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Non-peptidergic primary afferents are presynaptic to neurokinin-1 receptor immunoreactive lamina I projection neurons in rat spinal cord

Non-peptidergic primary afferents are presynaptic to neurokinin-1 receptor immunoreactive lamina... Background: Pain-related (nociceptive) information is carried from the periphery to the dorsal horn of the spinal cord mostly by two populations of small diameter primary afferents, the peptidergic and the non-peptidergic. The peptidergic population expresses neuropeptides, such as substance P and calcitonin gene-related peptide, while the non-peptidergic fibers are devoid of neuropeptides, express the purinergic receptor P2X3, and bind the isolectin B4 (IB4). Although it has been known for some time that in rat the peptidergic afferents terminate mostly in lamina I and outer lamina II and non-peptidergic afferents in inner lamina II, the extent of the termination of the latter population in lamina I was never investigated as it was considered as very minor. Because our preliminary evidence suggested otherwise, we decided to re-examine the termination of non-peptidergic afferents in lamina I, in particular with regards to their innervation of projection neurons expressing substance P receptors (NK-1r). We used retrograde labeling of neurons from the parabrachial nucleus combined with lectin IB4 binding and immunocytochemistry. Samples were examined by confocal and electron microscopy. Results: By confocal microscopy, we studied the termination of non-peptidergic afferents in lamina I using IB4 binding and P2X3 immunoreactivity as markers, in relation to CGRP immunoreactivy, a marker of peptidergic afferents. The number of IB4 or P2X3-labeled fibers in lamina I was higher than previously thought, although they were less abundant than CGRP-labeled afferents. There were very few fibers double-labeled for CGRP and either P2X3 or IB4. We found a considerable number of IB4-positive fiber varicosities in close apposition to NK-1r-positive lamina I projection neurons, which were distinct from peptidergic varicosities. Furthermore, we confirmed at the ultrastructural level that there were bona fide synapses between P2X3-immunoreactive non-peptidergic boutons and neurokinin-1 receptor-positive lamina I dendrites. Conclusions: These results indicate the presence of direct innervation by non-peptidergic nociceptive afferents of lamina I projection neurons expressing NK-1r. Further investigations are needed to better understand the role of these connections in physiological conditions and chronic pain states. Introduction neuropeptides, such as substance P (SP) and calcitonin Unmyelinated C afferents comprise approximately 70% gene related peptide (CGRP), expresses the high affinity of all primary afferents fibers [1]. The majority of such nerve growth factor receptor, trkA, and terminates fibers transmit nociceptive information from the periph- mainly in lamina I and outer lamina II of the spinal dor- ery to the spinal dorsal horn and have been divided into sal horn [3-5]. The non-peptidergic unmyelinated affer- two main populations, the peptidergic and the non- ents, although devoid of neuropeptides, express the peptidergic [2]. The peptidergic population synthesizes purinergic P2X3 receptor, bind the plant isolectin B4 (IB4), express receptors for glial cell-derived neuro- * Correspondence: alfredo.ribeirodasilva@mcgill.ca trophic factor (GDNF) and terminate mainly in inner Department of Pharmacology and Therapeutics, McGill University, Montreal, lamina II of the spinal dorsal horn [6-8]. These two Quebec H3G 1Y6, Canada Alan Edwards Centre for Research on Pain, McGill University, Montreal, populations can also be differentiated based on the ultra- Quebec H3A 2B2, Canada structural properties of their central terminals in the Full list of author information is available at the end of the article © 2012 Saeed and Ribeiro-da-Silva; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Saeed and Ribeiro-da-Silva Molecular Pain 2012, 8:64 Page 2 of 10 http://www.molecularpain.com/content/8/1/64 spinal dorsal horn.; The peptidergic terminate mostly as and P2X3-IR fibers at low magnification, it was apparent simple axo-dendritic boutons, containing several large that the band of intense IB4+ labeling extended more granular vesicles (LGV), and occasionally as central ventrally than the P2X3 band (Figure 1). Overall, some terminals of type Ib glomeruli (CIb), which are rich in IB4+ and P2X3-IR terminals co-localized CGRP immu- LGV [9,10]. In contrast, the non-peptidergic afferents noreactivity (in yellow in Figures 1 and 3), although the terminate most often as the central bouton of type Ia majority did not. A surprising observation was that the glomeruli, which are poor in LGV, have a very dense number of boutons in lamina I that were IB4+ or P2X3-IR matrix and are often postsynaptic to GABA-positive and did not co-localize CGRP immunoreactivity was con- neurons at axo-axonic or dendroaxonic synapses [10]. siderable, and much higher than what could be expected Regarding peptidergic afferents, their dorsal horn based on the literature. Some IB4+ and P2X3-IR boutons termination has been investigated in particular in rela- which did not co-localize CGRP immunoreactivity could tion to neurons which express the SP receptor, the be observed in transverse sections (Figure 1 C-D; arrow- neurokinin-1 receptor (NK-1r) [11-13]. In particular, it heads). However, these boutons were particularly apparent has been shown at the confocal and electron micro- in parasagittal (Figure 2) and horizontal sections (Figure 3). scopic levels that SP-immunoreactive (IR) terminals in- In horizontal sections, we could be absolutely certain that nervate preferentially neurons which display NK-1r the boutons from non-peptidergic afferents were located immunoreactivity [11,13]. In lamina I, an abundant in- in lamina I since we used serial sections, and the confocal nervation by SP-IR primary afferents of projection neu- images were obtained with a very small pinhole corre- rons, which are immunoreactive for the NK-1r and are sponding to an optical slice of ~0.5 μm adjacent to the activated by noxious stimuli, has been dected [13]. Un- white matter. These sections revealed a considerable in- fortunately, our knowledge concerning the central termi- nervation of lamina I by boutons immunoreactive for nations of the non-peptidergic C fiber population is P2X3, although they were clearly less abundant than the limited. In rat, as mentioned above, they have been CGRP-IR (Figure 3A). The comparison with optical slices shown to terminate mostly in inner lamina II as central from inner lamina II allowed us to assess how much more terminals of type Ia glomeruli. However, besides the fact abundant P2X3-IR boutons were in that layer (Figures 1D, that they are postsynaptic to GABAergic interneurons, 2, and 3B), confirming that inner lamina II is the main ter- their synaptic connections are not well known. However, mination site for non-peptidergic afferents. studies in rat utilizing whole-cell recordings suggest that these non-peptidergic afferents form indirect