control over subicular principal neurons mediated by intrinsic GABAergic parvalbumin neurons. This is in line with reports on circuitry in neocortex as well as in the entorhinohippocampal system, where parvalbumin neurons have been identified and characterized as GABAergic inhibitory interneurons acting at the local circuit level (Kawaguchi and Hama, 1987; Hendry and Jones, 1991; Wouterlood et al., 1995; Freund and Buzsáki, 1996; Kawaguchi and Kubota, 1997). As the perforant pathway provides excitatory, glutamate-mediated input, excitation of subicular interneurons by perforant pathway activity might lead to the early suppression of activity in return projections or suppression of activity of subicular neurons involved in intrahippocampal transfer of information. Given the great variety in subicular neurons involved in various projections (Witter, 2006; Menendez de la Prida, 2006) and the great variety in physiological properties of the subicular principal cells it could be of interest to establish the precise anatomical relationships between the subicular parvalbumin neurons and characterized output neurons. Acknowledgments—We acknowledge the assistance by Luciënne Baks-te Bulte and Michel van den Oever in the preparation of the histological material. Nico Blijleven provided much appreciated technical assistance with the confocal microscope, cheerfully dealing with the plethora of associated computer interfaces and programs.
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(Accepted 11 August 2008) (Available online 22 August 2008)
CONTACTS BETWEEN MEDIAL AND LATERAL PERFORANT PATHWAY FIBERS AND PARVALBUMIN EXPRESSING NEURONS IN THE SUBICULUM OF THE RAT F. G. WOUTERLOOD,a* A. J. BOEKEL,a V. ALIANE,a J. A. M. BELIËN,b H. B. M. UYLINGSa,c AND M. P. WITTERa,d
Structures in the medial temporal lobe, in particular the parahippocampal region and the hippocampal formation, are strongly implicated in learning and memory (Eichenbaum, 1999, 2002; Suzuki and Eichenbaum, 2000; Witter and Amaral, 2004). The relevance of this part of the brain is emphasized by changes that occur in cases of mild cognitive impairment, temporal lobe epilepsy and Alzheimer’s disease (Behr and Heinemann, 1996; De Toledo-Morrell et al., 2000; Schwarcz and Witter, 2002; Van Hoesen, 2002). Based on its connectivity the subiculum can be perceived as a crossroad of hippocampal in- and output. The subiculum receives via the perforant pathway a dense and topographically organized projection from layer III neurons in the entorhinal cortex (Naber et al., 2000; Burwell and Witter, 2002). Subicular output projects to forebrain and hypothalamic targets (Shibata, 1989; Wouterlood and Tuinhof, 1992; Naber and Witter, 1998; review in Witter, 2006) and, via a feedback circuit, back to the parahippocampal cortex (Sørensen and Shipley, 1979; Finch et al., 1986; Kloosterman et al., 2003; van Haeften et al., 2003). Yet, in spite of this gross knowledge of connectivity, descriptions of cytological and physiological details (Stewart and Wong, 1993; Harris and Stewart, 2001; Harris et al., 2001; Ishizuka, 2001; Menendez de la Prida et al., 2003; Menendez de la Prida, 2006), and even the discovery of spatially modulated firing of subicular cells (Sharp and Green, 1994; Sharp, 2006), much of the architecture of the neuronal network in this remarkable region is unknown and needs further clarification. In terms of its afferent connectivity, parallels exist between the subiculum and other hippocampal regions. For instance, boutons of perforant pathway fibers form synaptic contacts in the dentate gyrus with dendrites of both principal neurons (granule cells) and interneurons (Freund and Buzsáki, 1996). In field CA1 a similar arrangement has been reported (Freund and Buzsáki, 1996; Kajiwara et al., 2008), and a strong argument in favor of extending this organizational scheme to the subiculum is the finding of feed-forward inhibition here (Finch and Babb, 1980; Finch et al., 1988). As GABA is the most abundant inhibitory neurotransmitter in the hippocampus, subicular GABAergic interneurons are most likely involved. Chemically distinct types of subicular neurons contain either calbindin D-28K (mouse, monkey; Seress et al., 1994; Fujise et al., 1995), nitric oxide synthase (rat; Lin and Totterdell, 1998), or several neuropeptides (rat; Köhler and Chan-Palay, 1982, 1983; Roberts et al., 1984). Recently we observed in the electron microscope that entorhinal input to the subiculum targets to a large extent principal neurons (Baks-te Bulte et al., 2005). However,
a
Graduate School Neurosciences Amsterdam, Research Institute Neurosciences Vrije Universiteit, Department of Anatomy and Neurosciences, Vrije University Medical Center, Room MF-G-136, P.O. Box 7057, 1007 MB Amsterdam, The Netherlands
