Department of Anatomy and Histology, Faculty of Veterinary Medicine, Aristotle University of Thessaloniki, 54006 Thessaloniki, Greece
Address correspondence to Dr G.C. Papadopoulos, Department of Anatomy and Histology, Faculty of Veterinary Medicine, Aristotle University of Thessaloniki, 54006 Thessaloniki, Greece. Email: gpapadop{at}vet.auth.gr.
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Abstract |
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Introduction |
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Local circuit nonpyramidal neurons, largely GABAergic- inhibitory in function, have been shown to be differentially affected after NA application (Gellman and Aghajanian, 1993; Kawaguchi and Shindou, 1998
). In addition, NA shares some cellular actions or acts synergistically with particular peptides contained within certain subpopulations of GABAergic neurons (Ferron et al., 1985
; Magistretti and Morrison, 1988
). Given the physiological, pharmacological and biochemical evidence for NA and local circuit neuron interactions, and the extensive compartmentalization of the cortical interneurons on the basis of their neurochemical content (Emson and Hunt, 1984
; Jones and Hendry, 1986
; DeFelipe, 1993
; Kubota et al., 1994
), the study of the ultrustructural relationships between the NA coeruleo-cortical fiber system and the distinct cell types of GABAergic neurons, although a demanding task, is a prerequisite for understanding the physiology of NA neurotransmission in the cerebral cortex. In the present study, we sought to determine the cellular substrates for the functional interactions between NA and the peptides somatostatin (SRIF), neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) in the rat visual cortex. The anatomical relationships between the NA fibers and the peptide-characterized neurons were studied by applying double preembedding immunocytochemistry. The gold-substituted silver peroxidase method was used to intensify and differentiate the NA profiles from the non-intensified peptidergic neurons.
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Materials and Methods |
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Twenty-four adult Wistar rats (body wt 200240 g) were used in this study. The animals were deeply anesthetized with ether and perfused through the ascending aorta, first with saline, containing 0.1% sodium metabisulfite (SMB), and then with 400 ml of 5% glutaraldehyde and 1% SMB in 0.05 M sodium cacodylate buffer at pH 7.4. Brains were promptly removed, blocked and postfixed in the perfusate for 30 min at 4°C. Coronal sections (50 µm) through the visual cortex were obtained with a Vibratome and collected in 0.05 M Tris-buffered saline (TBS), plus 0.1% SMB (TBSSMB, pH 7.4). Sections were treated for 20 min with sodium borohydride (0.5% in TBS), to inactivate excess of glutaraldehyde, thoroughly washed in TBSSMB and preincubated for 1 h in a blocking solution, consisting of 5% normal goat serum (NGS) and 0.1% Triton X-100 (TX) in TBSSMB, and subsequently transferred to the primary antiserum of the first immunocytochemical series. All incubations, treatments and washing steps were performed at room temperature, unless otherwise stated, and under continuous mild agitation on a shaking platform.
Double Immunocytochemistry
The following immunocytochemical procedures were applied for immunolabeling sections destined for ultrastructural analysis. Another group of sections, destined for semiquantitative estimates by bright-field microscopy (see Data Analysis, this section) was processed identically, except from adding 0.2% TX in all incubation media.
Tissue sections were incubated in a polyclonal antibody against NA (kindly supplied by Dr M. Geffard, University of Bordeaux II), diluted 1:20 000 in TBSSMB, plus 2% NGS, 0.01% TX and 0.01% sodium azide, for 48 h at 4°C. Following washing in TBS, the sections were transferred in biotinylated IgG (1:400 in TBS, plus 2% NGS) and avidinbiotin peroxidase complex (ABC; 1:200 in TBS), for 2 and 1 h respectively. Tissue-bound peroxidase was visualized in 3,3'-diaminobenzidine tetra-hydrochloride (DAB; 0.015% in Tris buffer, pH 7.6) with the addition of 0.05% hydrogen peroxide. The immunoperoxidase reaction was terminated after 46 min, resulting in a dark yellow end-product.
Single immunolabeled sections were silver/gold-toned according to the gold-substituted silver-peroxidase (GSSP) intensification method, originally described by Gallyas and co-workers (Gallyas et al., 1982). The optimal incubation period in the silver developer was determined by pilot electron microscopic studies to be 45 min at 15°C. After the end of the intensification procedure, every immunoreactive profile appeared invariably black in color. Sections were washed extensively in TBS for 2 h and processed for the second immunoreaction.
Antibodies against SRIF (1:2000), NPY (1:2500) and VIP (1:3000), generously provided by Dr R.M. Buijs (Netherlands Institute for Brain Research, Amsterdam), were diluted in TBS, plus 2% NGS and 0.01% sodium azide. No TX was added. Sections from each animal were randomly divided into three groups and incubated (72 h at 4°C) in the respective sera. Thereafter, tissue was washed in TBS and succesively incubated for 2 h in biotinylated IgG (1:200) and ABC reagent (1:100), both diluted in TBS. Finally, the sections were immersed in DAB solution (0.05% in Tris buffer, pH 7.6), containing 0.1% hydrogen peroxide, for 1215 min.
Processing for Light and Electron Microscopy
Sections destined for light microscopy were mounted on gelatin-coated glass slides, gradually dehydrated through ethanol and coverslipped with DPX. For electron microscopy, sections were postfixed in 1% osmium tetroxide for 30 min, dehydrated through ascending series of ethanol, infiltrated with propylene oxide and epoxy resin (Araldite CY212), and next day flat embedded in Araldite. The material was systematically examined in the light microscope and selected areas of the visual cortex were photographed before being reembedded in resin capsules and thin-sectioned with a diamond knife. Ultrathin sections were cut from the surfacemost portions of the Vibratome sections and collected on Formvar-coated slot grids. After counterstaining with lead citrate and uranyl acetate, they were serially examined at the electron microscope.
Antibodies
All primary antisera were produced in rabbits. The procedures for raising and purifying the antibodies, and all specificity tests have been described elsewhere (Buijs et al., 1983; Geffard et al., 1986
; Mons and Geffard, 1987
). Biotinylated goat anti-rabbit IgGs and ABC reagent were purchased from Vector Laboratories (Burlingame, CA). Omission of the primary and/or secondary antisera from the immunocytochemical procedure resulted in abolition of staining. Similarly, when the primary sera were replaced by nonimmune rabbit serum, no reaction product was detected by either light or electron microscopy.
Data Analysis
A light microscopic semiquantitative approach was followed to compare the relative incidence of the NA-contacted SRIF, NPY and VIP neurons. Coronal strips of primary visual cortex (layers IVI; 200 µm wide), double immunostained for NA-SRIF, NA-NPY or NA-VIP, were selected from the optimally immunoreactive sections of six separate animals (four strips per animal, for each case). In these areas, the SRIF-, NPY- and VIP-immunolabeled neurons were drawn using a photomicroscope equipped with camera lucida. Each cell was carefully examined for putative contacts (i.e. no apparent gap interposed, at x800 magnification) by varicose regions of NA fibers, and accordingly marked. Close appositions, in which peptidergic elements and NA puncta appeared in slightly different focal planes, were not marked.
Methodological Considerations
Data presented in this study were obtained from double-immunolabeled material in which NA immunodetection was accomplished first and immunoreaction for the peptides followed. However, in pilot experiments, material was either single labeled for each antigen or double labeled following the reverse immunoprocedure, i.e. peptide first, amine second. Neuropeptides, which are abundant and robust antigens, were found to survive relatively better than NA the first immunocytochemical and the subsequent intensification reaction, and thus they were selected for immunolabeling in the second series. Furthermore, this combination permitted the examination of the NApeptide interactions at the light microscopic level, since the NA puncta, in black, were unequivocally identifiable against the brownish peptidergic elements.