connec- Innervation of NK-1r-IR lamina I projection neurons by tions with lamina I projection neurons through inter- non-peptidergic afferents neurons in lamina II [14,15], while other studies, using To study the innervation of NK-1r-IR lamina I projec- transgenic mice, proposed that the non-peptidergic tion neurons by non-peptidergic afferents, we labeled afferents synapse onto inner lamina II interneurons neurons retrogradely by means of a stereotaxic cholera which in turn would connect with deep lamina V projec- toxin subunit b (CTb) injection in the lateral parabra- tion neurons [16]. However, it was never investigated chial nucleus. The injection site covered most of the whether lamina I projection neurons receive direct parabrachial complex, including the lateral parabrachial synapses from non-peptidergic afferents, likely because nucleus, an observation that is comparable to distribu- it was assumed that the termination of such afferents in tions previously reported by us [17,18]. The majority of lamina I was negligible. In this study, we examined the retrogradely labeled lamina I neurons were found on quantitatively the innervation of NK-1r-positive lamina I the contralateral side, although a few were also present projection neurons by non-peptidergic unmyelinated on the side ipsilateral to the CTb injection. CTb labeling primary afferents, using confocal microscopy, and also of spinoparabrachial lamina I neurons included the cell provided ultrastructural evidence of direct synapses of body and primary dendrites (Figures 4 and 5). these afferents on NK-1r-IR lamina I neurons. We also performed a labeling of these cells with anti- bodies against the SP, receptor, the NK-1r. Indeed, it has Results been previously described [17,18] that lamina I, fusiform Termination of non-peptidergic afferents in lamina I and multipolar spinoparabrachial neurons, identified by We examined the termination of non-peptidergic affer- retrograde tracing of CTb, often express NK-1r immu- ents using either IB4 binding or P2X3 immunoreactivity. noreactivity, while lamina I pyramidal neurons seldom ex- We used sections cut in the three planes: transverse, press NK-1r immunoreactivity. These neuronal populations parasagittal and horizontal (Figures 1, 2, and 3), and fo- can be identified based on their dendritic arborization and cused on the region equidistant from the lateral and cell body shape when viewed in the horizontal plane. Multi- medial limits of the dorsal horn. In transverse sections, polar neurons have four or more dendrites arising from an when comparing the distribution of IB4-binding (IB4+) irregularly-shaped soma (Figure 4A). Fusiform neurons Saeed and Ribeiro-da-Silva Molecular Pain 2012, 8:64 Page 3 of 10 http://www.molecularpain.com/content/8/1/64 Figure 1 CGRP, IB4 and P2X3 staining in transverse spinal cord sections. A and B show low magnification confocal images of CGRP-IR and IB4 positive (A) or P2X3-IR (B) fibers. C and D represent high magnification confocal images from the middle third of the latero-medial extent of the superficial dorsal horn. In C, note that there is limited co-localization of IB4 and CGRP (in yellow). Arrowheads show axonal varicosities (boutons) from non-peptidergic fibers in lamina I, which do not co-localize CGRP immunoreactivity. The framed regions in A and B indicate the approximate regions from where C and D, respectively, were obtained (the latter originate from other sections). CGRP (in green); IB4 (in red); P2X3 (in red). Scale bar (A, B) = 200 μm; scale bar (C, D)=20 μm. possess two dendrites, each arising from one end of a was performed for the peptidergic innervation on lam- spindle-shaped soma (Figure 4B and 5). Lastly, pyramidal ina I neurons, which has been previously investigated neurons have a triangular soma with one primary dendrite [11,22,23]. arising from each of the three tips of the soma (Figure 4C). In this study, we observed IB4+ non-peptidergic varicosities Ultrastructural demonstration of synapses between non- in apparent direct apposition to all three populations of peptidergic primary afferents and lamina I neurons lamina I projection neurons, multipolar (Figure 4A), Because the resolution of confocal microscopy is insuffi- fusiform (Figure 4B) and pyramidal (Figure 4C). These bou- cient to demonstrate the presence of bona fide appositions tons were distinct from CGRP-positive peptidergic bou- tons, and from boutons of the small proportion of primary afferents which are simultaneously CGRP-positive and bind IB4, as described previously [19,20] (Figure 5). Quantification of non-peptidergic varicosities apposed to NK-1r-positive lamina I projection neurons As the objective of this study was to investigate the non-peptidergic innervation of NK-1r-IR projection neurons, our quantitative analysis focused on the in- nervation of multipolar and fusiform neurons. Indeed, pyramidal neurons are seldom immunoreactive for the substance P receptor [21]. Our quantitative analysis Figure 2 Confocal images at high magnification obtained from revealed a substantial innervation of NK-1r-IR multi- parasagittal spinal cord sections showing CGRP-IR (green) and polar and fusiform populations of lamina I projection P2X3-IR (red) varicosities in the superficial dorsal horn. P2X3-IR varicosities were present in considerable number in lamina I (LI) but neurons by non-peptidergic afferents (Figure 6). Since were more highly concentrated in inner lamina II (LIIi). Scale the main objective of this paper was to give an account bar = 20 μm. of the non-peptidergic innervation, no quantification Saeed and Ribeiro-da-Silva Molecular Pain 2012, 8:64 Page 4 of 10 http://www.molecularpain.com/content/8/1/64 Figure 3 Confocal images at high power obtained from horizontal spinal cord sections. In a confocal optical section from lamina I adjacent to the white matter (A), note the relatively abundant P2X3-IR fibers with varicosities (boutons). CGRP-IR fibers and boutons were considerably more abundant in this lamina. In a confocal optical section from inner lamina II (B), note the very high density of P2X3-IR fibers and varicosities, higher than that of CGRP-IR fibers in lamina I. Note that most varicosities display either P2X3 or CGRP immunoreactivity, although some co-localization is observed (yellow). Scale bar (A, B)=20 μm. of two structures and synapses between them, we per- binding the lectin IB4. We also provide evidence at the formed an ultrastructural study using P2X3 immunoreac- confocal microscopy level that IB4+ boutons are in ap- tivity. We had to omit glutaraldehyde from the fixative position to lamina I projection neurons immunoreactive because it completely blocked the immunostaining, even for the NK-1r. Lastly, we provide ultrastructural evi- in very low concentration, which affected the quality of dence of synapses between P2X3-IR boutons and lamina the ultrastrucure. In lamina I, all P2X3-IR boutons estab- I dendritic profiles immunoreactive for the NK-1r. lished simple axo-dendritic or axo-somatic synapses (Figure 7A). In inner lamina II, most P2X3-IR boutons Technical considerations were involved in complex synaptic arrangements, as pre- Because most of this study was carried out using con- dicted from the literature [24], forming the central elem- focal microscopy, we could not fully ensure that boutons ent of type Ia glomeruli (Figure 7B). from non-peptidergic afferents were presynaptic to neu- We used a double labeling for P2X3 and NK-1r to rons in lamina I. For this reason, we carried out an ultra- demonstrate direct appositions and synapses between structural study using antibodies against P2X3 and P2X3 immunoreactive boutons and NK-1r dendritic pro- provided direct evidence of synapses between P2X3-IR files (Figure 8). axonal boutons and dendrites and cell bodies in lamina I. We also performed at the ultrastructural level a double Discussion labelling for P2X3 and the NK-1r that demonstrated that In this study, we demonstrate the presence in lamina I some of the structures innervated by these boutons of a significant number of boutons originating from expressed the NK-1r, in agreement with what was non-peptidergic afferents immunoreactive for P2X3 or assumed from the confocal data. We could observe Figure 4 Confocal triple-labeling image showing the innervation of lamina I spinoparabrachial (A) multipolar, (B) fusiform and (C) pyramidal neurons by IB4+ varicosities (indicated by arrowhead). IB4 (red); NK-1r (green); spinoparabrachial neurons labeled with CTb (white). Scale bar (A-C)= 20 μm. Saeed and Ribeiro-da-Silva Molecular Pain 2012, 8:64 Page 5 of 10 http://www.molecularpain.com/content/8/1/64 Figure 5 Example of a quadruple labeling observed at the confocal level using a multi-track approach. In this image the following signals were simultaneously detected: CGRP (green); IB4 binding (red); CTb transported retrogradely from the parabrachial nucleus (blue); NK-1r (white). A fusiform neuron, double labeled with CTb and NK-1r, is innervated by CGRP-IR boutons (arrowhead) and IB4+ (arrow) boutons, which represent distinct populations. However, a small population of varicosities co-labeled for CGRP and IB4 (curved arrow) was detected. Scale bar (A-E)= 20 μm. unequivocal evidence of synaptic contacts, in spite of the fact that we had to use a fixative without glutaraldehyde and pre-treatment of the sections with a detergent for a short period to obtain P2X3 staining at the ultrastruc- tural level. We were unable to use IB4 binding for elec- tron microscopy because of the bad penetration of the lectin in the tissue. Conversely, we were unable to use P2X3 immunoreactivity for the study of the innervation of the neurons at the confocal level because of antibody incompatibilities when performing the required triple la- beling. It should be pointed out that Naim et al. [25] Figure 7 Electron micrograph showing: in A, a P2X3-IR bouton Figure 6 Quantification of IB4-positive boutons in direct (arrow) forming an axo-somatic contact onto a lamina I cell apposition to the cell body and primary dendrites of NK-1r-IR body (CB), which is also postsynaptic to unlabeled axonal lamina I spinoparabrachial neurons. Results are expressed in boutons (V); in B, a P2X3-IR central bouton of a type Ia number of appositions from boutons per 100 μ of membrane glomerulus (CI P2X3+) presynaptic to 3 lamina I dendrites (D) length of NK-1r-IR cell. N = 6 animals. and apposed to an axonal bouton (V2). Scale bar = 0.5 μm. Saeed and Ribeiro-da-Silva Molecular Pain 2012, 8:64 Page 6 of 10 http://www.molecularpain.com/content/8/1/64 Figure 8 Electron micrographs showing axo-dendritic contacts between P2X3-positive boutons (arrows) and NK-1r-positive dendrites (D) (A and B). Note the silver-intensified gold particles along the plasma membrane of the dendrites, representing NK-1r-IR sites. B, unlabeled axonal bouton. Scale bar = 0.5 μm. examined at the ultrastructural level ultrathin sections NK-1r) because of photobleaching during the perfor- recut from thicker sections previously examined by con- mace of the Z-stacks. However, we examined enough focal microscopy. Naim et al.'s study showed that immu- cells using the quadruple labeling to ensure that the pro- noreactive varicosities seen in close apposition to the portion of axonal boutons colocalizing IB4 binding and membrane of NK-1r-IR under the confocal microscope CGRP immunoreactivity in apposition to lamina I neu- actually formed synapses when viewed under the elec- rons was low. An example of the quadruple labeling is tron microscope. Therefore, we are confident that the given on Figure 4. IB4+ varicosities in apposition to NK-1r-IR lamina I pro- jection neurons should establish synapses in a high pro- Innervation of lamina I projection neurons by non- portion of cases. peptidergic C fibers It is known that in rat there is a small proportion of The main objective of this study was to investigate the peptidergic sensory fibers in lamina I and II that coloca- issue of the innervation of NK-1r-IR lamina I projection lize CGRP and somatostatin, do not respond to NGF neurons by non-peptidergic C fibers because in our and bind IB4 [19,20]. This obliged us to investigate the knowledge a systematic study combining labeling of co-localization of IB4 binding and P2X3 immunoreactiv- these sensory fibers with labeling of these lamina I neu- ity, our markers of non-peptidergic nociceptive C fibers, rons had never been done. This is an important issue for with CGRP immunoreactivity. Indeed, as predicted, we the reasons given below. Previous work by Lu and Perl found a limited level of co-localization of either marker [14], using simultaneous whole-cell recordings from of non-peptidergic afferents with CGRP immunoreactiv- pairs of neurons, provided some evidence that input ity. However, most varicosities that we observed in lam- from primary afferent C fibers terminating in inner lam- ina I which were IB4+ or P2X3-IR did not co-localize ina II may reach lamina I projection neurons via inter- CGRP immunoreactivity, what reassured us regarding posed interneurons. Since the great majority of C fibers the validity of our findings regarding the overall innerv- innervating inner lamina II are non-peptidergic, it has ation of lamina I by non-peptidergic afferents. But it was been later suggested that the above pathway may be im- still possible that a subpopulation of these afferents in- portant for pain-related information conveyed by non- nervating NK-1r-IR lamina I projection neurons would peptidergic C fibers to reach lamina I projection neurons represent exactly this minor subpopulation that is simul- which then project to supraspinal levels. If this is true, taneously CGRP-IR and IB4+. Therefore, we carried out lamina I nociceptive projection neurons would receive a quadruple labeling in which we detected CTb (the direct input from peptidergic afferents and polysynaptic retrograde label), NK-1r, CGRP and IB4 binding. Unfor- input from the non-peptidergic afferents [24]. tunately, because of antibody incompatibilities, we could Alternatively, it has been proposed that the non- not perform P2X3 staining and had to use the lectin IB4 peptidergic afferents are part of a distinct and parallel conjugated to a fluorochrome. Although our confocal pathway from that of their peptidergic counterpart. This microscope can identify 4 separate signals reliably using view obtained some support from a study by Braz et al. the multi-track approach, it was technically impossible [16] in a transgenic mouse, which demonstrated that the to perform a quantitative analysis of the innervation of termination of non-peptidergic afferents on lamina II lamina I cells using more than 3 signals (IB4, CTb and would be on excitatory interneurons, which in turn Saeed and Ribeiro-da-Silva Molecular Pain 2012, 8:64 Page 7 of 10 http://www.molecularpain.com/content/8/1/64 would synapse on deep lamina V projection neurons to give the reader an idea of the order of magnitude of with ascending connections to the amygdala, hypothal- the two types of innervation. Indeed, Todd at al. [13] amus and bed nucleus of stria terminalis. As they found have detected a density of peptidergic innervation of minimal connection with lamina I neurons expressing 16.2 contacts/100 μm of NK-1r-IR lamina I projection the NK-1r, the above group proposed the involvement of neuron membrane. Therefore, the non-peptidergic in- the non-peptidergic afferents in the affective/emotional nervation is of lamina I NK-1R-IR neurons is substantial, component of pain. Alternatively, other studies also in although less abundant than the peptidergic. Although the mouse, proposed that distinct subsets of primary the focus of this study was mainly on NK-1r-IR projec- sensory afferents selectively mediate responses to differ- tion neurons in lamina I, there is also a population of ent stimulus modalities. These studies provided some NK-1r-positive neurons in deep laminae III-V, which evidence suggesting that, in the mouse, non-peptidergic also project supraspinally and send processes to laminae afferents play a particular role in transmitting mechan- I-II [25]. These neurons have been shown to receive on ical pain, as opposed to the peptidergic which would be average 18.8 contacts per 100 μm of dendrite length involved in conveying thermal pain [26,27]. This idea of from substance P-IR boutons in these superficial layers divergent pain pathways can be criticized since all the [25]. Another study has reported that these deeper neu- studies supporting it were performed in mice, which rons receive contacts from IB4-binding non-peptidergic have been shown to demonstrate an explicit dichotomy afferents in laminae I-II, although in much lower density between the C fiber populations that does not apply to (just 2 appositions per 100 μm of length of NK-1r-IR rats and higher order species such as primates. In par- dendrite membrane) than for the peptidergic afferents ticular, the vanilloid TRPV1 receptors are localized only [34]. These values of non-peptidergic innervation of in the peptidergic fibers in mice [28,29] whereas they are laminae III/IV neurons are considerably lower than present in both peptidergic and non-peptidergic affer- those we found in the current study. ents in the rat and higher species [30,31]. Our data provides evidence that non-peptidergic pri- Conclusion mary afferents establish direct connections with NK-1r- These results expand our previous knowledge concern- IR lamina I projections neurons, in addition to the pos- ing the termination in dorsal horn of non-pepdergic pri- sible indirect connections via an interposed interneuron mary afferents by providing evidence that they terminate suggested in previous studies [14]. Although the non- directly on lamina I neurons expressing the NK-1r. peptidergic primary afferent termination in lamina I may seem as a minor contribution to the lamina I synaptic Materials & methods circuitry when compared to their termination in inner The guidelines of the Canadian Council on Animal Care lamina II, they may prove to have a significant role in for the care and use of experimental animals and of the the transmission of nociceptive signals. Indeed, the International Association for the Study of Pain were fol- terminals of these fibers in inner lamina II represent the lowed in all the experiments. Furthermore, all studies central bouton of type Ia glomeruli, and are involved in were previously approved by the McGill University complex modulatory circuits involving GABAergic pre- Faculty of Medicine Animal Care Committee. synaptic inhibition [24]. A direct termination of non- A total of 26 male Sprague Dawley rats (Charles River, peptidergic afferents on lamina I cells would bypass such Quebec, Canada) weighing 225-250 g were used for the modulation. Since non-peptidergic afferents have a more experiments. The number of animals used and their suf- extensive distribution in the epidermis than peptidergic fering were kept to the minimum necessary for the con- afferents [32], show larger and more sustained responses duction of the study. Animals were exposed to 12 hr to capsaicin than peptidergic afferents [33] and signal light/dark cycle and given food and water ad libitum. mainly via glutamate without the presence of neuropep- The cages were fitted with soft bedding and a plastic tide co-transmitters, the activation of NK-1r-IR lamina I tube for an enriched environment and the animals were projection neurons by these non-peptidergic afferents housed in cages of four. would result in a different signal transmitted than when activated by the peptidergic afferents. Animal preparation Our results revealed that, on average, there were 6.7 Injection of tracers appositions from non-peptidergic boutons per 100 μm For the immunohistochemistry experiments requiring of length of NK-1r-IR lamina I projection neuron mem- retrograde tracing, six animals were anesthetized using brane. How does this compare to the peptidergic innerv- 5% isoflurane with oxygen and placed in a stereotaxic ation of these cells as shown in other studies? Although apparatus (David Kopf Instruments, Tujunga, CA) and such comparison is not fully legitimate because of meth- stabilized with non-perforating ear bars. The coordinates odological differences with our study, we report it here for the parabrachial nucleus (rostral/caudal: -9.12; Saeed and Ribeiro-da-Silva Molecular Pain 2012, 8:64 Page 8 of 10 http://www.molecularpain.com/content/8/1/64 medial/lateral: -2.1; dorsal/ventral: -6.3) were calculated different species), or IB4, in PBS + T containing 5% NDS. from the Paxinos & Watson Rat Brain Atlas [35] with Next, the sections were washed in PBS + T and then incu- Bregma as the reference point. A small hole was bored bated in species-specific secondary antibodies that were through the skull at the target point, exposing the dura raised in donkey and conjugated to either AlexaFluor 488, mater and a glass micropipette (Wiretrol II, Drummond AlexaFluor 405, Rhodamine RedX or biotin. The sections Scientific Company, Broomall, PA) was lowered to the were incubated in 3 different cocktails: #1) rabbit anti- stereotaxic position of the parabrachial nucleus. Two μl CGRP at a 1:200 dilution (Sigma, St Louis, MO) and lectin of 1.0% solution of CTb (List, Campbell, CA) was slowly IB4 conjugated to AlexaFluor 568 at a 1:200 dilution (Mo- injected into the parabrachial nucleus over a period of lecular Probes); #2) rabbit anti-CGRP and guinea pig anti- 20 minutes, followed by a waiting period of 10 minute P2X3 at a 1:25,000 dilution (Neuromics, Edina, MN); #3) before the micropipette was retracted from its position goat anti-CTb at a 1:5000 dilution (List Biological), rabbit to minimize leakage of the tracer. CTb was injected anti-NK-1r at a 1:10000 dilution (Sigma, St Louis, MO), seven days prior to sacrificing the animals. guinea pig anti-CGRP at a 1:8000 dilution (Peninsula, San Carlos, CA) and lectin IB4 conjugated to AlexaFluor 647 Animal perfusion at a 1:200 dilution (Molecular Probes). All the sections For immunohistochemistry, eighteen animals were were washed with PBS + T and then (for #1) incubated for deeply anesthetized with 0.3 ml/100 g of body weight of 2 hours at room temperature with donkey anti-rabbit Equithesin (6.5 mg chloral hydrate and 3 mg sodium AlexaFluor 488; (for #2) incubated for 90 minutes in a bio- pentobarbital i.p.). They were then perfused through the tin conjugated donkey anti-guinea pig IgG (Jackson left cardiac ventricle with perfusion buffer (for compos- Immunoresearch, West Grove, PA, 1:200). Further ampli- ition see [36]) for one minute, followed by 4% parafor- fication of the P2X3 signal was achieved by treating the maldehyde (PFA) in 0.1 M phosphate buffer (PB), pH sections with 1 hour incubation in an avidin-biotin (A + 7.4, for 30 minutes. The spinal cord segments L4-L5, as B) complex (Vectastain Elite ABC kit, Vector Laborator- well as the brains from animals injected with the retro- ies) followed by tyramide (Perkin-Elmer, Norwalk, CT, grade tracer, were extracted and post-fixed for 1 or 1:75) for 7 minutes. Sections were then incubated in strep- 2 hours, respectively, in the same fixative. tavidin conjugated to AlexaFluor 568 (Molecular Probes, For examination under the electron microscope, eight Eugene, OR, 1:200) and donkey anti-rabbit AlexaFluor animals were anesthetized as mentioned above, and then 488. For #3, sections wereincubated for 2 hours at room perfused with perfusion buffer for one minute, followed temperature with secondary antibodies: donkey anti-goat by 30 minutes perfusion with a mixture of 4% PFA and Rhodamine Red X, donkey anti-rabbit AlexaFluor 488, 15% picric acid in 0.1 M PB. The spinal cord segments and donkey anti-guinea pig AlexaFluor 405. Finally, all L4-L5 were taken out and post-fixed for one hour in the sections were washed with PBS, mounted on gelatin- same fixative. subbed slides and coverslipped with an anti-fading All specimens were immersed overnight in 30% su- mounting medium (Aqua Polymount; Polysciences, crose in PB at 4°C for cryoprotection. Warrington, PA). Slides were stored at −4°C pending further processing. Tissue processing To evaluate the injection site in the parabrachial nu- Confocal microscopy cleus, brainstem sections were incubated with anti-CTb The injection site at the level of the parabrachial nucleus antibody followed by a biotinylated donkey anti-goat IgG was examined by cutting serial, 100 μm-thick coronal sec- and streptavidin conjugated to AlexaFluor 568. They tions of the relevant brain region. The dorsal aspect of the were then mounted and coverslipped as described above. L4-L5 spinal cord segment was cut into serial, 50 μm- thick horizontal sections (n = 10), 50 μm-thick parasagittal Electron microscopy sections (n = 4) or 50 μm-thick transverse sections (n = 4). Spinal cord specimens from the L4-L5 region were All sections were cut using a freezing sledge microtome freeze-thawed in liquid nitrogen for 30 seconds, then cut (Leica, Richmond Hill, Ontario) and collected as free- into 50 μm-thick transverse sections on a Vibratome floating in phosphate-buffered saline (PBS) with 0.2% (TPI, St. Louis, MO, USA) and collected as free-floating Triton-X 100 (PBS + T). To block unspecific staining, all in PBS. To increase further antibody penetration, sec- spinal cord sections were incubated, for one hour, in 10% tions were incubated for 15 minutes in PBS + T, but all normal donkey serum (NDS) (Jackson, West Grove, PA) further incubations were carried out in PBS without Tri- in PBS + T at room temperature. Subsequently, the sec- ton. Following a short PBS wash, the sections were incu- tions were placed in primary antibodies (or conjugated bated with 50% ethanol for 30 minutes. They were then lectin IB4 - see below) for 48 hours at 4°C. We used a incubated for 30 minutes in 0.5% BSA (bovine serum al- mixture of 2 or 4 primary antibodies (each raised in a bumin) and subsequently incubated for one hour in 10% Saeed and Ribeiro-da-Silva Molecular Pain 2012, 8:64 Page 9 of 10 http://www.molecularpain.com/content/8/1/64 NDS. Sections were incubated for 48 hours, at 4°C, ei- separate detections of AlexaFluor 488, streptavidin con- ther with a primary antibody mixture of guinea pig anti- jugated to AlexaFluor 568 or Rhodamine Red X, IB4- P2X3 at dilution 1:200,000 and rabbit anti-NK-1r at dilu- conjugated to AlexaFluor 647 and/or AlexaFluor 405, re- tion 1:4000, or the guinea-pig anti-P2X3 antibody (at the spectively. Images used for quantification represent ser- same dilution) alone, in 5% NDS and 0.1% BSA. ial optical sections obtained along the z-axis (z-stacks), Sections were then washed several times with PBS and using a 63X plan-apochromatic oil-immersion objective. then incubated for 90 minutes in a biotinylated donkey Furthermore, for an unbiased representation of the anti-guinea pig IgG antibody at dilution of 1:1000 in PBS images taken, all the parameters of laser power, pinhole and processed for tyramide amplification as described size and image detection were kept constant for all sam- above. Afterwards, the sections were incubated, for one ples. The images obtained were converted to TIFF files. hour, in A + B enzyme complex. Labeling, for the P2X3 Our criteria of identification and quantification of lam- antibodies, was revealed using following the incubation of ina I neurons have been described extensively in previ- the section with intensified DAB (3’,3’diaminobenzidine ous publications from our laboratory [18] (see also with 1% cobalt chloride and 1% nickel ammonium sulfate) Results section). To calculate the density of IB4-positive to which 1% hydrogen peroxide was added [37]. The reac- boutons per unit length of neuronal membrane, the tion was stopped by two washes in PBS. Sections pro- length of membrane in the soma and proximal dendrites cessed for P2X3 staining were then osmicated (see below). of NK-1r-IR neurons was measured with the help of an Sections previously incubated in two primary antibodies MCID Elite Image Analysis System (Imaging Research, (anti-P2X3 and anti-NK-1r) were placed for 10 minutes in St.Catharines, ON, Canada) in each optical section and washing buffer (0.8% BSA and 0.5% fish gelatin in PBS; the number of appositions from IB4-positive boutons pH 7.4) and then incubated overnight at 4°C with anti- onto it was counted. This was done on alternate optical rabbit IgG conjugated to gold particles (1.4 nm diameter, sections from the z-stack, to avoid counting the same Nanoprobes, Yaphank, NY) in washing buffer, at dilution boutons twice. of 1:200. The following day, all sections are washed for Competing interests 3 minutes in washing buffer, rinsed for 3 minutes in PBS, The authors declare they have no competing interests. and then immersed for 10 minutes in 1% glutaraldehyde in PBS. The sections were washed with de-ionized water Authors’ contributions for 2 minutes and then incubated for 8 minutes with an AWS designed and performed all experimental protocols described in this HQ silver enhancement reagent following manufacturer's manuscript as well as the writing of the initial draft of the manuscript. ARS provided supervision for data analysis, study direction, image acquisition, instructions (Nanoprobes, Yaphank, NY). The sections manuscript design and revisions. Both authors have read and approved the were then thoroughly rinsed with de-ionized water and final draft of this manuscript. then washed with 0.1 M PB. The sections were then post-fixed in 1% osmium tet- Acknowledgements roxide in PB for 1 hour at room temperature and, subse- Supported by Canadian Institute of Health Research (CIHR) grant MOP-79411 (to A.R.-da-S). The authors would like to thank Manon St-Louis for laboratory quently, dehydrated in ascending concentrations of expertise, Jacynthe Laliberté for confocal microscopy assistance and Johanne ethanol and propylene oxide, followed by flat-embedding Ouellette for electron microscopy assistance. the sections in Epon [37]. The Epon-embedded sections Author details were observed at low power with a light microscope and Department of Pharmacology and Therapeutics, McGill University, Montreal, selected areas were trimmed and re-embedded in Epon 2 Quebec H3G 1Y6, Canada. Alan Edwards Centre for Research on Pain, McGill blocks. The Epon blocks were then trimmed, cut into University, Montreal, Quebec H3A 2B2, Canada. Department of Anatomy and Cell Biology, McGill University, Montreal QuebecH3A 2B2, Canada. ultrathin sections (60 nm) on a Reichert-Jung ultrami- Department of Pharmacology and Therapeutics, McGill University, 3655 crotome (Nussloch, Germany) using a diamond knife Promenade Sir-William-Osler, Montreal, Quebec H3G 1Y6, Canada. and placed onto formvar-coated one-slot grids. Finally, Received: 4 April 2012 Accepted: 6 September 2012 sections were counterstained with uranyl acetate and Published: 10 September 2012 lead citrate and examined using a Philips/FEI CM120 electron microscope equipped with a digital camera. References 1. Nagy JI, Hunt SP: The termination of primary afferents within the rat Image acquisition and quantification dorsal horn: evidence for rearrangement following capsaicin treatment. J Comp Neurol 1983, 218:145–158. Sections were examined using a Zeiss LSM 510 confocal 2. 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Yu XH, Ribeiro-da-Silva A, De Koninck Y: Morphology and neurokinin 1 and take full advantage of: receptor expression of spinothalamic lamina I neurons in the rat spinal cord. J Comp Neurol 2005, 491:56–68. • Convenient online submission 22. Ma W, Ribeiro-Da-Silva A, De Koninck Y, Radhakrishnan V, Henry JL, Cuello • Thorough peer review AC: Quantitative analysis of substance P-immunoreactive boutons on physiologically characterized dorsal horn neurons in the cat lumbar • No space constraints or color figure charges spinal cord. J Comp Neurol 1996, 376:45–64. • Immediate publication on acceptance 23. Charlton CG, Helke CJ: Autoradiographic localization and characterization of spinal cord substance P binding sites: high densities in sensory, autonomic, • Inclusion in PubMed, CAS, Scopus and Google Scholar phrenic, and Onuf's motor nuclei. JNeurosci 1985, 5:1653–1661. • Research which is freely available for redistribution 24. Ribeiro-da-Silva A, De Koninck Y: Morphological and neurochemical organization of the spinal dorsal horn.In Pain. Edited by Bushnell MC, Submit your manuscript at Basbaum A. New York: Academic Press; 2008:279–310. www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Molecular Pain Springer Journals

Non-peptidergic primary afferents are presynaptic to neurokinin-1 receptor immunoreactive lamina I projection neurons in rat spinal cord

Saeed, Abeer; Ribeiro-da-Silva, Alfredo
Molecular Pain , Volume 8 (1) – Sep 10, 2012
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Springer Journals
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Copyright © 2012 by Saeed and Ribeiro-da-Silva; licensee BioMed Central Ltd.
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Medicine & Public Health; Pain Medicine; Molecular Medicine; Neurobiology; Neurosciences; Neurology
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1744-8069
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1744-8069
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10.1186/1744-8069-8-64
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22963197
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Abstract

Background: Pain-related (nociceptive) information is carried from the periphery to the dorsal horn of the spinal cord mostly by two populations of small diameter primary afferents, the peptidergic and the non-peptidergic. The peptidergic population expresses neuropeptides, such as substance P and calcitonin gene-related peptide, while the non-peptidergic fibers are devoid of neuropeptides, express the purinergic receptor P2X3, and bind the isolectin B4 (IB4). Although it has been known for some time that in rat the peptidergic afferents terminate mostly in lamina I and outer lamina II and non-peptidergic afferents in inner lamina II, the extent of the termination of the latter population in lamina I was never investigated as it was considered as very minor. Because our preliminary evidence suggested otherwise, we decided to re-examine the termination of non-peptidergic afferents in lamina I, in particular with regards to their innervation of projection neurons expressing substance P receptors (NK-1r). We used retrograde labeling of neurons from the parabrachial nucleus combined with lectin IB4 binding and immunocytochemistry. Samples were examined by confocal and electron microscopy. Results: By confocal microscopy, we studied the termination of non-peptidergic afferents in lamina I using IB4 binding and P2X3 immunoreactivity as markers, in relation to CGRP immunoreactivy, a marker of peptidergic afferents. The number of IB4 or P2X3-labeled fibers in lamina I was higher than previously thought, although they were less abundant than CGRP-labeled afferents. There were very few fibers double-labeled for CGRP and either P2X3 or IB4. We found a considerable number of IB4-positive fiber varicosities in close apposition to NK-1r-positive lamina I projection neurons, which were distinct from peptidergic varicosities. Furthermore, we confirmed at the ultrastructural level that there were bona fide synapses between P2X3-immunoreactive non-peptidergic boutons and neurokinin-1 receptor-positive lamina I dendrites. Conclusions: These results indicate the presence of direct innervation by non-peptidergic nociceptive afferents of lamina I projection neurons expressing NK-1r. Further investigations are needed to better understand the role of these connections in physiological conditions and chronic pain states. Introduction neuropeptides, such as substance P (SP) and calcitonin Unmyelinated C afferents comprise approximately 70% gene related peptide (CGRP), expresses the high affinity of all primary afferents fibers [1]. The majority of such nerve growth factor receptor, trkA, and terminates fibers transmit nociceptive information from the periph- mainly in lamina I and outer lamina II of the spinal dor- ery to the spinal dorsal horn and have been divided into sal horn [3-5]. The non-peptidergic unmyelinated affer- two main populations, the peptidergic and the non- ents, although devoid of neuropeptides, express the peptidergic [2]. The peptidergic population synthesizes purinergic P2X3 receptor, bind the plant isolectin B4 (IB4), express receptors for glial cell-derived neuro- * Correspondence: alfredo.ribeirodasilva@mcgill.ca trophic factor (GDNF) and terminate mainly in inner Department of Pharmacology and Therapeutics, McGill University, Montreal, lamina II of the spinal dorsal horn [6-8]. These two Quebec H3G 1Y6, Canada Alan Edwards Centre for Research on Pain, McGill University, Montreal, populations can also be differentiated based on the ultra- Quebec H3A 2B2, Canada structural properties of their central terminals in the Full list of author information is available at the end of the article © 2012 Saeed and Ribeiro-da-Silva; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Saeed and Ribeiro-da-Silva Molecular Pain 2012, 8:64 Page 2 of 10 http://www.molecularpain.com/content/8/1/64 spinal dorsal horn.; The peptidergic terminate mostly as and P2X3-IR fibers at low magnification, it was apparent simple axo-dendritic boutons, containing several large that the band of intense IB4+ labeling extended more granular vesicles (LGV), and occasionally as central ventrally than the P2X3 band (Figure 1). Overall, some terminals of type Ib glomeruli (CIb), which are rich in IB4+ and P2X3-IR terminals co-localized CGRP immu- LGV [9,10]. In contrast, the non-peptidergic afferents noreactivity (in yellow in Figures 1 and 3), although the terminate most often as the central bouton of type Ia majority did not. A surprising observation was that the glomeruli, which are poor in LGV, have a very dense number of boutons in lamina I that were IB4+ or P2X3-IR matrix and are often postsynaptic to GABA-positive and did not co-localize CGRP immunoreactivity was con- neurons at axo-axonic or dendroaxonic synapses [10]. siderable, and much higher than what could be expected Regarding peptidergic afferents, their dorsal horn based on the literature. Some IB4+ and P2X3-IR boutons termination has been investigated in particular in rela- which did not co-localize CGRP immunoreactivity could tion to neurons which express the SP receptor, the be observed in transverse sections (Figure 1 C-D; arrow- neurokinin-1 receptor (NK-1r) [11-13]. In particular, it heads). However, these boutons were particularly apparent has been shown at the confocal and electron micro- in parasagittal (Figure 2) and horizontal sections (Figure 3). scopic levels that SP-immunoreactive (IR) terminals in- In horizontal sections, we could be absolutely certain that nervate preferentially neurons which display NK-1r the boutons from non-peptidergic afferents were located immunoreactivity [11,13]. In lamina I, an abundant in- in lamina I since we used serial sections, and the confocal nervation by SP-IR primary afferents of projection neu- images were obtained with a very small pinhole corre- rons, which are immunoreactive for the NK-1r and are sponding to an optical slice of ~0.5 μm adjacent to the activated by noxious stimuli, has been dected [13]. Un- white matter. These sections revealed a considerable in- fortunately, our knowledge concerning the central termi- nervation of lamina I by boutons immunoreactive for nations of the non-peptidergic C fiber population is P2X3, although they were clearly less abundant than the limited. In rat, as mentioned above, they have been CGRP-IR (Figure 3A). The comparison with optical slices shown to terminate mostly in inner lamina II as central from inner lamina II allowed us to assess how much more terminals of type Ia glomeruli. However, besides the fact abundant P2X3-IR boutons were in that layer (Figures 1D, that they are postsynaptic to GABAergic interneurons, 2, and 3B), confirming that inner lamina II is the main ter- their synaptic connections are not well known. However, mination site for non-peptidergic afferents. studies in rat utilizing whole-cell recordings suggest that these non-peptidergic afferents form indirect connec- Innervation of NK-1r-IR lamina I projection neurons by tions with lamina I projection neurons through inter- non-peptidergic afferents neurons in lamina II [14,15], while other studies, using To study the innervation of NK-1r-IR lamina I projec- transgenic mice, proposed that the non-peptidergic tion neurons by non-peptidergic afferents, we labeled afferents synapse onto inner lamina II interneurons neurons retrogradely by means of a stereotaxic cholera which in turn would connect with deep lamina V projec- toxin subunit b (CTb) injection in the lateral parabra- tion neurons [16]. However, it was never investigated chial nucleus. The injection site covered most of the whether lamina I projection neurons receive direct parabrachial complex, including the lateral parabrachial synapses from non-peptidergic afferents, likely because nucleus, an observation that is comparable to distribu- it was assumed that the termination of such afferents in tions previously reported by us [17,18]. The majority of lamina I was negligible. In this study, we examined the retrogradely labeled lamina I neurons were found on quantitatively the innervation of NK-1r-positive lamina I the contralateral side, although a few were also present projection neurons by non-peptidergic unmyelinated on the side ipsilateral to the CTb injection. CTb labeling primary afferents, using confocal microscopy, and also of spinoparabrachial lamina I neurons included the cell provided ultrastructural evidence of direct synapses of body and primary dendrites (Figures 4 and 5). these afferents on NK-1r-IR lamina I neurons. We also performed a labeling of these cells with anti- bodies against the SP, receptor, the NK-1r. Indeed, it has Results been previously described [17,18] that lamina I, fusiform Termination of non-peptidergic afferents in lamina I and multipolar spinoparabrachial neurons, identified by We examined the termination of non-peptidergic affer- retrograde tracing of CTb, often express NK-1r immu- ents using either IB4 binding or P2X3 immunoreactivity. noreactivity, while lamina I pyramidal neurons seldom ex- We used sections cut in the three planes: transverse, press NK-1r immunoreactivity. These neuronal populations parasagittal and horizontal (Figures 1, 2, and 3), and fo- can be identified based on their dendritic arborization and cused on the region equidistant from the lateral and cell body shape when viewed in the horizontal plane. Multi- medial limits of the dorsal horn. In transverse sections, polar neurons have four or more dendrites arising from an when comparing the distribution of IB4-binding (IB4+) irregularly-shaped soma (Figure 4A). Fusiform neurons Saeed and Ribeiro-da-Silva Molecular Pain 2012, 8:64 Page 3 of 10 http://www.molecularpain.com/content/8/1/64 Figure 1 CGRP, IB4 and P2X3 staining in transverse spinal cord sections. A and B show low magnification confocal images of CGRP-IR and IB4 positive (A) or P2X3-IR (B) fibers. C and D represent high magnification confocal images from the middle third of the latero-medial extent of the superficial dorsal horn. In C, note that there is limited co-localization of IB4 and CGRP (in yellow). Arrowheads show axonal varicosities (boutons) from non-peptidergic fibers in lamina I, which do not co-localize CGRP immunoreactivity. The framed regions in A and B indicate the approximate regions from where C and D, respectively, were obtained (the latter originate from other sections). CGRP (in green); IB4 (in red); P2X3 (in red). Scale bar (A, B) = 200 μm; scale bar (C, D)=20 μm. possess two dendrites, each arising from one end of a was performed for the peptidergic innervation on lam- spindle-shaped soma (Figure 4B and 5). Lastly, pyramidal ina I neurons, which has been previously investigated neurons have a triangular soma with one primary dendrite [11,22,23]. arising from each of the three tips of the soma (Figure 4C). In this study, we observed IB4+ non-peptidergic varicosities Ultrastructural demonstration of synapses between non- in apparent direct apposition to all three populations of peptidergic primary afferents and lamina I neurons lamina I projection neurons, multipolar (Figure 4A), Because the resolution of confocal microscopy is insuffi- fusiform (Figure 4B) and pyramidal (Figure 4C). These bou- cient to demonstrate the presence of bona fide appositions tons were distinct from CGRP-positive peptidergic bou- tons, and from boutons of the small proportion of primary afferents which are simultaneously CGRP-positive and bind IB4, as described previously [19,20] (Figure 5). Quantification of non-peptidergic varicosities apposed to NK-1r-positive lamina I projection neurons As the objective of this study was to investigate the non-peptidergic innervation of NK-1r-IR projection neurons, our quantitative analysis focused on the in- nervation of multipolar and fusiform neurons. Indeed, pyramidal neurons are seldom immunoreactive for the substance P receptor [21]. Our quantitative analysis Figure 2 Confocal images at high magnification obtained from revealed a substantial innervation of NK-1r-IR multi- parasagittal spinal cord sections showing CGRP-IR (green) and polar and fusiform populations of lamina I projection P2X3-IR (red) varicosities in the superficial dorsal horn. P2X3-IR varicosities were present in considerable number in lamina I (LI) but neurons by non-peptidergic afferents (Figure 6). Since were more highly concentrated in inner lamina II (LIIi). Scale the main objective of this paper was to give an account bar = 20 μm. of the non-peptidergic innervation, no quantification Saeed and Ribeiro-da-Silva Molecular Pain 2012, 8:64 Page 4 of 10 http://www.molecularpain.com/content/8/1/64 Figure 3 Confocal images at high power obtained from horizontal spinal cord sections. In a confocal optical section from lamina I adjacent to the white matter (A), note the relatively abundant P2X3-IR fibers with varicosities (boutons). CGRP-IR fibers and boutons were considerably more abundant in this lamina. In a confocal optical section from inner lamina II (B), note the very high density of P2X3-IR fibers and varicosities, higher than that of CGRP-IR fibers in lamina I. Note that most varicosities display either P2X3 or CGRP immunoreactivity, although some co-localization is observed (yellow). Scale bar (A, B)=20 μm. of two structures and synapses between them, we per- binding the lectin IB4. We also provide evidence at the formed an ultrastructural study using P2X3 immunoreac- confocal microscopy level that IB4+ boutons are in ap- tivity. We had to omit glutaraldehyde from the fixative position to lamina I projection neurons immunoreactive because it completely blocked the immunostaining, even for the NK-1r. Lastly, we provide ultrastructural evi- in very low concentration, which affected the quality of dence of synapses between P2X3-IR boutons and lamina the ultrastrucure. In lamina I, all P2X3-IR boutons estab- I dendritic profiles immunoreactive for the NK-1r. lished simple axo-dendritic or axo-somatic synapses (Figure 7A). In inner lamina II, most P2X3-IR boutons Technical considerations were involved in complex synaptic arrangements, as pre- Because most of this study was carried out using con- dicted from the literature [24], forming the central elem- focal microscopy, we could not fully ensure that boutons ent of type Ia glomeruli (Figure 7B). from non-peptidergic afferents were presynaptic to neu- We used a double labeling for P2X3 and NK-1r to rons in lamina I. For this reason, we carried out an ultra- demonstrate direct appositions and synapses between structural study using antibodies against P2X3 and P2X3 immunoreactive boutons and NK-1r dendritic pro- provided direct evidence of synapses between P2X3-IR files (Figure 8). axonal boutons and dendrites and cell bodies in lamina I. We also performed at the ultrastructural level a double Discussion labelling for P2X3 and the NK-1r that demonstrated that In this study, we demonstrate the presence in lamina I some of the structures innervated by these boutons of a significant number of boutons originating from expressed the NK-1r, in agreement with what was non-peptidergic afferents immunoreactive for P2X3 or assumed from the confocal data. We could observe Figure 4 Confocal triple-labeling image showing the innervation of lamina I spinoparabrachial (A) multipolar, (B) fusiform and (C) pyramidal neurons by IB4+ varicosities (indicated by arrowhead). IB4 (red); NK-1r (green); spinoparabrachial neurons labeled with CTb (white). Scale bar (A-C)= 20 μm. Saeed and Ribeiro-da-Silva Molecular Pain 2012, 8:64 Page 5 of 10 http://www.molecularpain.com/content/8/1/64 Figure 5 Example of a quadruple labeling observed at the confocal level using a multi-track approach. In this image the following signals were simultaneously detected: CGRP (green); IB4 binding (red); CTb transported retrogradely from the parabrachial nucleus (blue); NK-1r (white). A fusiform neuron, double labeled with CTb and NK-1r, is innervated by CGRP-IR boutons (arrowhead) and IB4+ (arrow) boutons, which represent distinct populations. However, a small population of varicosities co-labeled for CGRP and IB4 (curved arrow) was detected. Scale bar (A-E)= 20 μm. unequivocal evidence of synaptic contacts, in spite of the fact that we had to use a fixative without glutaraldehyde and pre-treatment of the sections with a detergent for a short period to obtain P2X3 staining at the ultrastruc- tural level. We were unable to use IB4 binding for elec- tron microscopy because of the bad penetration of the lectin in the tissue. Conversely, we were unable to use P2X3 immunoreactivity for the study of the innervation of the neurons at the confocal level because of antibody incompatibilities when performing the required triple la- beling. It should be pointed out that Naim et al. [25] