Calbindin D-28k and parvalbumin in the rat nervous system.

0306-4522/90$3.00+ 0.00 Pergamon Press plc 0 1990IBRO

,VeuroscienceVol. 35, No. 2, pp. 375..475,1990 Printed in Great Britain

CALBINDIN

D-28k AND PARVALBUMIN NERVOUS SYSTEM* M. R.

IN THE RAT

CELIOt

Institute of Anatomy, University of Kiel, Olshausenstrasse 40, D-2300 Kiel, F.R.G. Abstract-This paper describes the distribution of structures stained with mono- and polyclonal antibodies to the calcium-binding proteins calbindin D-28k and parvalbumin in the nervous system of adult rats. As a general characterization it can be stated that calbindin antibodies mainly label cells with thin, unmyelinated axons projecting in a diffuse manner. On the other hand, parvalbumin mostly occurs in cells with thick. mvelinated axons and restricted. focused nroiection fields. The distinctive staining with antibodies against these two proteins can be ‘observed throughout the nervous system. Calbindin D-28k is primarily associated with long-axon neurons (Go&$ type I cells) exemplified by thalamic projection neurons, strionigral neurons, nucleus basalis Meynert neurons, cerebellar Purkinje cells, large spinal-, retinal-, cochlear- and vestibular ganglion cells. Calbindin D-28k occurs in all major pathways of the limbic system with the exception of the fornix. Calbindin D-28k is, however, also found in some short-axon cells (Golgi type II), represented by spinal cord interneurons in layer II and interneurons of the cerebral cortex. It is also detectable in some ependymal cells and abundantly occurs in vegatative centres of the hypothalamus. The “paracrine core” of the nervous system and its adjunct (1985, Nieuwenhuys, Chemoarchifecrure o.the Brain. Springer, Berlin) is very rich in calbindin D-28k. The dist~bution of calbindin D-28k-positive neurons is very similar to that of the dihydro~rydine subtype of calcium channels. Most of the cells containing calbindin D-28k are vulnerable to neurodegenerative processes. Pa~albumin-immunoreactive neurons have a different, and mostly complement~y dist~bution compared with those which react with calbindin D-28k antisera, but in a few cases (Purkinje cells of the cerebellum, spinal ganglion neurons), both calcium-binding proteins co-exist in the same neuron. Many pa~albu~n-immunoreactive cells in the central nervous system are intemeurons (Golgi type II) and, to a lesser extent, long-axon cells (Golgi type I), whereas conditions are vice versa in the peripheral nervous system. Intrinsic parvalbuminic neurons are prominent in the cerebral cortex, hippocampus, cerebellar cortex and spinal cord. Long-axon ~~albumin-immuno~active neurons are, for example, the Purkinje cells, neurons of the thalamic reticular nucleus, globus pallidus, substantia nigra (pars reticulata) and a subpopulation among large spinal-, retinal-, cochlear- and vestibular ganglion cells. Parvalbumin is rich in cranial nerve nuclei related to eye movements. In addition to nervous elements, pa~album~n immunoreactivity occurs in a few ependymal cells and in some pillar cells of the organ of Corti. In contrast to calbindin D-28k, parvalbumin is virtually absent from vegetative centres and pathways. Parvalbumin co-exists with GABA in cortical neurons and is, in general, prefe~ntially associated with “fast-tiring” neurons. Whole functional systems are revealed by either parvalbumin or calbindin D-28k immunohistochemistry. Pa~albumin, for example, occurs in the whole chain of neurons of the epicritic sensibility in the somatosensory system. Calbindin D-28k, on the other hand, occurs in the whole taste pathway. With antibodies against calbindin D-28k and parvalbumin it is possible to reveal at least two as yet undescribed brain nuclei: one in the hypothalamus ~a~albumin I), the second in the medulla (~lbindin D-28k 1). Calbindin D-28k and parvalbumin are evenly distributed within the various domains of the same neuron, but in some instances their levels vary between soma and cell processes. The immunostaining with calbindin D-28k and pa~albumin antisera show gradations in intensity among neurons belonging to different populations, a phenomenon compatible with the presence of varying concentrations of these proteins. Calbindin D-28k and pa~albumin are excellent new neuroanatomi~l markers which can be utilized to selectively visualize certain neurons and pathways in the central nervous system and peripheral nervous system.

Calcium ions were discovered by Ringeti3’ to be a necessary component in the bathing fluid to preserve myocardial contractibility in explanted frog hearts. *Dedicated with love to my wife Monica. tPresent address: Institute of Histology,

University

of

Fribourg. CH-1700 Fribourp. Switzerland. Abbreviah.&:

CaBP, calbind:; D-28k; CAMP, cyclic adenosine-3’,5’-monophosphate; DAB, 3,3’-diaminobenzidine; EGTA, ethyIeneglycolbis(aminoethylether)tetra-acetate; IR, immunoreactive; PAP, peroxidaseantiperoxidase; PBS, phosphate-buffered saline; PV, parvalbumin; SDA, sexually dimorphic area; TBS, Tris-

buffered saline.

Ten years later Locket63 found that neuromuscular transmission in frog skeletal muscles subsided in the absence of extracellular calcium. In the following 60 years a large number of observations on the crucial role of calcium in the most distinct fields of biology accumulated.‘47 A major conceptual advance was the gradual reaiization that Ca*’ elicited it’s effects intracellularly.3Q Of further relevance were the suggestions that Ca2+ couples stimulation and contraction in skeletal

muscles,237 and takes part in stimulation and exocytosis in endocrine glands and nervous tissue.77.2” 375

376

M. R. CELIO

Ca2+ is indispensable for axoplasmic transport”‘~‘*’ and may even be involved in leaming’37 and memory formation.*0~‘67 On the other hand, un~ontroiied elevation of the intracellular Ca2+ concentration leads to excessive cell activation, injury and, ultimately, cell death.39 Based on the observation that Ca*+ selectively triggers a wide variety of biological responses, Rasmussenzz4 advanced the idea that Ca*+ may serve as a universal second messenger, analogous to cyclic adenosine-3’S’-monophosphate (CAMP). Although Rasmussen’s224 article fails to explain how the wide variety of Ca*+ effects can be performed by one and the same messenger, the concept of “transmembrane information transfer” by means of Ca’+ is an established hypothesis in modern biology. The discovery of a specific, high-a~nity intracellular acceptor protein, troponin-C,78,79 which binds Ca2+ to induce skeletal muscle contraction, initiated a new epoch in the field of catcium research. But for a period of time, the interaction of Ca*+ with an intracellular calcium-binding protein was regarded as exclusive for skeletal muscles. With the isolation of a widely occurring calciumbinding protein, ~almodulin,% the general importance of calcium-binding proteins became evident. Caimodulin has been found to bind Ca*+ and to transduce--through graded conformational changes-the signal carried by this ion in at least 15 different biologically selective effects,“’ leading to its designation as a “trigger protein”.66 How calmodulin alone may give rise to all these effects is still not completefy understood. The problem of the specificity of the Ca2+-signal has been moved to another level, but in principle, it remains unresolved. Perhaps the differential intracellular distribution or the combinations and ~~utations of many different calcium-binding proteins in a given cell and their subtle competition for CaZ+ help to establish an ordered sequence of reactions. In addition to calmodulin and troponin-C, some other proteins are known to bind Ca*+ with highaffinity; these are the vitamin D-dependent calciumbinding proteinz7’ (now called calbindin D-28k, CaBP), S-1003* and parvalbumin (PV).“6,“7,209New calcium-binding proteins are being discovered in rapid suceession,‘77~199~202~2”~276 and most of them belong to a single family of proteins, which have evolved from a common precursor.‘47 The function of these calcium-binding proteins is less versatile than that of calmodulin; PV and CaBP either act in calcium transport or as intracellular calcium butTers,14’ so called “transport/buffer” proteins.& PV in fast muscle Gbres is thought to transiently bind Ca*+ and to shuttle them back to the sarcoplasmic reticulum, thus increasing the speed of muscle reiaxation.43.96*209 On the same line of thought, CaBP may be involved in the transI~ation of Ca2 + through the intestinal mucosa.“4,277 Up to now all calcium-binding proteins, except

troponin-C’3~233 have been isolated from the brain of various species. While S-100 occurs in astrocytes,sg~‘” calmodulin 27~159~247uZVBl~~42.47 and ~a~p’6,‘7,‘8,19”.W.9’~~30,2W66

are

ali

present

in

neurons,

Calmodulin is ubiquitous”’ and occurs in all neurons (see, however, Refs 159,247), while CaBP and PV only occur in certain subsets of neurons. Information published on the anatomical distribution of the calcium-binding proteins are still fragmentary and sometimes controversial. Therefore, the aim of the present investigation is to analyse in detail the distribution of CaBP and PV immunoreactivities in cell bodies, processes and pathways in the adult rat nervous system by sensitive and specific immunohistological methods. This morphological study is a necessary step to allow future physiological, biochemical and pha~aco~o~~l studies of calciumbinding proteins in the brain. It was undertaken in order to gain insight into the role of these two calcium-binding proteins in the nervous system and to exploit the antibodies against ~lcium-binding proteins as new neuroanatomical markers. This report is complete but not exhaustive and shall help to raise interest in these two proteins. The results are presented according to to~~aphical aspects, while the discussion is organized according to functional entities. EXFRRIM~TAL

PROCEDURRS

The brain of 67 adult rats (42 Wistar, 20 ZUR-Siv, four Sprague-Dawley, one Long-Evans) of both sexes (37 males and 30 females), the spinal cord and the spinal ganglia and various peripheral nerves of five more animals, the eyes of four animals and the inner ear of three other creatures were used for this study. In addition we studied 10 incomplete series of rat brains-and other tissues as well as single sections of the brain of other rats. For Fig. 12 we employed a CB 57 mouse, processed by perfusion fixation with‘lO% formalin “embedded” in 50% bovine serum albumin. For Figs 7 1, 72 and 73, a seven-day-old rat was used, processed by perfusion with Bouin fluid. In addition we studied sections of the neurological mice mutants “staggerer”24* and “quaking”X8 (Jackson Laboratories, U.S.A.). All animals were kept under a constant dark-Tight schedule (7 h light on, 19 h light off) with food pellets (NAFAG, St Gallen, Switzerland) and tap water provided ad libitunz. They weighed between 250 and 350 g at the time of the tissue collection, which took place at different times of the day.

Fifty microlitres of colchicine solution in phosphatebuffered saline (PBS) (1 mg/ml) were injected intraventricularly to nine rats (Sprague-Dawley and Wistar). After two days the paraplegic animals were perfused and processed by perfusion with 4% (w/v) pa~fo~~dehyde in 0.1. M phosphate buffer, pH 7.4. Tissue processing

Unless stated otherwise, the animals were perfused through the ascending aorta with c. 300 ml of fixative. The perfusion was followed by excision of the tissue and 2 h nostfixation. The followina fixatives were used. (1) Perfusion with Bouin fluid [saturated aqueous picric acid; forrnalin 40% (Merck); concentrated acetic acid, 15: 5:2 by volume]. The tissue was dehyd~ted and embedded in paraffin foliowing routine methods. For the inner ear, the whole petrosal bone was decalcified with 5% formic acid in water for two

Calcium-binding proteins in the rat brain days previous to embedding. (2) Perfusion fixation with 10% formalin (made from 40%. Merck) in 0.1 M cacodylate buffer pH 7.4. The tissue was either: (a)“‘embedded” in -50% bovine serum albumin for Vibratome sectioning; or (b) soaked in 18% sucrose (w/v) for 24 h at 4°C and frozen on pulverized dry ice for cryostat sections. (3) Perfusion with 4% (w/v) paraformaldehyde (Merck or Aldrich) in 0.1 M phosphate-buffer, pH 7.4. The tissue was processed as in 12). (41 Perfusion with 2.5% alutaraldehyde (v/v) (25% amp&l&, Polysciences), 2.5%- (w/v) paraformaldehyde (Aldrich) in 0.1 M cacodylate buffer pH 7.4 (+ 50 mM CaCl,). For the detection of PV and GABA on consecutive sections, 1 g/l Na-metabisuhite was added to the lixative which was prepared at pH 7.8. Alternatively, blocks were embedded in ~Araldite. and sections depolymerixed with Na-alcoholate.‘76 (5) Banid free&a of 4-5-mm-thick dimes of unfixed tissue in isopentane, co&d by liquid nitrogen, freeze substitution with acetone at -70°C for one week; freeze fixation at the same temperature with a mixture containing acetone (90 ml), formalin 40% (4 ml), concentrated acid (1 ml), distilled water (5 ml) for one week; embedding in paraplast. The thicker (50 urn) Vibratome or cryostat sections l(2) and (3) above] were used for the survey and counts of the relative and absolute cell body density and fibre systems and for electron microscopic pi-e-embedding staining, while the 6-pm-thin paraliln sections [( 1) above] provided finer details of perikaryal and dendritic morphology. Method (5) above was only included to provide evidence that no artificial redistribution of CaBP and PV took place before embedding and immunostaining (see Ref. 268, on this subject). It was, however, not used as a routine because of the laborious procedure.. The spinal cord was at best immunostained after processing with methods (I), (2b) and (3) above, whereas the eye and particularly the inner ear could only be studied after using method (I), because of obvious technical constraints. Method (4) was used to test the resistance of the antigens studied to the effects of strong fixation and also, slightly modified, to permit the visualization of GABA and PV on consecutive sections. The pa&in (5-pm-thick) and cryostat (16~pm-thick) sections were collected on chrome-alum gelatine-coated slides, dewaxed with xylol and mhydrated (for paraffin processed for immunohistochemistry and sections), mounted in Et&i@. The Vibratome (50 pm) sections were incubated floating in diluted antiserum solution on a shaker, collected on coated slides and mounted in Eukitt’a after short dehydration in ethanol and xylol. In three cases the Vibratome sections were mounted undehydrated with glycerine gelatine (1: 1). Four rat brains (all males) were processed by method (1) (Bouin/paratBn) and cut serially in coronal (twice), longitudinal or horizontal sections, respectively. Every 100 sections, three consecutive sections were collected on three different slides. All three were immunostained; two were counterstained for cells with Cresyl Violet (1% in acetate buffer pH 3.4 for 20 min at 4OC), respectively for fibres with Luxol Fast Blue (12 h at 60°C). .I

_

Antibodies Three different polyclonal antibodies against rat muscle PV (identical to brain PV25) were used first. They satisfy various criteria of specificity as demonstrated by Ouchterlony immunodiffusion and preadsorption experimentsz6”*“’ and immunoblotting. ‘w’~ In addition we used three well characterized monoclonal antibodies against carp PV (235, 239, 267) reacting with mammalian PV.x’ The polyclonal antibodies against chicken intestinal CaBP have been characterized by radioimmunoassays7 and have been used by various groups for the localization of the antigen in various tiss~es.~*~~~~~~~*‘~ Later we used two well characterized monoclonal antibodies against chicken gut CaBP (nos 300, 316) reacting

311

with mammalian calbindin.sl Their specificity has been tested in immunoblots. No cross-reaction between PV and CaBP antibodies is evident according to cross-adsorption tests They also do not recognize calmodulin, oncomodulin’73 and S-100. The antiserum agaist GABA (coupled to albumin) was purchased from Immunonuclear Corporation, Stillwater, MN, U.S.A. and the monoclonal antibody against GABA was kindly provided by Dr Streit, Ziirich.‘75 The specificity of the antiserum was independently proven by various tests!’ PrearLrorption tests The CaBP and PV antibodies used in this study have been preadsorbcd with their respective antigens: high performance liquid chromatography, purified rat muscle PV and chicken intestinal CaBP (I-10pm). The adsorption was carried out in the following manner: the given amount of antigen was mixed with 100 ~1 of antibodies diluted 1: 100 to 1:50,000. Tubes containing exactly the same dilutions of antibodies but without any addition of antigen were incubated in parallel and served as controls. The adsorbed and unabsorbed antibodies were incubated with the paraffin sections for 48 h at 4°C and further processed with the standard peroxidase-antiperoxidase (PAP)-technique (see above). Immunohistochemistry A synthesis of the method used is given in a separate publication.“* The sections on the slides (respectively floating) were incubated for 48-72 h at 4°C in a moist chamber with the primary antibodies diluted 1: 1000 to 1: 50,000 in Ca2+- and M$+-free Tris-buffered saline (TBS). The best signalto-noise ratio was at a dilution of I:2000 to 1: 10,000 for PV and 1: 5000 to 1: 20,000 for the CaBP antibodies. For the detection of PV and GABA on consecutive sections, the antibodies were diluted in TBS with 1 mg/ml Nametabisulfite and 38Omg/l Na-borohydride. After 3 x 5 min, TBS rinsing (3 x 15 min for the Vibratome sections) the sections were incubated with goat-anti-rabbit IgG (Miles), 1: 200 for 30 min (1: 200 for 2 b) at room temperature. After a further wash with TBS (3 x 5 min and 3 x 15 min. resoectively) the sections were incubated with rabbit PAP’complex (Stemberger-Meyer Inc., Jarretsville Pike, MD, U.S.A.) 1: 500 for 30 min (1: 500 for 4 h) at room temperature. The locations of the antibody-bound peroxidase were then visualized by incubation with the substrate 3,3’-diaminobenzidine (DAB)-HCl-hydrogen peroxide under visual control. The monoclonal antibodies were localized by an indirect procedure which involved the use of an affinity purified goat-anti-mouse IgG coupled to peroxidase (Miles, 1: 500), or by using the avidin-biotin method (Vector Laboratories, U.S.A.). Some sections, particularly those treated according to methods (2a) and (2b) above, were osmicated with 0.01% osmium tetroxide (0~0,) after completion of the immunostaining. Quant$caiion of immunoreactive neurons The size of the immunostained perikarya in selected areas was determined by measuring the major and minor diameter in the mid nucleolus plane by means of a calibrated eyepiece grid (see Table 2). Bouin’s flxation followed by paratlin embedding is known to produce a 20% shrinkage of the tissue.206With the exception of the small cells in the external plexiform layer of the olfactory bulb and of spinal ganglion cells, cell measurements have therefore been performed on Vibratome sections [method (2a) above] mounted in glycerine gelatine. The total number of PV-immunoreactive (-IR) cells in the fascia dentata of two animals were counted in complete series of Vibratome and paraflin sections, those in the caudatoputamen by counting PV+ neurons in every second Vibratome section of a whole sagittal series.

M. R.. CELIO

378 Camera lucida drawings

Drawings were performed using a drawing tubus and a x 40 plan or a x 100 planapo (oil) Zeiss objective. The size of the cells under study was determined using a calibrated eye-piece grid. The r~onst~~tion could only be carried out on Vibratome sections (50 ,um) which permit visualization of the dendritic network; all sections of this series were dehydrated and embedded in Eukitt’s. RESULTS*

General

remarks

This paper deals with the distribution of CaBP and PV in the normal adult rat central nervous system, the specialized primary sensory end organs and the peripheral nervous system.

*Terminology and summary of the most important physiological and biochemical data regarding the ions and the proteins discussed in this paper

PV immunoreactive, PV-IR, PV-positive structure, PV+, parvalbuminic are used as synonymes, as are calbindin-positive CaBP+, CaBP-IR, CaBP-immunoreactive. Extracellular Ca*+ and Mg’+ in the central nervous

svstem value to 1-2 mM.‘95 ’ Ca*+ and Mg2+ : free in~a~llular

calcium and magnesium, as measured with ion selective microelectrodes are 1.7 x lo-‘M, 6.6 x 10w4M in invertebrate giant neurons, respectively. r3 Values of free intracellular Ca2+ for vertebrate neurons are of the order of lo-’ M.‘** Rat PV (molecular weight of 12,000, isoelectric point 4.9): this designation reflects the small size, the high solubilitv in water and the high electrophoretic mobility of this protein (high diffusion constant and low vik cositvj.“3J’6 Besides Ca*+. PV also binds M~z*+.~’At physiolo~~al levels of Mgi+ (1 mM) and K+ @X0 mM), and at levels of Ca2+ corresponding to those of resting cells (approximately lo-’ M;see below), 1 mol PV binds 2 mol of Me2+ and none of Ca*+. A rise in intracellular calcium level causes calcium binding, which is accompanied by a release of Mr$+.‘r’ PV has an af%nity for ?a*+ of the order of lO’M&d for Mg*+ of about l@ M. PV miaht reeulate the Ca*+ (and Ma*+ ) fluxes in the cell (“bu~er/t&sport”) and participa; in Ca*+ and/or Mg2+ activated processes.66 The synthesis of PV in muscles is neurally regu---.I

lat&153J89

CaBP, the new name for the vitamin D-dependent calcium-binding protein,2’7~278~279~2a’ (molecular weight 28,000; isoelectric point 4.8): CaBP seems to influence the efficiency of the Ca*+ transport mechanism in the chicken intestine. It’s synthesis in the chicken gut, kidney and peripheral nervous system’” is dependent on the presence of vitamin D.‘97~2”~280 In the brain, however: such a vitamin D-dependence could not be found.‘S~242**67 CaBP binds four Ca*+ with high affinity f& = 2 x lo6 M), not to be confused with the 9000 mol. wt CaBP, isolated from rat intestine,279 which is not detectable in nervous tissue (Ref. 269 and own unpublished observation).

In general terms, no obvious differences can be found between the pattern of immunostaining due to various procedures of tissue fixation and embedding. In accordance with the observations of others2@ with CaBP, no immunoreaction can be carried out with unfixed tissue sections. We also suppose that PV, as soluble protein not anchored to any subcellular organelle (at least in muscles99) can be easily displaced and redistributed. In the following description, dot-like structures will be referred to as nerve terminals (boutons terminaux) and the smooth, immunoreactive fibres as axons. Sometimes, especially in the thalamus and reticular formation, the distinction between axons and nerve terminals is difficult at light microscopic level and our interpretation has to be assumed as only tentative. In some cases the intra~llular dist~bution of CaBP and PV varies reproducibly between the various parts of a given neuron. The soma in general contains the highest density of CaBP- and PV-IR sites as seen in the antibody dilution tests and as inferred from the resistance of its staining towards the action of strong fixatives. There are instances, however, where PV occurs only in the axon and terminals and CaBP only in the perikaryon. Examples for the first case are the neurons of the deep cerebellar nuclei, for the second pyramidal cells of the hippocampal CAl-region. In various regions of the brain, the CaBP antibodies produce an homogeneous and diffuse reaction as if all neural elements present in the tissue section, neurons and all their processes, had been stained. Colchicine application does not change the staining pattern of cell bodies reacting with PV antibodies in cerebral cortex, hippocampus, thalamus and cerebellar cortex. However, interference with axoplasmic transport produces an accumulation of PV in certain long-axon neurons. Examples include the deep cerebellar nuclei, the vestibular nuclei, the retinal ganglion cells. This aspect of the distribution of PV is described in a separate publication’ but the most important exceptions are included in Table 6. Control sections of brains incubated in preimmune serum or antigen (1O-9 M) adsorbed antiserum do not exhibit specific immunostaining. Oniy unequivocally stained structures are included in the description and discussion; certain obviously negative, but important exceptions will be pointed out from time to time. The description of the results for CaBP in regions (e.g. cerebellum, hippocampus) already well described by other groups’8X90*95 will be kept at a minimum.

Figs l-76. Many of the following figures have been obtained with the technique of “histography”. By this method the sections on the slide are directly projected on photographic paper, which is subsequently developed and fixed. The image, therefore, is a negative of the original and the immunos~~n~ structures (brown in the original) appear white. This old technique produces sharper images compared with methods which make use of an internegative. For abbreviations see Table 6.

C~cium-binding

proteins in the rat brain

Fig. 1. (A) Coronal section of the olfactory bulb at the level of Fig. 83 incubated with calbindin antiserum. A prominent terminal field is seen in the central half of the accessory olfactory bulb (AOB). Immunoreactive perikarya are very numerous in the glomerular layer (IGr), but also occur in all other layers of the main olfactory bulb. Notice the stripe of thin terminals in the inner half of the external plexiform layer (EPL). No axons course in the lateral olfactory tract (lo). The labelling with the monoclonal antibody against CaBP differs in not showing the terminal field in the AOB, which, therefore, probably derives from the cross-reaction of the antiserum with calretinin. x 50. (B) Consecutive section to (A) incubated with PV antibodies. Terminal fields are virtually absent. Interneurons occupy the external plexiform layer (EPL) and some are scattered in the inner granular layer (I&). x 50. Fig. 2. (A) Photograph of a portion of a coronal section of the olfactory bulb labelled with a CaBP antiserum. A band of strong immunoreactive cell bodies with short processes, representing periglomerular cells, is seen in the upper third of the figure (GL). Scattered neurons are found in the external plexiform layer (EPL) at the boundary between mitral (M) and internal plexiform layer (IPL) and in the granular layer (bottom of the figure). Some of the positive neurons at the boundary between mitral and internal plexiform layer are drawn with the camera lucida Fig. 77A. Notice bundles of thin axons (arrowheads) in the olfactory nerve layer (ON). One of this bundle impinges upon a ~omer~um (star). x 120. (B) Section adjacent to that of Fig. lA, but incubated with a PV antibody. Neurons predominantly in the external plexiform layer (EPL) are tagged. These cells have slender cell processes radiating in all directions, but respecting the laminar borders. Some of these cells are drawn in Fig. 778. Few periglomerular neurons are tagged in the glomerular layer (arrows). Notice axons in the granular layer (arrowhead). Abbreviations as in Fig. 1A. x 120.

380

M. R.

Telencephalon Olfactory bulb (Figs 82 and 83) C~~b~d~n D-28k. A lot of cells in the ~omeru1~

layer are intensely stained as are scattered neurons at the boundary between the mitral and inner plexiform layer (Figs 1A, 77A). The neurons in the periglomerular region have one, or seldom, two cell processes entering one glomerulus (see Table 2 for sizes). An exact arborization pattern of the dendrites of single cells is difficult to discern because of intermingling cell processes. The inner half of the external plexiform layer shows an homogeneous band-like staining (Fig. lA, EPL) but terminals cannot be individually discerned. The neurons in the inner plexiform layer are of two types, as described in the legend to Fig. 77A. At higher antiserum concentration CaBP+ neurons also appear in the external plexiform layer and in the internal granular layer (Figs 1A and 2A). Giant, faintly CaBPf neurons with coronally oriented cell processes are frequently seen around the olfactory ventricle. With polyclonal antibodies, cross-reacting with calretinin on immunoblots, CaBP immunoreactivity also occurs in the axons of the Jacobson’s nerve innervating the vomeronasal organ. The CaBP+ terminal fields in the accessory bulb (Fig. 1A) form a sharply demarcated sphere of interlacing axons and te~inals. No CaBP+ neurons are found. Fascicles of extremely thin, probably unmyelinated axons impinge upon some glomerula (five to six in a 50+mthick section) of the main olfactory bulb (Fig. 2A). My observations with mon~lon~ ~ti~dies are in complete agreement with those of some authors,‘* but differ from those of others,95 who probably used CaBP antibodies cross-reacting with calretinin. Some glomerula probably receive calretinin-positive axons. Perhaps these fibres carry odour-rn~~ity specific info~atio~. Therefore, our observation gives further support to the spatial pattern hypothesis of olfactory processing.* The cells stained in the periglomerular layer preponderantly are periglomerular cellist’ because of their size (Table 2) and their typical dendritic r~ifi~tion pattern. Some larger cell bodies (15 x 10pm) may belong to external tufted cells. Neurons 1, 3, 4 at the boundary between mitral cell layer and inner plexiform layer (Fig. 77A may all represent horizontal cells; Fig. 2, cells 15 and 16)24’whereas cell 2 in the camera lucida drawing in Fig. 77A resembles more the Cajal cells (Fig. 2, cells 9, 10 of the aforementioned paper). Cell 4 actually resembles the cell depicted on the right of the photomontage of Fig. 4 in Ref. 241; this cell is called “superficial short-axon cell”, but according to these authors only occurs in the externat plexiform layer. It is not

CELIO

possible to depict neurons of deep layers with the drawing tubus but I have the impression that representatives of all interneuronszi6 are immunolabelled. The general staining pattern of CaBP in the olfactory bulb resembles that seen with ~et~nk~halin antisera.‘% Parualbumin. In the rostra1 part (Figs 1B and 2B) small (c. 12pm) PV-IR neurons are limited to the external plexiform layer. The soma are preferentially located at the inner and outer periphery of the external plexiform layer (railroad track) and the cell processes are mostly directed towards the centre of the external plexiform layer. The immunoreactive cell processes arborize in close proximity to the cell body and are typically fairly contorted (Figs 2B and 77B). Scattered PV-IR cell bodies can also be found in the mitral cell layer and in the inner plexiform layer, but their processes ramify in the external plexiform layer. Single, very smalf immunoreactive cells (approx. 10 pm) are situated parallel to the tractus olfactorius lateralis, but only in its rostra1 part. In addition, ~~glomer~ar cells in the glomerular layer and a few elongated neurons, squeezed against the convex medial surface of the accessory olfactory bulb, are stained with the PV antibodies (Fig. 2B). PV + axons, running horizontally in coronal sections, can be seen in the internal granular layer and, seldomly, in the external plexiform layer. No PV+ axons can he discerned in the lateral olfactory tract (Figs 1B, 3B). The neurons in the external pIexiform Iayer, immunostained with PV antibodies, belong to various classes, Cell 3 in Fig. 77B presumably represents a Van Gehuchten cell (Fig. 3, cells 17 and 18).*4’ Cells 1, 2, 447 represent superficial short-axon cells (Fig. 3, cells 20-23).*“’ At this point I have to correct a statement made in our previous communication,42 namely that al1 GABAergi@ periglomerular cells are PV-IR. As born out later, I misinterpreted the layering of the olfactory bulb and located PV+ cells in the periglomerular region instead of the external piexiform layer. No~iths~nding, a subpopulation of ~~glomeNJar c& indeed display PV. Hama’s group’45 also detected PV immunoreactivity in the external plexiform layer. In the literature I found no remarks on the electrophysiology or chemistry of intemeurons of the external plexiform layer. Anterior olfactory nucleus (Figs 84 and 85) Ca~bindin D-28k. CaBP+

subdivisions

of the anterior Parvalbumin. Large cells antibodies in the polymorph of the ventral, lateral and

neurons are found in all olfactory nucleus. are stained by the PV layer of the rostra1 part dorsal divisions of the

Fig. 3. Series of eight consecutive coronal sections through the basal ganglia and the amygdala from rostra1 to caudal, alternatively incubated with CaBP (A, C, E, G) and PV antibodies (B, D, F, H). The section plane corresponds to: (A, B) Fig. 86, (C, D) Fig. 88, (E, F) Fig. 89 (G, H) Fig. 90. Notice the striosomes in the striatum (arrows in A) and the continuity between caudatoputamen and olfactory tubezcle (cell bridges, CB) in the CaBP-incubated sections. CaBP+ strands are inserted between unlabelled portions of the olfactory tubercle. The amygdala is parcellated and is poor in CaBP in the basolateral (BL) and intercalated cell mass (I), whereas these zones are rich in PV (E, F). PV-IR axons are prominent in the optic nerve (F, H). Notice drop-like unla~lled regions in the lateral nucleus (La) of the amygdaia (G; island of Calleja?). An, as yet undescribed, PVt neuronal aggregation media1 to the optic tract (opt) and embedded in axons of the median forebrain bundle (ml%) is encircled (PRVI) (H), cf. Fig. 318. x 20.

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Fig. 3.

in the rat brain

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anterior olfactory nucleus, but not in the medial division (Fig. 84). At more caudal levels however, the medial anterior olfactory nucleus contains parvalbuminic cells. Preco~i~~urai hippocampus (tenia recta] indusium griseum (Figs 85 and 86) Calbindin D-28k. A subpopulation of neurons of the indusium griseum and tenia tecta is slightly

stained with calbindin antibodies. The neurons have beaded dendrites, spanning the whole width to the medial surface of the brain (Fig. 29A). Parua[bum~~. Interneurons displaying PV immunoreactivity are numerous in the indusium griseum (both tenia tecta, and tenia tecta, d~visions~~). Their axons impinge upon the soma of pyramidal cells and give an appearance similar to that of the hippocampus proper to this layer (Fig. 29B). Endopirifurm nucleus (Figs 86-90) Calbindin D-28k. Only a few, small neurons with short processes can be visualized. Within the endopiriform nucleus CaBP-~mmunostaining shows fluffy and intensely immunostained terminals (Fig. 3G). P~~a~~urn~~. Only a few immuno~active cell bodies and processes occur in the endopiriform nucleus, particularly at caudal levels. The neurons have a slender tree of dendrites running in all directions, but the axons cannot be discerned. The endopiriform nucleus is delimited from the surrounding by its paleness due to the absence of a terminal field (Fig. 3H).

Primary olfactory cortex (piriform cortex)

(Figs 85-90) Ca~b~~d~~ D-28k. A multitude of scattered CaBPi-

perikarya with slender spineless processes are seen in the depth of the polymo~h layer (Figs 3C-G, 4A, 7A). The plexiform layer lacks CaBP+ cell bodies and most, if not all, faint CaBPi neurons are detectable in the pyramidal ceil layer. The neuropil of the poiymorph and of the pyramidal cell layer is occupied by a large amount of extremely thin terminals, which give an homogeneous appearance to this region. Parvalbumin. Positive neurons are numerous, of varying sizes and have divergent, spineless dendritic processes. They are mostly located in the upper

and lower part of the polymorphic layer or at the boundary between polymorphic- and pyramidal cell layer (Figs 3B,D,F,H, 4B, 7Bf. Few, smaller PV-IR cells occur in the plexiform layer and single neurons in the pyramidal layer. PV+ cell processes (axons?) deriving from these cells engulf the perikarya of pyramidal cells. Extensive dendritic arborizations are detectable in the polymorphic cell layer (Fig. 7B). The richness in forms, sizes and locations of CaBP- and PV-IR cells in the piriform cortex is overwhelming. Comparing my slides with the Golgi drawings,‘0s*‘79it becomes apparent that both, CaBP and PV, occur in representatives of ail subsets of smooth dendritic neurons. No particular difference in the morphology between CaBP+ and PV+ neurons can be detected, although there are si~ifi~ut~~ more CaBPf than PV+ neurons. The majority ofimmunoreactive cells are found in the potymorph cell layer and resemble the neurons depicted in Figs 15 and 16 of Ref. 108. inhibitory interneurons with high discharge frequencies in layer III (polymorph layer) of the rabbit piriform cortex (Table 3) have been described,238 but unfortunately the authors do not provide us with the morphology of the impaled cells. Most of these neurons display glutamate decarboxylase immunoreactivity.‘*” ~~ppo~ampus a& dentate gyros (Figs 89-92) Co~~ind~nD-28k. The CaBP antibodies stain with moderate intensity all portions of the granule cells of the dentate gyrus (soma, dendrites, axon, te~inals). Therefore, overall (Fig. 4A), the dentate gyrus and the mossy fibres converging to the CA3 subfield of the hippocampus are selectively visualized. The granular layer of the dentate gyrus is stained stronger than the molecular layer and the mossy fibres (arrows in Fig. 6A,C,E). The labelling of the molecular layer is homogeneous and trilaminar, with the middle portion being more coloured. This middle band of terminals coalesces with a band of terminals in the lacunosum-molecuIare layer of CA3 (Figs 4A, arrows in 6A,C,E). An unreactive zone is observed just subjacent to the granular cell layer. In Wistar rats we only see a infrapyramidal mossy fibre projection (Figs 4A, 6A,C,E), whereas both infra- and suprapyramidal mossy fibre projections are characteristic for Long-Evans rats. Pyramidal neurons located in the inner half of the CA1 and CA2 pyramidal cell layers have their soma and, less intensely, the stem dendrites moderately stained with the CaBP antibodies (Fig. 8A) but pyramidal neurons in the CA3 and CA4 subfieids remain unreactive (Figs 4A, BA,C,E). A band of

Fig. 4. Horizontal section of the whole rat brain at the level of Figs 58-59 of a rat brain atlas.“% Overview of the staining pattern from the olfactory bulb to the cerebellum. (A) incubated with calbindin antibody; (B) with PV antibody. This figure instructively demonstrates the complementary nature of the staining pattern of CaBP and PV in the basal ganglia (CPU and GP), in the thaiamus, in the medial genie&ate body (MC) and in other brain regions. Other areas are rich in both CaBP and PV, e.g. the cortical mantle, hippocampus, cerebellum (Ce) and vestibular area (Ve). The second point of interest of these two images is that both proteins respect quite accurately cytoarchitectonic boundaries and selectively visualize certain brain portions (not always the case for CaBP). Notice the stratification of labelling in the neocortex and hippocampus. The star in the caudatoputamen of A marks the laterodorsal zone of lower CaBP immunoreactivity. The pial labelhng in both sections is artefactual. x 12.

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Fig. 4.

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Fig. 5. Low-power histography of two consecutive cross-sections at the level of Fig. 27 in Ref. 208 incubated with CaBP (A) and PV antibodies (B). Notice the thalamic and hypothalamic labelling which are virtually complementary in the two sections. The hypothalamic labelling with PV antibodies (B) is probably artefactual. x 12.

Cal~j~-bin~ng

proteins in the rat brain

385

Fig. 6. Sequence of sections through the hippocampus and adjoining cortex at various levels from ventral to dorsal incubated with antibodies against CaBP (A, C, E) and PV (8, D, F). C and D correspond to the level depicted in Fig. 2 of a review article. 263Notice the patchy distribution of labelling in layer 2 of the entorhinal cortex (Ent) with both antisera and the sharp boundaries between CAI, subiculum (S), presubiculum (Prs), entorhinal (Ent) and perirhinal cortex (PRh). In the hippocampus, on the other hand, only CaBP reveals the cytoarchitectonic boundaries, whereas PV fairly similarly stains CAI-CA4. The white arrow in A, C and E marks the mossy fibre projection. The open arrow marks the probable projection from the entorhinal cortex to the molecular layer of the dentate gyrus (L‘perforant path”). x 15.

axons and terminals, lying parallel to the pyramidal cell layer, is seen in the stratum lacunosummoleculare of CAl, CA2 and CA3 (Figs 4A, arrows in 6A-C). It might represent part of the projection to the dentate gyrus from the entorhinal cortex. Bundles of extremely thin CaBP+axons course in the alveus CaBP+

and ventral hippocampal commissure and a few in the fimbria hippocampi. Single, intensely stained cells are regularly dispersed in the various layers of CA1 (Fig. 8A). These “interneurons” cluster at the CA2-CA3 border, (Fig. 8C) whereas they are extremely rare in the dentate gyrus and CA4.

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Fig. 7. (A) Picture of a coronal section of the primary olfactory cortex incubated with CaBP antibodies. Positive multipolar cells are concentrated in the polymorph layer (PO]). Note the homogeneous terminal labelling in all layers with the exception of a juxtapyramidal band in the plexiform layer (Pie). x 120. (B) The PV antisera reveals populations of somewhat larger multi~lar neurons, located more su~~cially in the polymorph (Pol) and in the pyramidal cell (Py) layer. These cells have dendrites ascending in the plexiform layer (Ple). Note the punctate labelling. x 120.

My observations in ammons horn are consistent with over work published the years by some arouns lb.l7.18~19.9O.~3O~22l~222.25~ but not with those of others95 who do not find pyramidal cell staining. In general, I have nothing to add to the descriptions of the aforementioned authors, with the small exception that CaBP-IR interneurons are often observed at the CAlCA3 border and that they form a population distinct from the PV-IR interneurons (not shown). Some CaBP+ neurons in the molecular layer probably correspond to cell b of Fig. 477 in Ref. 37. The thin CaBP- infragranular band corresponds to the adenosine triphosphatase-rich zone of the hippocampus.2R2 The band of terminals in the lower part of the stratum lacunosum-moleculare is in size and location comparable with that seen in acetylcholinesterase27’ and in Timm-stained material (Lipp, persona1 communication). It might derive from neurons of the nucleus reuniens thalami.‘” Most of the terminal field in the molecular layer of the dentate gyrus arises in the entorhinal cortex, containing CaBPf projection neurons in layers II and III. Electronhysiologically, it is well known that pyramidal cells of the -hippocampus display dendritic CaZ+ spikesz4’ a nronertv bv the CaBP+ Purkinje cells . . . * also dismayed and inferior olivary neurons.-I” Like the granule cells of the dentate gyrus, the pyramidal cells show long-term potentiation which may be a Ca?+-dependent phenomenon.*” The seizure threshold of cells in the hippocampus directly correlates with the amount of CaBP? the fascia dentata shows the highest threshold and the highest CaBP content. The CA2CA3 region has the lowest threshold4,*” and no CaBP. Ca2+-conductances are less prominent in the dentate gyrus.“’ Pur~albu~j~. PV+ cell bodies are detected in the stratum oriens, stratum pyramidale, and stratum radiatum of the various hippocampal subfields and in

the stratum granulare, moieculare and hylus (str. polymorphe) of the dentate gyrus (Figs 4B, 6B,D,F, SB). The cell bodies are mostly polygonal and belong to two different size classes (see Table 2), in both the hippocampus and in the fascia dentata (Figs 8B, 78). The processes (axons) emanating from the neurons enmesh the cell bodies of pyramidal and granule cells. The largest PV+ cells of the dentate gyrus have slender, delicate dendrites penetrating the molecular layer. The punctate structures abutting on pyramidal and granule cells are thin and extremely closely packed (Fig. 8D) and give to the lamina pyramidalis and granularis a darker and diffuse look overall (Figs 4B, 6B,D,F, 8B). The lacunosum-moleculare layer of the hippocampus is occupied by a variety of beaded and straight, coarse and thin PV+ cell processes, mainly coursing ~r~ndicularly to the pyramidal cell layer (Fig. 79). The density of PV+ neurons in the hippocampus is differing in the various subfields (e.g. dorsoposterior CA 1 has less than other parts of CA1) but a quanti~cation is not attempted. Most of the PV+ cells in the fascia dentata have their perikarya situated at the boundary between granular layer and hylus; fewer occur embedded in the granular layer, and PV+ cefl bodies in the molecular layer are an exception. A camera iucida drawing of the different cell types in the hilar region of the hippocampus and dentate gyrus is presented in Fig. 78. In the molecular layer of the DG the PV + processes are coarse and smooth, and beaded dendrites are rarely observed. The ratio of PV-IR cells to granule cells


387

Fig. 8. (A) Immunohistochemical demonstration of CaBP in a longitudinal section of the adult rat hippocampus (CAl-field). The soma and dendrites of pyramidal cells (Py) are lightly labelled, whereas single neurons lying in the striatum oriens (Or), radiatum (Rad) and lacunosum-mole~lare (LMol) are intensely labelled in their entirety. Notice that only the innermost row of pyramidal cells displays CaBP, whereas the outer pyramidal cells are unstained. CaBP+ axons run in the alveus (alv) and in the stratum lacunosum-moleculare (black arrow). x 120. (8) lmmunohistochemical demonstration of PV in a coronal section of the adult rat hippocampus. The bipartite dark, fluffy layer, occupying the middle part of the picture correponds to the pyramidal cell layer (Py), which is ensheathed by innumerable fibres and terminals. Perikarya are labelled in the stratum oriens (Or), pyramidale (Py) and radiatum (Rad) and an array of processes perpendicular to the pyramidal cell layer can be seen in the stratum oriens (Or) radiatum (Rad) and lacunosum-moleculare (LMol). Some of the processes are beaded (see Fig. 79). x 190. (C) CAZ-3 border in a Vibratome section incubated with CaBP antisera. The mossy fibre (MF) staining tapers out (arrow). Various interneurons are concentrated at this point in the stratum radiatum (Rad). In the upper part of the figure, the CaBP+ pyramidal cells of the CA2 region are visible (arrowheads). x 120. (D) High magnification of a semithin (1 hrn) cryo-section incubated with PV antibodies. The surface of pyramidal cells (Py) is tapestried with PV+ terminals. A PV-IR interneuron is marked by an arrow. x 750.

within the dentate gyrus is about 1: 200, with regional variations. The absolute number of parvalbuminic cells in the fascia dentata is of approx. 4000 + 500 as determined in series through two rat brains. Single PV+ axons are observed in the fimbria hip~campi and in the dorsal hippocampal commissure but none in the ventral hippocampal commissure. PV+ cell processes and cells are found in the septohippocampal nucleus too (Figs 4B, 29B). The cells staining with the PV antibodies in the rat ammons horn evidently represent interneurons. This statement is based on several lines of evidence; firstly, virtually no axons are seen leaving with the fimbria hippocampi, thus PV-IR cells are intrinsic to the ammons horn. Secondly, only basket cells have their cell bodies in the stratum oriens.

Thirdly, the pyramidal cell perikarya are ensheathed by a plexus of PV-IR terminals, obviously representing the basket-like endings of the axis cylinder collaterals of basket cells. Fourthlv. the number of PV+ cells (at least in the fascia dentat;) is comparable with that of interneurons counted according to pure mo~hologi~l criteria by others.246 The morphology of PV-IR cells in the rat hippocampus corresponds closely to those of the interneurons depicted in a classical paper on the mouse ammons horn’65 (Fig. 6, nos 1. 2 and 3, Fig. 7, no. 4. Fig. 8, no. 2). The PVC neurons in the dentate-gyrus corres&nd to representatives of most cells de&ted in Ficl. 28 of another oaoer’ (cf. Fie. 78 of this paper).‘Therefore, >V is a selective’marke; for iiterneurons in the hippocampus of adult rats. Preliminary evidence suggests, however, that only a minor subpopulation of interneurons is PV + In consecutive sections GABA and PV, as well as GABA and CaBP, co-exist in the same basket cell, but there are more GABA than PV or CaBP neurons.

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Hama’s group has recently published an interesting series of papers ‘38~‘39.‘40.‘41 in which they demonstrate occurrence of PV in 20% of the GABA cells. These authors are also inclined to interpret PV+ neurons as representing basket cells. Additional publicatio&’ confirm and extend these observations. Interneurons are also known to display immunoreactivity towards glutamate decarboxylase’88,“9 and various peptides’84.‘6’.‘75 and to be cytochrome-oxidase-

positive.“’ Table 3 summarizes the scanty data about the known electrophysiology of hippocampal interneurons. They are characterized by a high firing rate similar to many other PV+ cells. Interestingly, their action potential duration is half that of pyramidal cells243and they seem to be a primary target for excitatory (oxytocin + vasopressin)“’ or inhibitory

(opioid

peptides)“’

peptide

effects.

Subiculur comp1e.u (Fig. 92) Both, CaBP and PV, allow a sharp demarcation of the boundaries between the various parts of the subicular complex (Fig. 6). Calbindin D-28k. The immunolabelling varies somewhat between dorsal and ventral (see Fig. 6A,C,E). The pyramidal cell layers and the neuropil in str. lacunosum-moleculare of the prosubiculum are more strongly immunoreactive (Fig. 6A,C,E) than those in CAI and in the subiculum. Between subiculum and presubiculum there is a CaBP-poor band. The presubiculum displays a strong CaBP-labelling of the superficial, densely packed small cells, whereas the parasubiculum contains only scattered CaBP + elements in its whole thickness. Parralbumin. The distribution of PV+ neurons in the prosubiculum does not deviate from the description given above for the CAI region of the hippocampus. In the subiculum. the PV+ cells are loosely packed and form a wider band (Figs 4B, 6B,D,F). PV labels innumerable neurons in the superficial and lower layers of the presubiculum. The presubiculum lower layer is stuffed with PV+ neurons and terminals, whereas the superficial layer is so to a lesser degree. Neocortex (Figs 84-93) A parcellation and stratification according to cortical areas is evident in the distribution of elements containing PV and, less so, in those containing CaBP. A detailed description of the “cytoarchitectonics according to calcium-binding proteins” goes beyond the scope of this paper. Therefore, a general account is given, limited to the parietal cortex and Fig. 9 depicts representative views of some cortical regions. described in the rat brain.‘” Virtually all neurons displaying CaBP and PV immunoreactivity are GABAergic4’ In the somatosensory cortex some rare cases of co-existence of both PV and CaBP with GABA in the same neuron can be determined by studying consecutive cryo-sections. Although the CaBP+ and PV+ neurons constitute the majority of GABAergic neurons, a minor subpopulation of GABA cells remains unreactive towards CaBP and PV antisera.47 It is as yet unknown

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if this subpopulation corresponds to a particular cell type. It is worth noting that GABA+ neurons in layer I never contain calcium-binding proteins. Calbindin D-28k. The immunostaining with the CaBP antibodies in the cerebral cortex shows a fairly homogeneous and strong labelling of the upper three layers (I, II and III) and a moderate homogeneous staining of layer V (Fig. 91,H). In these layers most, but not all pyramidal cells, interneurons and the neuropil, are stained. Interneurons in the upper layers are strongly immunoreactive and are of the bipolar and bitufted type (Fig. 1 IA). Layers V and VI of the cortex display single, scattered, multipolar CaBP-IR interneurons. whereas IV is poor in CaBP+ elements. The interneurons in layer VI are spaced enough to permit identification of their spineless dendritic ramifications. They represent mainly interneurons, probably those having ascending axons (Fig. 73. cells 19, 20, 21 in Ref. 164). All CaBP+ interneurons of the cerebral cortex are GABAergic, but represent a minor subpopulation. Often CaBP-IR dendrites come in close contiguity to each other, or to CaBP+ perikarya. Positive axons are difficult to discern because of their thinness, but they leave the cortical mantle, e.g. through the corpus callosum (Fig. 9A). The total number of CaBP+ neurons in the cerebral cortex is impossible to ascertain because of the diffuse neuropil labelling in the upper three layers. The number of strongly stained. scattered interneurons is less than 5%. The substrate for the homogeneous staining of the upper cortical layers, also seen in monkey?* and cat”“’ visual cortex remains as yet undetermined and only immunoelectron microscopy may shed light on this problem. The homogeneous labelling suggests a diffuse CaBP+ cortical innervation. possibly originating in the intralaminar thalamic nuclei. in the nucleus basalis of Meynert”” and in the dorsal raphe nuclei. region The “barrel-field ” ,zxxa well-defined koniocortical of the rodent cerebral cortex is characterized by “puffs” of intense. homogeneously immunoreactive terminal fields (“barrels”). demarcated by calbindin-poor “septa”.

Purralbumin. Relative number and size of PV-IR cells vary considerably throughout the depth of the cortex and from area to area (Fig. 9). These neurons are mainly located in layers II and IV, but are present in all cortical layers, except for layer I. Layer I appears as a white band in low-power pictures (Fig. 4). At higher magnification only some randomly dispersed immunoreactive cell processes can be seen. PV-reaction products appear within somata, dendrites. axons of neurons and within punctate structures. Neurons of different morphologies and sizes contain PV. The PV+ cell body is mostly multipolar and, less often, bitufted (Fig. I IB). In this last case, the cell axis is perpendicular to the pia mater: a few neurons with horizontal cell axis are observed. Some PV+ perikarya, particularly in the frontal cortex. have the shape of an inverted pyramid. However. dendritic spines are never seen on PV + cortical cells. In most layers the PV+ cells are packed in such a


389

Fig. 10. Co-existence of PV and GABA in some interneurons of the somatosensory cortex. Consecutive, adjacent sections incubated with antibodies against GABA (A} and PV (8). Arrow points out the cell harbouring the two substances. The slight variation in cell morphology is due to differentiai stretching of the frozen sections during the thawing process. Asterisks mark two landmark blood vessels. x 750. Fig. I I. Interneurons of the cerebra1 cortex reveaied with CaBP (A, B) and PV (C, D) antibodies. (A) Notice the gradations in staining intensity between different CaBP+ neurons. The strongly stained represent interneurons, probably of the bipolar (Bip) and bitufted (Bit) sort. The moderately labelled ones are small pyramidal cells. Photograph was taken at the boundary between layers II and III. x 300. (B) Semithin cryo-section (0.5-l pm) of a region similar to A. The pyramidal shape of the moderately CaBP-labelled cells is evident (arrows). Both the cytoplasm and nuclei are immunoreactive. Intemeurons are marked by arrowheads. Dorsal is left. x 300. (C) PV-IR multipolar neurons at the boundary between layers I and II and in layer II. Notice the terminal field in layer II. x 300. (D) PVt multipolar neurons in lower cortical layers. Both cytoplasm and nuclei (without nucleoli) are labelled. Notice the absence of spines from dendrites. x 300. 390


391

Fig. 12. Low-power view of a tangential section through layer IV of the barrel-held of the rat somatosensory cortex incubated with PV antibodies. The basic organization in barrels (B) and septal meanders (S), characteristic of this region, is visualized. The black dots are PV+ interneurons. x 120. Fig. 13. Higher magnification of a field-of-view of the rat basal ganglia showing a large immunoreactive neuron, a delicate meshwork of Iibres and terminals and an immunola~ll~, sohtary axon, coursing in the bundles of the internal capsule (arrow). x 480. (B, C) Multipolar, PV + interneurons are clustered together in the dorsolateral part of the caudatoputamen. Fibres of the internal capsule (arrows). Notice a discrete background labelling of terminals in the neuropil. x 120.

density as to make a ~~onst~ct~on of their dendritic tree impossible. In layers IV and lower V, terminals

impinge upon the surface of almost every perikaryon. They are so numerous, that they appear to form a continuous sheet around the soma.” PV+ terminals can also be found abutting on PV+ cell bodies and proximal dendrites. Roughly estimated, PV+ neurons in the parietal cortex represent approximately 10% of the total neuronal number (see, however, Ref. 29). The size, shape and morphology of PV+ neurons are similar to those of interneurons as described in Golai36~37~‘~~i66~Z~ and electron microsconic studiesztO The absence of PV+ fibres in the corpus callosum, anterior commissure and crura cerebri suggests that PV occurs mainly in neurons intrinsic to the rat neocortex.” However, PV + fibres course in the bundles of the internal capsule through the caudatoputamen, while others perforate the corpus callosum. A precise association of PV + neurons to Go&i-classified intemeuronal types cannot be readily achieved because PV-immunost~n~ cells are not spatially isolated, and therefore interfere with the reconstruction of PV-IR cell

processes. However, with the exception of short-axon interneurons located in layer I (Figs 71, IOF, IlA, UK),‘@ representatives of all other types of intemeurons,‘~,~~ with the possible exception of bipolar cells, seem to display PV immunoreactivity. In the “barrel-field” the neuropil is subdivided in nearly circular PV-rich zones of approximately lOO-pm diameter (“barrels”) and in PV-poor “septal”-meanders, Small, multipolar, spineless PV neurons are scattered at the barrel side or in the septa, and their cell processes are polarized towards the centre of the barrel (“hollow”) (Fig. 12). The PVC neurons in the SMl barrel-field are spine-free and may correspond to those described as type II non-pyramidal cell~.*~~~al~oco~i~~ tibres extensively terminate on the soma and proximal dendrites of these smooth stellate cells in the barrels.283J*4These neurons are thought to be the “substrate for the fast-spike units, characterized by the rapid course of their bioelectric waveforms, their high rates of spontaneous activity and their ability to respond to higher freouencv stimulation of the vibrissae”249(see also Table 3). The d&ibution of PV-IR neurons has been compar& with the published localizations of neuropeptides’~s~28152~‘8s~2’3 or neurotmnsmitter synthesizing enzymes (glutamate decarboxylase). The best match in the distribution of PV is found with the inhibitor neurotransmitter GABA as revealed by GAD-immunohistochemistry. ‘*8~22’~228~229 In consecutive sec-

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tions we have shown that both substances co-exist in a subpopulation ofintemeurons.47 However, there are cortical GABA cells, which lack PV. It is not astonishing that a calcium-binding protein, which has the property of ~ntroll~g the ex~i~biiity of a neuron, is particularly accumulated in inhibitory neurons, which are in charge of controlling the excitability of other nerve cells. Basal gangiia Clauslrum (Figs 86-89) Cafbindin D-28k. The claustrum itself is almost devoid of CaBP (Fig. 3C, 14A). On the other hand, the claustrocortex is extremely rich in positive tern+ nals which occupy the upper layers (Fig. 3C). This zone is easily seen in coronal sections and delimits the piriform cortex from the parietal cortex. Pa~albumi~. Immunoreactive eel1 bodies and processes occur in the whole extent of the claustrum (Figs 3B,D, 14B). The cells have a slender tree of dendrites running in all directions, but the axon cannot be discerned. PV+ neurons in the most rostra1 regions of the claustrum have highly beaded cell processes. The claustrum is delimited from the surroundings by its fluffy, diffuse immunostaining, deriving from terminal fields (Fig. 14B). The claustrocortex is defined by a sharp band of immunoreactive neurons and terminals, probably located in layer V (Figs 3B,D, 14B). Caudatoputame~ (Figs 86-90) Calbindin D-28k. The immunostaining in the caudatoputamen and nucleus accumbens is one of the most impressive observations made wrth these antibodies. The CaBP-labelhng is homogeneously distributed in the innermost core of the neostriatum, but leaves a dorsolaterally crescent-shaped shell, virtually unstained {Figs 3C, stars in 4A, 29A). This portion contains the highest concentration of PV + terminals (see later). Particularly strong is the staining in the fundus striati (Fig. 3E,G). In the medial caudatoputamen only the fan-shaped fibre bundles of the internal capsule and the anterior commissure remain untagged by the CaBP antibodies. It is worth noting that small oval islands of tissue (striosomes) oriented parallel or perpendicularly to the course of the internai capsule, lack CaBP immunoreactivity (arrows in Fig. 3A). At higher magnification, the immunostain-

ing is observed in great numbers of densely packed, medium-sized, spiny neurons and in a fine texture of cell processes, terminals and fibres. The CaBPimmunostained region gradually merges with the relatively unstained dorsolateral portion of the caudatoputamen, but ends sharply at the boundary with the globus pallidus (Figs 3E,G, 4A, 14A, 29A). The dorsolateral portion of the striatum has no CaBP+ cell bodies, but harbours a moderate density of terminals. In parasagittal sections, a tongue-like protrusion, directly behind the external capsule, displays higher CaBP immunoreactivity than the surroundings. From the neostriatum and accumbens

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arises a very consistent CaBP+ projection to the mediodorsal substantia nigra (pars reticulata) (Fig. 14A). Although the size of CaBP+ cells in the caudatoputamen is very homogeneous (15 x IS pm), some larger neurons (20 x 20 pm) can be seen, The striking immunostaining in the caudatoputamen has been depicted schematically,gO and exactly described by Gerfen et al.” CaBP antibodies visualize a very prominent strionigral pathway, and in this respect they resemble antibodies against calcineurinto2 and DARRP-32, a neuronal phosphoprotein, occurring in the whole caudatoputamenu4 Yet the caudatoputamen staining with CaBP antisera is of further interest because of the visuali~tion of “striosome”like patches,97J05~‘fo which may correspond to the zones of low acetylchohnesterase activity, being avoided by the afferent terminals from the intralaminar nuclei.10s Although drawing of the CaBP-immunostained cells in the striatum cannot be performed because of the interlacing of ceil processes, it is assumed that they are medium spiny neurons, which represent about 95% of striatal neurons.r4r Parvalbumin. Spatially separated, strongly PV-IR neurons of various sizes are observed in the caudatoputamen, mainly in its dorsolateral, CaBP-poor part (Figs 3B,D, 4B, 14B). The cell bodies of the larger neurons have a smooth surface and polygonal or fusiform shapes (Figs 13A,B,C, 80,81). From here a tree of spine-free dendrites radiates over long distances (200pm) and intermingles with the processes, belonging to adjacent PV + cells, The axons of these cells are difficult to visualize, but they appear to ramify locally. In a few cases, axons entering the bundles of the internal capsule heading toward the globus pallidus can be seen. A terminal field, which can be best traced at low magnification (Figs 4B, 5B), lies in the same area where PV + neurons are located. The morphology of the largest cells at best resemble that of non-spiny type I large neurons$) and the “spine lose makroneurone”.67.**’ Their number and location do not match precisely that described by the above and other authors with the Golgi-method. but we have to keep in mind that PV antibodies may only stain a subpopulation of these neurons and that the capricious Golgi-method may deliver erroneous distributions. Large cells in the striatum have been shown by others to be acetylchotinesterase- and cholineacetyItransferase-1R.‘5 The smaller PV + neurons of the neostriatum appear cytologically identical to the larger cells in not having somatic or dendritic spines and only a few beaded dendrites; the axon has never been traced. The smaller PV+ neurons in the caudatoputamen of the rat at best correspond to type III medium neurons of Chang et al.” and may represent projection neurons. In the gray matter of the neostriatal dorsocaudal region, a higher density of fibres and terminals is seen. Single PV+ axons are detected in the fibre bundles

of the internal capsule, running through the caudatoput~en; their number increases laterally and in rising proximity to the globus pallidus. PV-IR cells account for less than 2% of caudatoputamen neurons as inferred from counting the total number of neurons in adjacent Cresyl Violetstained sections. Their number in a 40-urn-thick sagittal section is approx. 480 k 30 (n = 12).


393

Pig. 14. Horizontal section through the substantia nigra, visualizing the whole extent of the CaBP-IR striatonigral pathway arising in the “matrix” compartment of the caudatoputamen (CPU) (A). Notice the clustering of CaBP striatal terminals in the medial SNR and of PV+ neurons (B) in the lateral SNR. At this level the SNC has no CaBP+ neurons (cf. Fig. 32A). Striosomes are evident in the caudatoputamen of A (arrows). Another complementary feature of labelling is found in the subthalamic nucleus (S’lh), which has many PV+ terminals (B), probably deriving from the globus pallidus, but no CaBP-IR structures (A). x 10.

Accumbens (Figs 86 and 87) Calbindin D-28k. The general morphology is similar to that described for the caudatoput~en. Again there are zones of decreased immunoreactivity, particularly mediodorsally (shell of the aceumbens; arrows in Fig. 3A). Rostra1 to the anterior commissure, the CaBP+ accumbens is sharply demarcated basally but displays “finger-like” protrusions of CaBP+ neurons (cell bridges) penetrating the olfactory tubercle and reaching the inferior pial surface (Fig.

3A). The morphology of the CaBP+ neurons of the accumbens and of the caudatoputamen is identical. Par~alb~min. The dist~bution and ~oncent~tion of PV+ neurons in the accumbens is similar to that seen in the medial caudatoputamen. However, only the small cell type occurs. @Ifactory tubercle (Figs 86-88) Calbindin D-28k. “Bridges” of CaBP+ neurons descend perpendicularly from the accumbens and

394

M. R.

span the whole width of the olfactory tubercle (cell bridges in Fig. 3A). These protrusions of labelled neurons alternate regularly with CaBP-unstained portions of the olfactory tubercle. The CaBP + neurons in the olfactory tube&e cannot be differentiated morphologically from those in the caudatoputamen or accumbens (Fig. 15A). P~~aIb~~. The large, multipolar PV+ neurons are mostly found in the polymorph layer and resemble pallidal neurons (Fig. 15B). Smaller PV+, multipolar cells occur in the pyramidal cell layer of the olfactory tubercle, and only a few in the plexiform layer. Islands of Calleja Parvalbumin. Large PV+

cells, in their morphology resembling pallidal neurons, are dispersed between the Islands of Calleja. Their shape is somewhat obscured by the large amount of thick PV-t axons, coursing in the medial forebrain bundle. Globus pallidus (Figs 88 and 89) and entopeduncular nucleus (Fig. 90) Calbindin D-28k. Only a sheet of the outermost and a funnel-shah (in parasagittal sections) portion of the innermost part of the lentiform nucleus are slightly CaBP+ (Figs 4A, 14A). These terminal fields probably arise from collaterals of CaBP+ striatonigral fibres. The large central portion of the globus palhdus is poor in such terminals and may receive a separate input from the dorsolaterally located, CaBPstriatal region. This further chemical subdivision of the globus pallidus has not been seen either with substance P,“’ or d~orphin. ” In contrast to the ~udatoputamen, perikarya of the globus pallidus and entopeduncular nucleus never exhibit CaBP immunoreactivity. Pawalbumin. A great number of neurons of the globus pallidus (Figs 4B, 14B) and ento~un~uiar

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nucleus (Fig. 14B) display PV-IR, associated with their soma and processes. The cell bodies are polymorph, but mostly quadrangular (Fig. 18) and belong to two different size classes (see Table 1). The PV+ dendrites form a coarse network traversed by the unstained fibres of the internal capsule. PV+ axons leave the GP with the ansa lenticularis, lenticular fasciculus and stria medullars thalami. Pallidal neurons exhibit phasic, high frequency, firingT2 (see also Table 3). Ventral pallidus (Fig. 88) ~albindin D-28k. Against the background

of the caudatoputamen, the ventral pallidus looks rather pale with only a few terminals. In coronal sections these terminals are clustered in a “coma-shaped” lateral region. Parvalbumin. PV+ neurons are similar to those described in the globus pallidus, but occur in lower numbers. Most of the pallidal, entopeduncular and ventral pallidus neurons are GABAergictBS and some probably contain substance P.‘u Basal nucleus of Meynert (Fig. 89) Calbindin D-28k. The rat nucleus basalis is populated by large CaBP+ multipolar neurons with elongated soma (20-30 x 10pm) and thick and widely branching dendrites (Fig. 16). Earlier we described the presence of CaBP in the monkey basal nucleus.46 Subthalamic nucleus C~lb~nd~~ D-28k.

CaBP+ fibres and terminals avoid the subthalamic nucleus (Fig. 14A). Parvalbumin. The nucleus is crowded with a high number of thin PV+ axons and terminals, particularly in the lateral part (Fig. 14B). In adequate

Fig. 15. Horizontal section through the olfactory tubercle. The distribution of CaBP+ (A) and PV+ (B) elements is simiIar; both antigens mark neurons in the polymorph layer (Pol). The PV-staining (B) is analogous to the one observed in the globus pallidus. Notice in both cases the homogeneous staining of the pyramidal cell layer (Py) (stronger in B) and the absence of staining in the plexiform layer (Pie). Nomarsky optics. x 200. Fig. 16. Horizontal section of the nucleus basalis of Meynert (B) at the level of Fig. 57 in a brain atlasZoH CaBP+ neurons (arrows) have their perikarya and their coarse dendrites ordered parallel to the posterior border of the globus pallidus (GP). x 120. Fig. 17. Horizontal section of the olfactory tubercle at the level of Fig. 54 in Ref. 208. PV-immunoIabelling. Notice the positive fibres of the medial forebrain bundle and the unstained islands of Calleja (arrows). x 120. Fig. 18. Horizontal section of the globus pallidus (GP), incubated with PV antibodies. Large, triangular perikarya with coarse, interlacing cell processes are visualized. Nomarsky optics. x 350. Fig. 19. Horizontal section of the reticular nucleus of the thaiamus (Rt), incubated with PV antibodies. The intensity of staining is higher than in the GP (Fig. 18). Axons (arrows) leave the reticular nucleus of the thalamus in the direction of ventroposterolateral thalamic nucleus (VPL). Nomarsky optics. x 350. Fig. 20. Horizontal section of the amygdala incubated with CaBP antibodies. Large, multipolar neurons are visualized in the basolateral amygdaloid group (BL). x 200. Fig. 2 1.Horizontal section of the amygdala incubated with PV antibodies. The morphology of the positive neurons in the basolateral amygdaloid group (BL) is very similar to that of Fig. 20. x 200.


Figs 15-21.

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Table I. Relative staining intensities of some selected neurons towards calbindin D-28k and parvalbumin antibodies CaBP -_._ ~__._._..... . -.____ . ...________ Purkinje cells +++-I_ Basket- and stellate cells of the cerebellum Interneurons of the cerebral cortex +++ Periglomerular cells +++ _ Cells in the lamina plex. ext. Cells around the olfactory ventricle ++ Fascia dentata granule cells ++* CA1 pyramidal cells +* Hippocampal intemeurons ++e Medial habenular nucleus ++++ ~audatoputamen +++/++* _ Giobus palhdus Substantia nigra (pars reticulata) (pars compacta) +++ Thalamic nuclei ++* Retina +++ Inner hair cells (Corti’s organ) t++ Spinal ganglion ceils +++;++ Plexus myentericus +++ Plexus submucosus +++

PV __~_._ ++++ +++ +++i++ - (+) fff _ fff +++ -I+,/+ fff +++ +++ -I-i--t-i++ _ _

*Diffuse staining. + t + + ~very high; + + + , high; + + , moderate: +, low; -, absent.

Table 2. Size (minor and major diameter in microns) of pa~albuminand calbindin D-28k-immunoreactive cells in selected brain regions

Olfactory bulb Cortex cerebri Cortex cerebelli (Purkinje cells) Hippocampus (interneurons) Fascia dentata (interneurons) Nucleus reticularis thalami Caudatoputamen Globus pallidus Colliculus inferior Spinal ganglia (Ll) Ggl. spirale cochleae Nucleus basalis Meynert

CaBP

PV

10 x 10 15x24 35 x 35

12.5 x 10 12 x 30118 x 35 35 x 35

15x25

17 x 23 17 x 2312.3x 35 20x30/15x 15 17x30/20x20

15x15/20x20

30 x 50

35 x 47* 30 X 55120 X 20 15 x I5

20(30Q x 10

*Other different classes. tThe coarse primary dendrites make a delimitation of the somatic boundary dihicult. The tissue has been treated with (CaBP) or without (PV) albumin embedding and coverslipped in glycerine gelatin (PV) or Eukitt after dehydration (CaBP). The only exceptions are the spinal ganglia, which have been treated with Bouin + paraffin.

Table 3. El~tropbysiological

characteristics of some pa~~bum~n-positive

Effect _-_______--._.__I_ ______ .-...-. __ ___._,__” ____I__ inhibitory Layer III lnterneurons in pyriform cortex Barrels (IV layer) Inhibitory Neurons in SMI cortex inhibitory Nucleus reticularis thalami Inhibitory CA1 pyr. layer Hippocampus (basket-cells) Inhibitory Purkinje cells cerebellum Localization

Renshaw ceils Pallidal neurons and neurons in the SNR

Spinal cord

Inhibitory Inhibitory

cells in the brain Discharge rate and type -l_l___l_-l__ --.. Repetitivez3* Repetitive”’ S~ntaneous: 120/s (bursts of 3 s?” Spontaneous: .50/s”” 70/s quietly sitting monkey’“‘-‘*’ 400/s during movements (3060/s spontaneous)“2 Gery high-frequency~3’ Phasic, high frequency firing”


397

sections, the projection is seen to originate from the globus pallidus. Many moderately ~mmunoreactive neurons populate the subthalamic nucleus as well.

in subregions. In general the content of CaBP is very high, whereas PV is moderately rich in the amygdala.

Substantiu nigra (Figs 91 and 92)

Calbindin D-28k. The medial amygdaloid group is occupied by an extremely rich complement of CaBP+ neurons, embedded in a rich network of CaBP+ fibres and terminals (Fig. 3E,G). The posteromedial cortical amygdaloid nucleus is rich in neurons and endings which merge dorsally with the amygdalohippocampal transition area. Parvalhumin. The amygdalohippocampal transition area has neurons and terminals which are ordered similarly as in the hippocampus.

Calbind~ D-28k. A massive terminal field contain-

ing CaBP, is found in the mediodorsal portion of the substantia nigra, pars reticulata (Figs 14A, 22A). The staining is less pronounced or even absent in the laterodorsal aspect of the substantia nigra, pars reticulata (Figs 14A, 22A). The dorsal sheet of the substantia nigra, pars compacta, the substantia nigra, pars lateralis, the retrorubral area and the peripeduncular nucleus are populated by a moderate number of CaBP-IR neurons. In the substantia nigra, pars compacta, those located rostroventrally (Fig. 32A) are smaller, clustered and more numerous than those located distally. In the peripeduncular nucleus, CaBP+ neurons are embedded in a rich background of thin CaBP+ terminals and fibres. Parvalbumin. PV antibodies label many neurons in the substantia nigra, pars reticulata, in the substantia nigra, pars lateralis and in the ~~~duncular nucleus and in the pars compacta (Figs 14B, 22B). In the substantia nigra, pars reticulata, parvalbuminic neurons are of various sizes, but mainly large (20 x 25 pm) and unevenly distributed between the various quadrants. Coarse PV+ processes are intermingled with PV+ neurons in the more lateral portions, whereas the medial part of the SNR contains fewer PV+ neurons and thinner fibres. The immunostained cells have slender somata and coarse dendrites and resemble in shape those of the globus pallidus. The nigral cells merge with other PV+ neurons, belonging to the peri~duncular nucleus. PV + axons leave the substantia nigra in mediodorsal and rostrolateral direction. Ventral tegmental area (Figs 91 and 92) Caibind~n D-28k.

Widely spaced, small CaBP+ neurons are typical for the ventral tegmental area and adjacent portions of the ventral tegmentum (Fig. 22A). Others have first described these CaBP+ neurons and proved them to be dopaminergic.97 The linear raphe nucleus contains nothing but fibres and terminals. In addition to having a complement of small neurons, the interfascicular nucleus displays a dense, homogeneous terminal field as well. Parvalbumin. The ventral tegmental area is pierced by an innumerable quantity of PV + , coarse axons, but virtually does not display PV+ neurons (Fig. 22B). Robust parvalb~ini~ fibre tracts, surrounding the ventral tegmental area are the medial lemniscus, the medial longitudinal fascicle and the mammillary peduncle. Amygdala (Figs 89 and 90)

The use of CaBP- and PV-immunohistochemistry results in a sharp discrimination of the topography of the amygdala. Subnuclei are being further subdivided

Medial amygdaloid group

Basolateral amygdaloid group Calbindin D-28k. The lateral nucleus possesses a fair amount of terminals and displays CaBP+ neurons as well (Fig. 3E). On the other hand the rostra1 portion of the basolateral and basomedial show up as rather pale nuclei, surrounded by a dense terminal field region (Fig. 3E). They harbour a few, spatially separated, multipolar neurons with slender, nonspiny, cell processes. At more caudal levels (Figs 3G, 20, 30A) the basolateral amygdaloid group, particularly its lateral half, the basomedial and the ventral basolateral amyg~loid group are intensely tagged by CaBP antisera. The intercalated nuclei (I) have neither cells nor terminals inside their boundaries (Fig. 3E). Parvalbum~n. The drop-shaped basolateral and lateral nuclei are selectively visualized by the presence of a fine meshwork of PV+ fibres (Fig. 3F). Large neurons with slender, spineless cell processes occupy the basolateral (Figs 21, 30B) (ventral and dorsal) and the lateral amygdaloid nuclei. The PV+ neurons in the ventral basolateral ventral nucleus intermingle with those of the polymorph layer of the primary olfactory cortex (Fig. 3F,H). PV+ fibres arising in the globus pallidus enter the mediocranial aspect of the basolateral nucleus. The distribution of PV+ neurons and terminals is uneven as if a parceilation existed. The intercalated amygdaloid nuclei are devoid of PV immunoreactivity. Central amygdaloid group Caibindin D-28k.

The central nucleus shows a parcellation in a mediocranial region, very rich in calbindin-positive neurons and terminals and a lateral region with only a few cells and terminals (Fig. 3). Olfactory amygdala Calbindin D-28k, The anterior cortical amygdaloid

nucleus has only a few CaBP+ neurons and moderate terminal labelling. The posterolateral cortical amygdaloid nucleus is occupied by innumerable small, weakly labelled neurons embedded in a strong terminal field. The amygdalopi~fo~ transition area

398

M. R. CELIA

is characterized by a huge terminat field but relatively few strongly labelled neurons. Septal compiex (Figs 87 and 88) Calbindin D-28k. Probably all ceils of the triangu-

lar septal, (Fig. 28A) of the septohippocampal (Fig, 29A) and most of those in the lateral septal (with exception of the dorsal portion} nuclei, (Figs 28A, 29A) are CaBP+. The ventral and intermediate portion of the lateral septum are further parcellated into subzones by the presence or absence of CaBP+ multipolar neurons and terminals (Figs 28A, 29A). CaBP + terminals form an oblique band from dorsomedial to ventrolateral. The CaBP+ triangular septal cells infiltrate the ventral hippocampal commissure, the fornix and the stria medullaris thalami and reach as far as the fimbria hippocampi (Fig. 28A). The vertical limb of the diagonal band contains a few CaBP+ neurons. The septohippocampal nucleus shows a high density of CaBP f terminals and many CaBP+ axons (Fig. 29A). CaBP+ axons in the fornix are sharply segregated to the posterior third. Thin CaBPf axons course in the anteromedial aspect of the stria medullaris thalami. The neurons of the septofimbrial and medial septal nuclei do not express CaBP (Fig. 4A). Parvalbumin. In general the labelling is very discrete (Fig. 4A), or even absent (Fig. 28B). The occurrence of a few multipolar PV + neurons in the medial septal nucleus and subpially in clusters of two

to three in the lateral septal nuclei is regular (Fig. 4A). PV -I- cells in the triangular and in the septofimbrial nucleus can rarely be seen. Some PV + axons leave the septum in a dorsomedial direction and proceed to the stria medullaris thalami. Some large neurons, oriented sagittally, occupy the most medial portion of the nucleus of the diagonal band. Bed nucleus of the anterior commissure Parvalbumin. A few. medium-sized PV + cells with their cell processes inte~ingl~ are invariably found in the bed nucleus of the anterior commissure (Fig. am/ L3).

Bed nucleus of the stria terminalis (Fig. 88) Calbindin D-28k. The immunolabelling is very similar to that observed in the lateral septum: a myriad of small neurons embedded in a rich fibre and terminal matrix (Fig. 3C). The lateral and ventral nuclei have less positive sites than the medial nuclei.

Diencephalon Epithalamus Habenular nucIei (Fig. 90) Calbindin D-28k. Cell bodies in the lateral division of the medial (and a few in the ventral medial) habenular nucleus display a strong immunoreactivity towards CaBP antibodies. In a coronal section of the diencephalon this is the most striking staining (Fig. 27A). The nucleus is also more or less homogeneously and diffusely stained by the antibody, but the terminals and cell processes never attain the staining intensity reached by the soma. CaBP+ axons course in the habenular commissure. The habenulointerpeduncular tract’*’ is known to consist of substance P and acetylcholinestera~-containing fibres’j’ and is possible to represent the only efferent connection of the medial habenula. The medial habenula is another location, beyond the nucleus basalis of Meynert,46 where CaBP occurs in a cholinergic cell group, although cholinergic neurons are more common in the ventral medial habenular nucleus (see Fig. 4 in Ref. 253). Parvalbumin. PV+ fibres in the stria medullaris thalami form a rim in its lateral portion, enter the lateral habenular nucleus and impinge upon its neurons. PV + terminals are detectable only in the lateral habenular nucleus, also displaying some immunoreactive neurons (Fig. 27B). ~ha~omu~*6*1~~,‘33 (Figs 89-91)

The complementary nature of staining between PV and CaBP antibodies can be best appreciated in the thalamus. PV primarily occurs in “specific” nuclei, whereas CaBP is rich in “unspecific” thalamic nuclei. The rich, diffuse and homogeneous CaBPimmunostaining creates a peculiar chemical parcellation of the rostra1 thalamus. As already pointed out by others,” the distribution of CaBP “does not strictly correspond to defined nuclear groups”. Interestingly, neurons, located at the boundaries between classical thalamic nuclei, express calbindin immunoreactivity (e.g. Figs 5A, 22A). On the other hand, the PV immunoreactivity is dominated by axons and terminals and respects nuclear boundaries (e.g. Figs 5B, 22B). Perikaryal iabelling is a rare exception. Notwithstan~ng the lack of PV in the cell body, some axons deriving from thalamic cells and projecting to the cortex through the capsula interna, show PV immunoreactivity. Thus, in the rat, some thaiamic neurons behave like other long projecting neurons by segregating this protein to axon and terminals.

Nuclei of the diagonal band (Fig. 87) ~a~bindin D-2&k. In the horizontal

limb of the nuclei of the diagonal band CaBP is poorly represented. Parvulbumin. In the horizontal and vertical limb of the diagonal band the antibodies against PV stain some large neurons with few thick dendrites. These neurons are often clustered together.

Anterior nuclear group Calbindin D-28k. The anteroventrai shows terminals and scattered positive neurons. The labelling fades gradually from lateral to medial (Figs 4A, 29A). Parv~~~~~n. The anterodorsal, anteroventral and anteromedial nuclear groups dispiay a rich complement of terminals and axons (Figs 4B, 29B) which are

399

Calcium-binding proteins in the rat brain sharply demarcated from the ventrolateral nucleus by a thin, unlabelled band (arrow in Fig. 29B). Of the three nuclei the anterodorsal nuclear group is the most strongly labelled.

and corresponds to the nucleus of the optic tract, and in a triangular medioventral extension of the posterior nucleus which points in the direction of the retroflex fascicle (arrow in Fig. 22A).

Medial nuclei

rntralami~ar nuclei

Calbindin D-28k. The mediodorsal nucleus has many CaBP+ polymorph neurons at its circumference, clustered as a shell around an unstained central core (Figs 5A, 29A). The CaBP+ neurons merge with similar positive neurons in the paracentral and in the intermediodorsal nuclei (Fig. 29A).

Calbindin D-28k. The central medial, paracentral and central lateral nuclei are consistently stained by antisera against calbindin (Figs 4A, SA, 29A). The small CaBP neurons (15 x 15 pm) merge caudally with the periventricular gray. Within the intralaminar nuclei, the neurons are immunostained all over including the afferent terminals. Future immunoelectron microscopic investigation will shed light on this phenomenon.

Ventral nuclei Calbindin D-28k. The ventromedial nucleus is rich in positive neurons and endings, whereas the other ventral nuclei display clusters of stained neurons, fibres and terminals (arrows in Fig. 29A). They are sharply demarcated medially, but not laterally (Fig. 29A). Particularly striking are bands of CaBP-reactive neurons located in the ventromedial and at the boundaries between ventrolateral and ventroposterolateral (Fig. 29A, left), ventrolateral and the posterior thalamic nuclei, as well as between the ventroposterolateral and posterior thalamic nuclei. Calbindin-IR fibres enter the thalamus from the tegmental tracts. Pa~u~bumin. A rich plexus of PV+ axons and terminals of various calibers is typical for the ventromedial, ventrolateral, ventroposterolateral and ventroposteromedial complex (Figs 4B, 5B, 29B). The highest density of PV+ fibres and terminals is found in the ventroposteromedial nucleus (Fig. 29B). The axon staining can be traced back to the medial lemniscus. The dist~bution of PV+ terminals in the ventroposteromedial nucleus is patchy (Fig. 29B), reflecting perhaps the terminal fields occupied by single whisker afferences. The major iibre tracts ending in the ventral thalamus are rich in strongly PV-IR axons: medial lemniscus, trigeminal tract, superior cerebellar peduncle, ansa lenticularis (see also under fibre tracts). Lateral nuclear group Caibindin D-28k. The lateral nuclear group has a

large number of moderately stained CaBP+ neurons, where only the perikaryon is fabelled. Pa~albumin. The lateral nuclear group possesses the highest density of PV+ terminals following the ventral group. No perikarya are labelled (Fig. SB). Posterior nucleus Calbindin D-28k. A unique band with patches of

calbindin-IR neurons occupies the boundaries of the posterior nucteus (Figs 22A, 29A). At more caudal levels (Fig. 22A), this antiserum reacts diffusely with neurons and processes of the posterior, lateroposterior and suprageniculate thalamic nuclei. The staining in the dorsomedial lip, is particularly intense

The intralaminar nuclei receive afferences from various brain regions and from the inner segment of the pallidus”’ and project to layers I and VI of the cortex and to the striatum. In the cortex, we actually observe terminal fields in layers I -III and V, although the intralaminar nuclei may take part in the projection to layer I, other projection systems (i.e. from the nucleus basalis of Meynert) converge to the same site. The thalamic intralaminar nuclei project to the caudatoputamen4i and this correlates with the homogeneously CaBP-labelfed central core of the caudatoputamen. Parvalbumin. Light terminal fields occur in central medial, paracentral and parafascicular cleus. PV-t neurons are not detected except in parafascicular nucleus. These terminal fields do respect nuclear boundaries.

the nuthe not

Midline nuclei Calbindin D-28k. The rhomboid nucleus is calbindin-negative throughout and its boundaries are clearly delineated. The reuniens, interanteromedial, intermediodorsal and paratenial nuclei contain a large amount of CaBP+ neurons embedded in strongly labelled terminal fields. The paraventricular nucleus is very rich in calbindin-positive terminals (Fig. 29A). Parvalbumin. PV+ fibres cross the midline in the region of the rhomboid nucleus. Terminal fields, axons and single PV+ neurons are found in the paratenial nucleus (Fig. 29B). Ventroposterior nuclei Medial geni~late

complex

Calbindin D-28k. The boundaries of the medial geniculate complex ,are sharply demarcated because of the strong iabelling in the posterior thalamic group (Figs 22A, 29A) and due to an island of neurons in the ventral geniculate nucleus. The ventral medial geniculate complex has many perikarya, being moderately stained. The medial portion has lightly stained, and the dorsal portion stronger, but diffusely stained neurons (Fig. 29A) with enmeshed axons and terminals. The lamella of white matter, covering the medial geniculate complex has many CaBP+ thin axons, running in the frontal plane,

400

M. R. CELIO

Par~o~b~rni~. Ail three subdivisions of the medial geniculate complex display a rich plexus of thin terminals and axons (Figs 22B, 29B). The meshwork is more dense in the medial and ventral portion, as well as in the intergeniculate nucleus. No celI bodies are PV+. Lateral geniculate nucleus Calbindin D-28k. The dorsal lateral geniculate nucleus and the magnocellular ventral lateral geniculate nucleus show similar, moderate amounts of positive neurons and terminals, which are markedly concentrated in the outer rim of these two regions (Fig. 22A). They are continuous with the strong labelling of the intergeniculate leaflet nucleus, located at their boundaries (Pig. 22A). The terminals in the dorsal lateral geniculate nucleus stem from a subgroup of CaRP+ axons in the optic tract. They are filiform, beaded or punctated. Terminal fields form “puffs” of more intense immunoreactivity, separated by areas with fewer immunoreactive processes. Pawalbumin. Positive axons deriving from the optic tract, as well as terminals, are more numerous in the dorsal than in the ventral lateral genicuiate body (Fig. 223), and the two regions are sharply demarcated by the unstained intergeniculate leaflet. Moderately labelled neurons are observed in the most lateral part of the magnocellular ventral dorsal geniculate body (Fig. 4A).

The terminals found in the ventral lateral geniculate nucleus may represent collaterals of small retinal axons (W cells) destined to reach the ventral lateral geniculate nucleus.i’0 The identity of the small PV i- cells in the ventral lateral geniculate nucleus is unknown but their size is compatible with that of interneurons (see also Ref. 258).

mingled PV+ fibres and punctuate structures are found (Fig. 19). The PV+ processes are mostly ordered perpendicular to the course of the thalamocortical and ~orticothalamic radiations traversing the reticular thalamic nucleus. The axons leaving the reticufar thalamic nucleus can be followed to the dorsal and other nuclei of the thalamus, where they end in a profuse way. The endings in the thalamic nuclei do not delineate the shape of thalamic neurons; therefore, they are probably not preferentially located on the soma. Rarely have I seen PV-IR processes piercing the external capsule and projecting rostrally towards the striatum. PV-IR axons deriving from the globus pallidus and coursing in the ansa lenticularis pierce the ventromedial reticular thalamic nucleus. Our observations on the reticular thalamic nucleus and the course of its axons completefy support the careful analysis of others.“‘*239All reticular thalamic nucleus nenr_-. ons stain with PV antisera, within which two different size classes can be distinguished (see Table 2). Some authorszss are even able to classify reticular thalamic nucleus neurons in three different classes by means of Golgi jmpregnation. From a physiological point of view reticular thalamic nucleus neurons belong to two distinct categories of cells’” and they have been found to have a very high firing rate and (see also Table 3). The reticubursting activity rhythmus far thalamic nucleus is immunoreactive towards antisera against the GABA cells marker enzyme glutamate decarboXy~ase’2s.‘88.2” and, in the cat, the “inhibitory” neuropeptide somatostatin. ‘&The targets of the reticular thalamic ._._ nucleus in the thalamus disnlav very high densities of GARA receptors2” The reticular nucleus ii poci. 4, 6588664. 206. Patten B. M. and Philpott R. (1921) The shrinkage of embryos in the process preparatory to sectioning. Anar. Rec. 20. 393413.


473

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Expression and colocalization patterns of calbindin-D28k, calretinin and parvalbumin in the rat hypothalamic arcuate nucleus.

Subpopulations of GABAergic neurons containing parvalbumin, calbindin D28k, and cholecystokinin in the rat hippocampus.

Cerebral cortical calbindin D28K and parvalbumin neurones in Down's syndrome.

Distribution of the calcium-binding proteins parvalbumin and calbindin-D28k in the sensorimotor cortex of the rat.

Parvalbumin- and calbindin D28k-immunoreactive neurons in the hippocampal formation of the macaque monkey.

Calcium-binding protein (calbindin-D28K) and parvalbumin immunocytochemistry in the normal and epileptic human hippocampus.

Immunocytochemical Localization of Calbindin D28K, Calretinin, and Parvalbumin in Bat Superior Colliculus.

Ontological study of calbindin-D28k-like and parvalbumin-like immunoreactivities in rat spinal cord and dorsal root ganglia.

Calbindin D-28k and parvalbumin immunohistochemistry in developing rat retina.

Distribution of GABAergic interneurons immunoreactive for calretinin, calbindin D28K, and parvalbumin in the cerebral cortex of the lizard Podarcis hispanica.

Parvalbumin-, calretinin- and calbindin-D28k-immunoreactivity and GABA in a forebrain region involved in auditory filial imprinting.

The immunocytochemical distribution of calbindin-D28k and parvalbumin in identified neurons of the pulvinar-lateralis posterior complex of the cat.

Parvalbumin and calbindin-D28K immunoreactivity as developmental markers of auditory and vocal motor nuclei of the zebra finch.

Different populations of parvalbumin- and calbindin-D28k-immunoreactive neurons contain GABA and accumulate 3H-D-aspartate in the dorsal horn of the rat spinal cord.

Ontogeny of the calcium binding protein parvalbumin in the rat nervous system.

Distribution of calbindin D-28K and parvalbumin immunoreactivities in the nucleus olfactorius anterior of the rat.

Distribution of calbindin D28k-like immunoreactivity (LI) in the monkey ventral horn: do Renshaw cells contain calbindin D28k-LI?

Age-related reduction in calbindin-D28K expression in the Sprague-Dawley rat lens.

Calbindin D-28K- and parvalbumin-reacting neurons in the hypothalamic magnocellular neurosecretory nuclei of the rat.

Calbindin D28k-containing splanchnic and cutaneous dorsal root ganglion neurons of the rat.

Immunohistochemical markers in rat cortex: co-localization of calretinin and calbindin-D28k with neuropeptides and GABA.

The inferior colliculus: calbindin and parvalbumin immunoreactivity in neural grafts.

Heterogeneous development of calbindin-D28K expression in the striatal matrix.

Calbindin-D28k in the basal ganglia of patients with parkinsonism.