Every effort was taken to minimize background staining in the first immunocytochemical series. First, in order to preserve the antigenicity for the subsequent immunoreaction, and second, to preclude any possibility of nonspecific silver precipitation. In this line, the following control experiments were performed. (i) The secondary antibody and/or the ABC reagent were omitted. Tissue was then processed for DAB histochemistry (15 min) and GSSP-intensified for 10 min. (ii) The immunocytochemical procedure was properly followed, except omitting the chromogen reaction, and the sections were GSSP-intensified for 10 min. In either cases, traces of metal intensification were not observed by high power electron microscopy [the sensitivity and specificity of this method have been described elsewhere (van den Pol and Decavel, 1990)].
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Results |
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Metal intensification of the NA immunoreaction revealed the existence of a dense fiber network and a plethora of strong, reactive puncta in coronal sections of the rat visual cortex. The overall distribution pattern of the NA fibers was consistent with that described in previous studies making use of the same antibody (Papadopoulos et al., 1989; Seguela et al., 1990
; Paspalas and Papadopoulos, 1996
). In layers I and VI, long varicose fibers were seen to run parallel to the pia and the white matter respectively. Layers II/III were traversed by radially oriented fibers, whereas layers IV and V were characterized by the presence of short and tortuous fibers. The subjacent white matter contained a few, relatively short and tangentially oriented immunoreactive fibers. Noradrenergic fibers and puncta were also encountered in the periphery of neuronal perikarya or outlining small blood vessel profiles. Electron microscopy showed that NA immunoreactivity was exclusively associated with unmyelinated axons and axon varicosities (Fig. 1AC
). The varicosities, ranging from 0.45 to 2.2 µm in diameter, contained clear round vesicles (40 nm), elongated mitochondria and occasionally large dense-cored vesicles (80100 nm). Immunoreaction end-product was found to rim the synaptic vesicles, mitochondria and the cytoplasmic plasmalemmal surface. Intervaricose segments usually appeared less immunoreactive or escaped immunolabeling. In all cases, electron-opaque metallic particles were scattered over the NA-positive elements. The specificity of the silver/gold precipitation was remarkable and fully matched the DAB end-product localization. Metallic particles were found on mitochondria outer membrane, but never in the nonimmunoreactive mitochondrial matrix (Fig. 1A,B
). In lightly reactive varicosities, the electron-lucent lumen of the small vesicles could be seen to be free of particles, while the dense core of the large vesicles contained relatively abundant metal precipitate (Fig. 1C
). Examination of single thin sections showed that only a small portion of the NA varicosities are engaged in overt synaptic contacts with pyramidal and nonpyramidal neuronal elements. However, synaptic incidence was notably higher in uninterrupted series of sections. Junctional specializations were either symmetrical or asymmetrical (Fig. 1AC
), but the symmetric variety appeared more frequently. The latter type was characterized by narrow active zone, occasionally punctate, present only in a limited number of consecutive sections. Usually, postsynaptic elements were also recipients of an asymmetric synapse by an unstained axon terminal (Fig. 1A
).
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SRIF-, NPY- and VIP-immunoreactive non-pyramidal neurons were encountered in the whole cortical depth and in the white matter of cerebral hemispheres. In keeping with earlier descriptions (McDonald et al., 1982a,b
,c
; Parnavelas, 1986
), the SRIF and NPY neurons were preferentially concentrated in layers II/III and VVI, and exhibited the morphological features of multipolar and bitufted, and, less frequently, of bipolar neurons. The VIPergic cells were predominantly distributed in layers II/III and exhibited, for the most part, typical bipolar morphology, with ascending and descending primary dendrites arising from the opposite poles of the radially oriented perikarya. Although somata and proximal dendrites appeared almost evenly immunostained under the light microscope, ultrastructurally the peptide immunoreactivity was most frequently associated with the endoplasmic reticulum and Golgi apparatus of perikarya, and microtubules and neurofilaments of dendritic and axonal profiles (Fig. 2AC
). Numerous vesicle-filled varicosities appeared also immunoreactive for either SRIF, NPY or VIP.
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Examination of the double-immunostained material in the light microscope demonstrated a variety of anatomical relationships between the NAergic afferents and the peptidergic neurons. The NA fibers and puncta, which were black after metal intensification, were easily distinguished against the brownish, nonintensified peptidergic neurons. The morphological and distribution patterns, obtained for each of the NA, SRIF, NPY or VIP elements in the double-labeling experiments, were identical to the patterns revealed after single immunostaining for each antigen. In the electron microscope, NA boutons were usually found in close apposition with peptidergic elements without overt synaptic specializations. In most cases, synaptic complexes were revealed only after the analysis of uninterrupted series of thin sections. Non-neuronal elements, immunoreactive for NA or any of the neuropeptides studied, were never observed.
NA-SRIF
Light microscopic examination revealed that ~27% (98/364) of the SRIF-immunoreactive neurons, distributed in layers IIVI, were in close contact with NA boutons. These anatomical relationships were more frequently encountered in the deeper than the superficial cortical layers. Examined at high magnification and different focal planes, NA varicose fibers were seen to embrace the perikarya and the primary dendrites of a number of SRIF cells. In these complex associations, the NA boutons usually formed arrays that clearly appeared to outline portions of the target-cell circumference (Fig. 3B). Frequently, however, the SRIF neurons were not seen to be associated with complex NA ramifications and only a few single varicosities could be seen to contact the perisomatic domain (soma and primary dendrites) of these cells (Fig. 3A,C
).
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In the light microscope, varicose NA fibers were seen to contact ~25% (48/195) of the NPY-immunoreactive neurons, distributed in layers IIVI. Recipients of the NA contacts were primarily NPY perikarya, preferentially distributed in deeper cortical layers, but also proximal and more distal parts of primary dendrites. NPY somata and primary dendrites were typically seen to be surrounded by pericellular arrays (Fig. 5A,B) or to be contacted by multiple varicosities provided by NA fibers en passant (Fig. 5C
).
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Light microscopy showed that NA fibers are in close apposition with a portion, ~20% (75/382), of VIP-immunoreactive neurons. In layers IIVI, VIP cell bodies and the base of primary dendrites were occasionally found in contact with NAergic puncta. However, VIP neurons were never seen to be surrounded by NAergic pericellular arrays or to receive multiple terminations. Single NA fibers were found instead to proceed towards VIP cells and to establish from one to three terminal contacts with the target-neuron periphery (Fig. 7A).
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Discussion |
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Noradrenergic varicose fibers and numerous puncta were frequently seen juxtaposed to the peptidergic neurons. Given the physical limitations of light microscopy, these anatomical relationships cannot be assumed to be functional [see, however, de Lima et al. (de Lima et al., 1988)], and thus specific, especially if one considers the conflicting evidence about the synaptology of the NA fiber system in the cerebral cortex (Beaudet and Descarries, 1978
; Papadopoulos and Parnavelas, 1991
). Nevertheless, we have used the optimally double-immunostained material to estimate that about one out of four SRIF and NPY neurons, and one to five VIP neurons were closely associated with NAergic puncta. Furthermore, NA afferents were found to differentially innervate particular peptidergic neurons. More specifically, while NPY cells, as well as a portion of the SRIF population, were typically surrounded by basket-like NA multiterminal arborizations, other SRIF neurons and the VIP population were completely deprived of such complex NAergic terminations. In order to better anticipate these findings, special reference to peptide coexistence within nonpyramidal neurons has to be attempted. Somatostatin and NPY are colocalized in cortical interneurons to such an extent (virtually all NPY cells contain SRIF) that NPY neurons are generally considered as a portion (SRIF/NPY) of the SRIF population. On the other hand, VIP neurons represent a distinct neurochemical subclass, since VIP generally does not coexist with either SRIF or NPY [reviewed by DeFelipe (DeFelipe, 1993
)]. Since SRIF and NPY cells represent 23% and 12% respectively of the whole neuronal population in the rat visual cortex (McDonald et al., 1982b
,c
), and 25%, at least, of both SRIF and NPY neurons are NA-associated (present study), it seems reasonable to assume that almost 50% of the NA-contacted SRIF neurons contain also NPY (and thus receive multiterminal NA input), whereas the other half does not contain NPY (and probably receives the less complex NA input). We may thus summarize that three distinct nonpyramidal populations containing SRIF, SRIF/NPY or VIP are targets of the NA afferents, in a manner that is specific for each population. It should be noted that this differentiation of the NA target groups fully coincides with functional groups established on the basis of firing patterns of the nonpyramidal neurons (Kawaguchi and Kubota, 1996
, 1998
).
Noradrenergic boutons were found in the present study to establish symmetric or asymmetric synapses with both pyramidal and nonpyramidal neurons. The synaptic incidence was notably low for single thin sections, increasing when analysis was performed in uninterrupted series of sections, a peculiarity that has been attributed by many authors to the large disparity between the active zone length and the bouton diameter (de Lima et al., 1988; Saha et al., 1991
). Targets of symmetrical NA synapses were frequently recipients of asymmetrical synapses formed by an adjacent nonimmunolabeled terminal. Paired terminations and synaptic triads' have been suggested for various cortical neurotransmitter systems to represent the ultrastructural substrate for both presynaptic and postsynaptic modulation of the adjoining inputs (Somogyi et al., 1998
). In the present study, the NAergic synapses encountered on the SRIF, NPY and VIP neurons were invariably symmetrical, and were found to target the perisomatic domain of the peptidergic neurons, suggesting small variability in synaptic efficacy.
Relevant studies dealing with the NA innervation of neurochemically defined cortical interneurons have not been appeared, to our knowledge, in the literature. Catecholaminergic synapses on the soma of NPY neurons in the rat neocortex, visualized with antibodies against tyrosine hydroxylase (Tsuchiyama et al., 1989), were most probably dopaminergic [Asan has discussed the methodological considerations (Asan, 1993
)]. Cortical NA fibers have been previously demonstrated to synapse with, among others, dendritic shafts and nonpyramidal perikarya (Papadopoulos et al., 1989
). The present study further reveals that SRIF, NPY and VIP neurons belong to the nonpyramidal targets of the NA fibers. It seems likely, however, that interaction of the NA afferents with the nonpyramidal neuronal population involves other subclasses as well, like the fast-spiking parvalbumin-containing interneurons (Kawaguchi and Kubota, 1996
; Kawaguchi and Shindou, 1998
), which do not colocalize SRIF, NPY or VIP (Kubota et al., 1994
) [reviewed by DeFelipe, (DeFelipe, 1997
)]. This neurochemical diversity of the nonpyramidal targets of the NA afferents also explains the heterogeneous responses of the GABAergic neurons to NA or NAergic agonists (Kawaguchi and Kubota, 1998
; Kawaguchi and Shindou, 1998
). On similar lines, a remarkable chemical heterogeneity has been reported for the nonpyramidal targets of the dopamine (Sesack et al., 1995
), and, especially, the serotonin cortical afferent systems, (Freund, 1992
; Halasy et al., 1992
; Hornung and Celio, 1992
; Morales and Bloom, 1997
).
Undoubtedly, the overall physiological role of the NAergic input in the cerebral cortex is far from what is generally considered as classical neurotransmission (Woodward et al., 1979; Waterhouse et al., 1988
). A gain setting function has been traditionally proposed, since NA modulates the ratio of stimulus-driven activity, relative to spontaneous activity of cortical neurons (Foote and Morrison, 1987
; Woodward et al., 1991
). Apart from the NAergic influence that is directly exerted on pyramidal targets (Nicoll et al., 1990
), several groups describe prominent indirect effects, mediated via GABA interneurons equipped primarily with
-adrenoceptors (Madison and Nicoll, 1988
; Gellman and Aghajanian, 1993
; Bergles et al., 1996
; Kawaguchi and Shindou, 1998
). We further suggest that the NAneuropeptide anatomical interactions presented here are embedded in the above microcircuitry, by which NA may indirectly modulate the pattern of activity and level of excitability in the cortex.
Noradrenaline facilitates both excitation and inhibition in the cerebral cortex (Bevan et al., 1977). In the present study, we report a direct NAergic input to peptidergic cells, presumably inhibitory, since it is mediated by symmetric synapses. Nevertheless, NA is generally producing GABA-mediated inhibitory postsynaptic potentials to pyramidal cells, suggesting a prominent depolarizing effect on interneurons. Accordingly, if the NA input to peptidergic cells is indeed inhibitory, a polysynaptic mechanism, by which one NA-inhibited peptidergic neuron may in turn disinhibit another interneuron and finally elicit IPSPs in pyramidal cells, should be considered [Roberts has discussed GABA disinhibition (Roberts, 1976
)]. Alternatively, depolarization of GABAergic subpopulations by NA may result from presently unidentified asymmetric NAergic synapses, located not on the perisomatic domain of the peptidergic cells, but preferentially onto distal branches of their dendritic tree. Such a domain specificity should also provide an additional neuromodulatory advantage for the subtle control of the peptidergic interneurons. The above scenarios are apparently simplified. GABA-mediated IPSPs to pyramidal neurons must not be viewed solely as responses to single, indirect NA inputs. Multiple interneurons converge upon particular pyramidal cells (Tamas et al., 1997
), while paired terminations of NA afferents and peptidergic neurons have been shown to innervate other nonpyramidal cells (Paspalas and Papadopoulos, 1998
). Yet, the same interneurons can be synchronously activated by other monoamines (Miles, 1990
; Gellman and Aghajanian, 1993
). Cortical circuitry is ideally suited to provide such cellular interactions. Within the circuitry, particular types of GABAergic neurons are selectively interconnected in a unidirectional fashion (Somogyi et al., 1998
) or further innervate divergently other interneurons and principal cells, influencing simultaneously the pattern of both inhibitory and excitatory activities (Cobb et al., 1997
).
The exact physiological role of the NA in the cortical circuitry remains as yet largely obscure, and the nature and extend of anatomical interactions among chemically defined interneurons and glutamatergic cells need to be elucidated (G.C. Papadopoulos et al., in preparation). It is, however, evident that NA can modulate principal cell activity by taking full advantage of the neuron properties (neurochemistry, synaptology and function) of the intrinsic modulatory circuitry in cerebral cortex. This provides ample opportunity for flexibility and adaptation (Katz and Frost, 1996), but also for propagation of the influence to directly or indirectly connected cells and amplification of the input itself. Indeed, through this network, interneurons that ultimately target pyramidal perikarya are able to convey a more powerful indirect NAergic input than through the direct NA synapses which are predominantly localized on distal dendrites and dendritic spines of the pyramidal cells (Beaudet and Descarries, 1978
; Seguela et al., 1990
). Moreover, the fact that modulation of principal cell activity via distinct interneurons has been previously recognized for the serotonin and dopamine afferents of both mammalian and nonmammalian cerebral cortices (Freund et al., 1990
; Acsady et al., 1993
; Benes et al., 1993
; Morilak et al., 1993
; Martinez-Guijarro et al., 1994
; Sesack et al., 1995
) suggests that extrinsic modulation of cortical activity through intrinsic neuromodulatory networks does represent a highly conserved mechanism through phylogenesis.
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Acknowledgments |
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