b
Department of Pathology, Vrije University Medical Center, Amsterdam, The Netherlands
c
Department of Psychiatry and Neuropsychology, Division Brain and Cognition, Maastricht University Medical Center, Maastricht, The Netherlands
d
Kavli Institute for Systems Neuroscience and Center for the Biology of Memory, Norwegian University of Science and Technology, Trondheim, Norway
Abstract—The entorhinal cortex (EC) projects via the perforant pathway to all subfields in the hippocampal formation. One can distinguish medial and lateral components in the pathway, originating in corresponding medial and lateral subdivisions of EC. We analyzed the innervation by medial and lateral perforant pathway fibers of parvalbumin-expressing neurons in the subiculum. A neuroanatomical tracer (biotinylated dextran amine, BDA) was stereotaxically injected in the medial or lateral entorhinal cortex, thus selectively labeling either perforant pathway component. Transport was allowed for 1 week. Transported BDA was detected with streptavidin-Alexa Fluor™ 488. Parvalbumin neurons were visualized via immunofluorescence histochemistry, using the fluorochrome Alexa Fluor™ 594. Via a random systematic sampling scheme using a two-channel, sequential-mode confocal laser scanning procedure, we obtained image series at high magnification from the molecular layer of the subiculum. Labeled entorhinal fibers and parvalbumin-expressing structures were three dimensionally (3D) reconstructed using computer software. Further computer analysis revealed that approximately 16% of the 3D objects (‘boutons’) of BDA-labeled fibers was engaged in contacts with parvalbumin-immunostained dendrites in the subiculum. Both medial and lateral perforant pathway fibers and their boutons formed such appositions. Contacts are suggestive for synapses. We found no significant differences between the medial and lateral components in the relative numbers of contacts. Thus, the medial and lateral subdivisions of the entorhinal cortex similarly tune the firing of principal neurons in the subiculum by way of parvalbumin positive interneurons in their respective terminal zones. © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: parahippocampal region, hippocampal region, confocal laser scanning, neuroanatomical tracing, inhibition. *Corresponding author. Tel: ⫹31-20-444-8049; fax: ⫹31-20-444-8054. E-mail address: [email protected] (F. G. Wouterlood). Abbreviations: BDA, biotinylated dextran amine; LEA, lateral entorhinal cortex; MEA, medial entorhinal cortex; 3D, three dimensionally. 0306-4522/08 © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2008.08.023
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Fig. 1. Electron micrograph of a BDA-labeled axon terminal (BDA) in the subiculum forming a synapse (arrows) with a thin and nonspinous dendrite. The BDA has been deposited in layer III of MEA (Baks-te Bulte et al., 2005). Nonspinous dendrites in the subiculum may belong to interneurons. Note that the shape of the postsynaptic dendrite is similar to that of thin parvalbumin-immunofluorescent dendrites in the present study. BDA injection in layer III of entorhinal cortex.
approximately 18% of the axon terminals of perforant pathway fibers in the subiculum had synaptic contacts with presumed subicular interneurons (Fig. 1). In the dentate gyrus, entorhinal axons have been shown to target parvalbumin positive neurons (Zipp et al., 1989), and since these cells provide powerful perisomatic inhibitory innervation, this projection has been implicated in the control of granule cell firing. In the subiculum, parvalbumin positive interneurons are likely candidates to receive entorhinal inputs because they have apical dendrites that extend into the perforant path terminal zone (our own unpublished data). The topographical organization of the perforant pathway in the subiculum is striking, i.e. inputs from the medial entorhinal cortex (MEA) innervate a portion of the subiculum not innervated by the lateral entorhinal cortex (LEA) and vice versa (Witter, 2006). In addition, the two components of the perforant pathway likely mediate the transfer of different sensory information and they may show different overall electrophysiological effects as shown in the dentate gyrus (Pöschel and Stanton, 2007). In the present study we therefore explored whether parvalbumin positive interneurons in the subiculum are indeed innervated by entorhinal inputs and if so, whether the lateral and medial components of the pathway differ quantitatively. We combined for this purpose neuroanatomical tracing, immunofluorescence, high-resolution confocal laser scanning, three dimensionally (3D) computer reconstruction and quantification. A preliminary report has been published in abstract form (Boekel et al., 2006).
EXPERIMENTAL PROCEDURES Surgery, injection of tracer, kill All experiments were in accordance with local, national and European Community regulations on animal well-being. The smallest number of animals possible was used to substantiate our findings; that is, four female Wistar rats. Four female young Wistar rats (weighing 200 –220 g; Harlan Centraal Proefdierbedrijf, Zeist, The Nether-
lands) were used. Animals were deeply anesthetized with a 4:3 mixture of 10% ketamine (Aesco, Boxtel, The Netherlands) and 2% xylazine (Rompun, Bayer, Mijdrecht, The Netherlands) (total dose of this mixture 1 ml/kg body weight) and mounted in a stereotaxic frame. Biotinylated dextran amine (BDA, MW 10,000, Molecular Probes, Eugene, OR, USA; 5% solution, in 50 mM Tris-buffered saline, TBS, pH 7.4) was injected bilaterally via a vertical (in the sagittal plane) approach into the superficial portion of typical MEA (case 2001215 and 2001216; Bregma ⫺7.80, lateral 4.80 and deep 6.40 from the point of entry of the pipette, dorsal pial surface) or LEA (cases 2001219 and 2002012; Bregma ⫺6.36, lateral 6.80 and deep 5.60 from the dorsal pial surface) of the entorhinal cortex (coordinates derived from the rat brain atlas by Paxinos and Watson, 2005). Injections were made through glass micropipettes (tip diameter 10 – 20 m), using a positive pulsed 6 A DC current (7 s on/off; 20 min). The animals were killed 1 week after the surgery via an overdose of sodium pentobarbital (Nembutal, i.p. 60 mg/kg body weight; Sanofi Sante, Maassluis, The Netherlands). They were subsequently perfused transcardially with saline followed by 500 ml of a mixture of freshly depolymerized 4% paraformaldehyde and 0.1% glutaraldehyde (Merck, Darmstadt, Germany) in 100 mM phosphate buffer, pH 7.4, room temperature. Brains were removed from the skulls and postfixed for an additional 1 h at 4 °C. Sections, 40 –50 m thick, were cut on a vibrating microtome (model VT1000S, Leica Microsystems, Heidelberg, Germany). Brains with an injection of BDA in MEA were sectioned in the horizontal plane whereas the brains with an injection of BDA in LEA were cut in the frontal plane. After cutting, sections were rinsed in 100 mM phosphate buffer, pH 7.4, and further processed.
Immunocytochemistry Sections were rinsed three times with incubation buffer (50 mM Tris/HCl buffer with 150 mM NaCl and 0.5% Triton X-100™ pH 8.0; this buffer was also used for all rinses between incubation steps) (Triton is a trademark of Sigma, St. Louis, MO, USA) and preincubated free-floating (gentle agitation on a rocking plateau) for 1 h at room temp in 5% normal goat serum (Nordic, Tilburg, The Netherlands) and 2% bovine serum albumin (BSA, Sigma). Incubation with the primary antibody (mouse anti-parvalbumin (1:1000, Sigma) took at least 48 h at 4 °C. The next step consisted of a 2-h incubation at room temperature with a cocktail of goat anti-mouse IgG conjugated to Alexa Fluor™ 594 (1:400, Invitrogen–Molecular Probes, Eugene, OR, USA) and streptavidin–Alexa Fluor™ 488 (1:400, Invitrogen–Molecular Probes). Finally, the sections were mounted on glass slides, dried and coverslipped with DPX (Fluka Chemie AG, Buchs, Switzerland). We also incubated sections with a cocktail of streptavidin–Alexa Fluor™ 488 and streptavidin–Alexa Fluor™ 594 for laser scanner calibration purposes. Sections were inspected in a standard fluorescence microscope to chart the sites of BDA injections (Fig. 2A, B).
Confocal laser scanning microscopy: preparation of composite images Sections containing BDA-labeled fibers in the target area (the subiculum) were analyzed with the aid of a Leica TCS-SP2-AOBS confocal laser scanning microscope (CLSM; Leica Microsystems) equipped with an argon/krypton gas laser (488 nm excitation) and a He–Ne laser (594 nm excitation). We selected these excitation wavelengths since fluorescence crosstalk between channels with the fluorochromes Alexa Fluor™ 488 and Alexa Fluor™ 594 can be completely ruled out (Wouterlood, 2006). A channel is a specific configuration of the CLSM designed to detect a particular fluorochrome: laser, emission wavelength, dichroic mirror, range of the emission spectrophotometer detection (‘emission filtering’) and detector. First, we took series of images at low magnification (20⫻ objective) and in ‘simultaneous’ mode, in the 488 and
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Fig. 2. Experimental approach. (A, B) BDA injection site, case MEA 2001215, horizontal section: BDA green, parvalbumin red; injection site centered in the superficial cortical layers (I–III). Fibers run to the subiculum (Sub), fields CA1 and CA3 and the middle one-third of the molecular layer of the dentate gyrus (DG). Very dense terminal fiber labeling is indicated with flat gray in frame B. Note the dense parvalbumin staining in the Sub concentrated in the cell layer. The asterisk indicates layer IV, the lamina dissecans. (C) Sampling scheme. A superimposed grid of 100⫻100 m squares [yellow lines] over the subiculum includes the selection area (molecular layer [ML], outlined with thick white border). Beginning with one randomly selected square, every tenth square was selected for sampling (indicated with numbers). PP⫽perforant pathway; PrSub⫽presubiculum. COM⫽commissural fibers.
594 nm channels. From these images we assembled mosaic composite images of the entire subiculum (e.g. Fig. 2C).
Random systematic sampling strategy BDA-labeled perforant pathway fibers were restricted to the outer molecular layer of the subiculum which contains the distal portions
only of dendrites of subicular neurons. The somata of the candidate recipient neurons were invariably present much deeper, in, or close to, the cell layer. Thus for the purpose of determining contacts we needed only samples of the molecular layer. We applied the following procedure which is called ‘random systematic sampling’ (illustrated in Fig. 2C) (cf. Gundersen, 1986). First we placed an overlay sampling grid with 100⫻100 m square units over the composite image and
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Table 1. Sampling and counting Case
MEA 2001215 MEA 2001216 Average MEA LEA 2001219 LEA 2002012 Average LEA Average LEA⫹MEA Totals
Qualifying squares
Samples
Boutons reference density
Boutons mean number⫾S.D.
Contacts normalized number⫾S.D.
Contact ratio: ratio of boutons engaged in contacts/‘free’ labeled boutons
9 6
1006 893
7 10
1406 593
146 119 132.5 147 224 185.5
76 20 48 37 38 37.5 42.75
24.19⫾18.65 6.01⫾3.19 16.03 16.63⫾12.75 22.22⫾16.16 20.01 18
2.80⫾3.05 1.16⫾0.99 2.06 3.25⫾3.15 2.69⫾4.30 2.92 2.56
0.12 0.19 0.15 0.20 0.12 0.16 0.16
Number of sections
3898
636
Two animals (2001215, 2001216) with injection of tracer in MEA and two animals (2001219 and 2002012) with injection of tracer in LEA. All results brought together and averaged if necessary. Note that one qualifying square measures 100⫻100 m. One square corresponds with four samples (all samples having X and Y dimensions of 29.7 m). Boutons and contacts are expressed as the number of 3D objects per 1000 m3 scanned tissue volume.
we outlined the selection area. This area (indicated in Fig. 2C with a thick white line) contained those grid squares over the molecular layer where BDA-labeled fibers were present. Starting from square 1, i.e. a square selected randomly in the first row of squares inside the selection area, we sampled every tenth square. The number of squares per section qualifying for sampling ranged between 95 and 256. From each brain we scanned samples in six to nine separate sections. A survey of the sampling is presented in Table 1.
to calculate from the acquired image the best estimate of the original image (Bertero et al., 1990; Snyder et al., 1992; van der Voort and Strasters, 1995; Hiesinger et al., 2001; Wallace et al., 2001). The images obtained from the double-stained calibration sections were used to check and eliminate sources of possible errors, e.g. pixel ‘shift’ between channels as a result of chromatic aberration or other, instrument-related causes (discussed in Wouterlood, 2006).
High-resolution confocal laser scanning
Automated contact recognition and quantification. Automated 3D object and contact recognition was performed with SCIL_Image software (TNO, Delft, The Netherlands). The basic premises and the procedures necessary to run this software in automated contact recognition are discussed elsewhere (Wouterlood et al., 2007, 2008). Briefly, the software first outlined 3D objects in the Z-series of images in each channel by applying isodensity envelopes: ‘boutons’ in the 488 nm channel and ‘dendrites’ in the 594 nm channel. Isodensity envelope parameters (thresholds) were acquired as follows. For the 488 nm channel we used an objective, automated procedure based on the plugin ‘3D object counter’ (Fabrice Cordelières, Institut Curie, Orsay, France; e-mail [email protected]) of the public-domain program ImageJ (Rasband, 1997–2006; automated procedure described in detail in Wouterlood et al., 2008). For the 594 nm channel we determined the isodensity envelope threshold manually with a ‘best-match’ procedure since big, single objects like dendrites with inhomogeneous intense fluorescence contents provide unsatisfactory results in the automated procedure (Wouterlood et al., 2008). SCIL_Image was instructed via macros to first identify 3D objects in images in both channels and to analyze subsequently which of the 488 nm channel 3D objects showed partial voxel overlap with identified 3D objects in the 594 nm channel (explained in Wouterlood et al., 2008). After running the SCIL_Image based software we verified all ‘large’ contacts manually since the software showed a tendency to report one big contact where, in manual 3D reconstructions, a BDA-labeled fiber appears to make series of two or more small contacts with a parvalbumin dendrite. For this purpose we 3D reconstructed boutons and dendrites by means of Amira™ visualization/modeling software (Mercury Computer Systems, Inc., website at www.tgs.com), using the same thresholds implemented in the 3D object recognition in SCIL_Image.
Proper visualization of the involved structures required a high final magnification: 63⫻ immersion objective lens, NA 1.3, electronic zoom setting of 8⫻. At this setting one 100⫻100 m sampling square in the sampling grid was too large to scan completely in one pass. Since at the final magnification the dimensions of individual samples were 29.7 m in the X- and 29.7 m in the Y direction, we scanned in each 100⫻100 m square four immediately adjacent Z-series of images. It should be mentioned at this point that the drying of the sections on the slide and the subsequent embedding in mounting medium had resulted in a shrinkage of the section in the Z-direction of approximately 70%. Thus, tissue deformation in the Z-direction is most likely to be present (see Gardella et al., 2003). Final scanning parameters were: pinhole 1.00 arbitrary units [Airy disk], Z-increment fixed at 80 nm, image frame 512⫻512 pixels at eight-bit sampling, Z-series covering the entire section thickness, 70 –90 successive image frames. We applied the option ‘sequential scanning, alternating between frames’ through which the instrument acquires one image frame in the 488 nm channel followed by one image frame in the 594 nm channel and then moves one increment along the Z-axis to repeat the scanning. For calibration purposes we took during each acquisition session several images of the sections in which BDA-labeled fibers had been double-stained on purpose with both fluorochromes (see above). The scanning parameters for the calibration series were the same as for the experimental series. Since the perforant pathway does not include parvalbumin expressing elements (Wouterlood et al., 1995), colocalization of both markers was ruled out.
Post acquisition image processing Image deconvolution. All images were processed by means of an iterative maximum likelihood estimation algorithm (Huygens II deconvolution software, Scientific Volume Imaging, Hilversum, the Netherlands, website at www.svi.nl) running on an Origin 300 computer (SGI, Mountain View, CA, USA). Deconvolution is a statistical procedure, using the instrument’s point spread function,
Normalization of fiber densities. Deposition of a small amount of a tracer in regions of the CNS (e.g. our injections in MEA and LEA), leads most often to labeling of only a fraction of the fibers projecting from that region. A typical termination zone
F. G. Wouterlood et al. / Neuroscience 156 (2008) 653– 661 (e.g. the molecular layer of the subiculum) then contains a very dense focus of labeled fibers that rapidly thins out toward the edges. As a consequence, the proportion of labeled versus unlabeled fibers varies between sections as well as topographically within individual sections. In Fig. 2A and B these phenomena are clearly visible. A detail that may look trivial while it definitely affects our results is that we notice in the microscope only labeled fibers, remaining literally in the dark with respect to unlabeled fibers. Variations in the proportion of labeled versus unlabeled fibers, e.g. at the edges of the selection area, have profound effect on the absolute number of observed contacts. To correct for this bias we adopted a normalization procedure based on two assumptions, first that the integral fiber density of the perforant pathway in the molecular layer, or the sum of labeled and unlabeled fibers, was constant. The second assumption was that in the center of the selection area, where labeled fiber density is highest, all perforant pathway fibers were labeled. The highest encountered actual density of labeled fibers was therefore considered to be our ‘reference density’ for that particular experimental case. Normalization included three steps. First we determined the actual and reference densities of BDA labeled boutons. Then we calculated for each sample the proportion of the actual density to the reference density, and finally we multiplied the number of actually found contacts with the inverse proportion factor. Actual and reference densities of BDA-labeled boutons had been obtained during the isodensity threshold analysis with the plugin ‘3D object counter’ of ImageJ (see above).
RESULTS BDA: injection sites, projection patterns and termination areas Each BDA injection resulted in the labeling of a cluster of neurons in a distinct site in the entorhinal cortex (e.g. case 2001215L shown in Fig. 2A–B). In all cases, irrespective of the injection in either LEA or MEA, several contingents of labeled fibers were visible coursing to the subiculum and the hippocampal formation. First, a robust projection consisting of relatively thick, labeled fibers with few varicosities (‘passing fibers’) descended radially through the adjacent deep layers of the entorhinal cortex, and either passed through the white matter into the subiculum or entered the angular bundle. Collectively these fibers form the perforant pathway. Second, a dense contingent of projection fibers was present coursing at the surface of the brain in the medial direction, through the molecular layers of the parasubiculum and presubiculum into the molecular layer of the subiculum, where the fibers blended with the fibers arriving via the perforant pathway. A third contingent occurred deep in the white matter of the temporal cortex, contributing to commissural and rostrocaudal intracortical projections. Finally, a few scattered fibers from the angular bundle entered the deepest layer of the subiculum and could be traced through the alveus of field CA1, curving perpendicularly at their final destination into CA1, traversing the stratum pyramidale and radiatum on their way to stratum lacunosum-moleculare of CA1 (the alvear or temporo-ammonic tract; cf. Deller et al., 1996). The molecular layer of the subiculum contained the terminal plexus: densely woven patterns of labeled fibers studded with numerous boutons. The position of the labeled terminal plexus, along the transverse or so-called proximo-distal axis of the subiculum, varied with respect to the origin of the entorhinal
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fibers. In case of an injection in MEA, BDA-labeled terminal fibers were most prominent in the distal subiculum (immediately adjacent to the presubiculum, cf. Witter and Groenewegen, 1990; Witter, 2006). In cases 2001219 and 2002012 (tracer injection in LEA), the position of the labeled terminal fibers was shifted toward a position close to the border with field CA1, i.e. the proximal part of the subiculum. In line with previously published accounts on the organization of the perforant pathway, we observed in cases 2001215 and 2001216 (injections in MEA) anterogradely labeled fibers in the middle one-third of the molecular layer of the dentate gyrus, in the inner half of the stratum lacunosum-moleculare of field CA3 and in stratum lacunosum-moleculare of the proximal part of field CA1. In the cases with LEA injections, labeling in the dentate gyrus was confined to the outer one-third of the molecular layer and, in field CA3, in the outer half of stratum lacunosummoleculare. In field CA1, labeling was present in stratum lacunosum-moleculare overlying the distal CA1, i.e. adjacent to the subiculum. In the subiculum, individual BDA-labeled fibers in the terminal plexus were very thin. Boutons on these thin fibers were very prominent in the confocal microscope at the high magnification applied for the sampling. By far most labeled terminal boutons occurred as spindle-like swellings in en passant positions on the fibers (Fig. 3). The dimensions of these boutons varied considerably. The smallest boutons had a diameter of approximately 0.5 m whereas the diameters of the largest boutons reached approximately 1.0 m. Parvalbumin neurons, dendrites and fibers in the subiculum The terms ‘parvalbumin neurons,’ ‘-cell bodies,’ ‘-dendrites’ and ‘-fibers’ are used as shortcuts for parvalbumin immunopositive neurons, and similarly, for parvalbumin immunopositive cell bodies, dendrites and fibers. Cell bodies of parvalbumin neurons occurred in the deep (polymorph) layer and in the cell layer of the subiculum. Rarely a parvalbumin cell body was present in the molecular layer. The latter layer contained numerous parvalbumin dendrites, many of which physically emerged from the parvalbumin cell bodies in the cell layer. Parvalbumin dendrites often had a beaded appearance (thickenings and constrictions along the dendrite in a pseudocyclical way) (Fig. 3A, B). Parvalbumin dendrites did not bear spines. Thick parvalbumin dendrites could be distinguished with ease because of their diameter, cylindrical shape and attachment to parvalbumin cell bodies. Located in between these traceable dendrites and thick dendritic fragments were numerous thin truncated dendrites, and parvalbumin fibers. Because of the relatively strong weight of the criterion ‘diameter’ on the decision to classify a parvalbumin structure as a ‘dendrite,’ correspondingly the thinner parvalbumin dendrites were increasingly harder classifiable with certainty as dendrites. An elongated aspect of a thin parvalbumin structure together with a relatively stable diameter compared with a typical bouton was decisive to classify such a structure as a dendrite. Conse-
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Fig. 3. (A) Z-projection overlay image of a deconvoluted image series. Arrows indicate boutons of BDA-labeled fibers (green) close to parvalbumin dendrites (red). Frame size was in all samples 29.7⫻29.7 m. The inset shows one of the BDA-labeled fibers with its boutons at high magnification. (B) 3D Reconstruction of these image series. (C) Detail of squared area of B. Several contacts are visible (arrows). (D) Graphical output of SCIL_image analysis. Z-projection image. Yellow indicates overlap of voxels belonging to BDA-labeled boutons (color coded green) and parvalbumin dendrites (color coded red).
quently, at the lower range of the diameter scale it was impossible to distinguish the thinnest parvalbumin dendrites from thick parvalbumin axons. Such small profiles were eliminated from computer analysis by introducing an offset of 100 voxels. Contacts between BDA-labeled boutons and parvalbumin containing dendrites Typically, BDA-labeled fibers showed along their course continuous variation of their diameters (Fig. 3A). Close apposi-
tions (i.e. contacts) occurred between boutons (swellings) of these fibers and the shafts of dendrites of parvalbumin immunopositive neurons. In all sections studied, we found in the sampled areas in the molecular layer numerous varicosities of BDA-labeled fibers in contact with the parvalbumin dendrites (Fig. 3). In total our software analyzed 265 3D reconstructions in the subiculum after injections of BDA in MEA, and 371 3D reconstructions in the subiculum after injections of BDA in LEA. The results are shown in Table 1. Examples of appositions complying with the nu-
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merical criteria for a ‘contact’ are shown in Fig. 3B–D. The reconstructed BDA-labeled fiber in Fig. 3C has three en passant boutons in contact with the parvalbumin dendrite. The density references between cases differed considerably. The upper and lower extremes were both found in the MEA group of cases: case MEA 200215 showing the highest density reference of 76 3D objects per 1000 m3 and case MEA 200216 expressing the lowest density reference with 20 3D objects per 1000 m3. These differences in density references reflect individual variability in the density of fiber labeling. The average number of contacts in case of the medial perforant pathway, as measured in the sections taken from MEA injection cases 2001215 and 2001216, was 2.06/ 1000 m3; in case of the lateral perforant pathway this number, based on the analyses of sections taken from cases LEA 2001219 and 2002012 brains, was 50% higher: 2.92/1000 m3. In all cases the ‘contact ratio,’ which is the ratio of identified contacts to labeled boutons was remarkably similar, on average 0.15– 0.16, which implies that on average 15% to 16% of the observed boutons in each sample was in contact with a parvalbumin dendrite, irrespective whether the labeled fibers originated from MEA or LEA.
DISCUSSION The present results reveal close spatial interactions in the subiculum between boutons of fibers originating in entorhinal cortex and dendrites of neurons expressing parvalbumin immunoreactivity. That the projections from the MEA and LEA ‘behave’ the same in this respect is expressed by the ‘contact ratio’: the number of boutons (‘3D objects’) in contact with parvalbumin dendrites divided by the number of contacts plus ‘free’ BDA-labeled boutons. This contact ratio was in the order of magnitude of 16% and it appeared to be independent of the reference density. Although in cases MEA 2001215 and 2001216 the respective reference densities differed considerably, the average reference density in the MEA projections to the subiculum (48/ 1000 m3) corresponded reasonably well with the average reference density of the entorhino-subicular projection seen in LEA cases (37.5/1000 m3). In spite of the differences in absolute numbers of labeled fibers and boutons, the contact ratios were remarkably stable across all cases. We regard the differences between the density references in the MEA cases therefore as extremes of individual variability associated with subtle differences in injection circumstances, injection parameters, sizes and positions of the injection sites, and final uptake of the neuroanatomical tracer by projection neurons. Also the differences between MEA and LEA projections in absolute numbers of fibers labeled, which are in the order of magnitude of 30%, are not unfamiliar in tracing experiments. At the level of resolution offered by our instrument, the reported interactions consist of boutons of the labeled perforant pathway fibers abutting the shafts of parvalbumin dendrites. From electron microscopic observations (e.g. Fig. 1) we know that boutons on fibers are swellings that
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contain synaptic vesicles and mitochondria, and that they are engaged in synaptic contacts (see also Wouterlood and Groenewegen, 1985; Wouterlood and Jorritsma-Byham, 1993). The observation under light microscopic conditions of two structures in close apposition does not necessarily imply a synaptic contact between those two labeled elements. However, previously we have observed numerous synaptic contacts of entorhinal efferents in the subiculum with dendritic spines and with shafts of nonspinous dendrites (Baks-te Bulte et al., 2005; cf. Fig. 1), while, from the point of view of light microscopy, the physical apposition of a fiber and a dendrite is a prerequisite for a synaptic contact. Furthermore, even if only a fraction of the observed appositions would in reality be engaged in synaptic contact, then still a considerable number of contacts would be present. What might happen, yet indistinguishable with the present method is that different ratios of synapses with parvalbumin dendrites might exist in MEA with LEA. The present data however do not suggest such a difference. The strength of showing appositions between cellular elements of different identity is at the same time the shortcoming of our method: we can only estimate the total which is the sum of casual contacts plus functional synapses. However, in a recent electron microscopical analysis we noted that entorhinal fibers originating from either MEA or LEA, in addition to terminating onto spiny dendrites, are also engaged in synaptic contacts with dendritic shafts most likely belonging to non-spiny, interneurons (Baks-te Bulte et al., 2005). The present results at the confocal level of resolution are in line with these findings. It is noteworthy that Baks-te Bulte et al. (2005) reported approximately 18% of the perforant pathway synapses on dendritic shafts of subicular neurons (14% asymmetrical and 4.2% symmetrical axodendritic synapses), a figure which is quite similar to the contact ratios seen in the present tracing study between boutons of entorhinal fibers and parvalbumin positive dendritic elements. It should be noted here that shrinkage of the tissue (cf. Gardella et al., 2003) which we have not further taken into account, does not affect the contact ratio. Because this ratio was calculated from the actual image series without taking the reference density into account, it is not affected by differences in reference density either. Interestingly, our data indicate that the connectivity of the medial and lateral perforant pathway with parvalbumin positive elements in the molecular layer of the subiculum is quantitatively reasonably similar. This quantitative similarity implies that notwithstanding the reported differences in types of information transferred by way of these two routes (Eichenbaum et al., 2007) the overall connectional schemes may be rather similar. Role of parvalbumin neurons in the subiculum Comparison of the present findings of perforant pathway contacts on parvalbumin positive dendrites (16%) with our previously published, more general EM findings in which the dendritic targets (18%) were not further characterized (Baks te Bulte et al., 2005), indicates that the parvalbuminpositive interneuron population most likely represents the
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main recipient of entorhinal input. Thus, although a variety of interneurons have been differentiated electrophysiologically (Menendez de la Prida et al., 2003; Menendez de la Prida, 2006) as well as anatomically (Köhler and ChanPalay, 1982, 1983; Roberts et al.,1984; Seress et al., 1994; Fujise et al., 1995; Lin and Totterdell, 1998), the parvalbumin-positive interneurons should be considered as the most likely mediators for the reported weak inhibitory response of principal neurons in the subiculum upon entorhinal stimulation (dorsal subiculum; Gigg et al., 2000). The latter authors suggested that the inhibition they found was inflicted via a monosynaptic excitatory influence combined with a multisynaptic inhibitory route via CA1 neurons. The contacts between entorhinal afferents and subicular parvalbumin neurons presently reported suggest a direct polysynaptic inhibitory control over subicular principal neurons mediated by intrinsic GABAergic parvalbumin neurons. This is in line with reports on circuitry in neocortex as well as in the entorhinohippocampal system, where parvalbumin neurons have been identified and characterized as GABAergic inhibitory interneurons acting at the local circuit level (Kawaguchi and Hama, 1987; Hendry and Jones, 1991; Wouterlood et al., 1995; Freund and Buzsáki, 1996; Kawaguchi and Kubota, 1997). As the perforant pathway provides excitatory, glutamate-mediated input, excitation of subicular interneurons by perforant pathway activity might lead to the early suppression of activity in return projections or suppression of activity of subicular neurons involved in intrahippocampal transfer of information. Given the great variety in subicular neurons involved in various projections (Witter, 2006; Menendez de la Prida, 2006) and the great variety in physiological properties of the subicular principal cells it could be of interest to establish the precise anatomical relationships between the subicular parvalbumin neurons and characterized output neurons. Acknowledgments—We acknowledge the assistance by Luciënne Baks-te Bulte and Michel van den Oever in the preparation of the histological material. Nico Blijleven provided much appreciated technical assistance with the confocal microscope, cheerfully dealing with the plethora of associated computer interfaces and programs.
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(Accepted 11 August 2008) (Available online 22 August 2008)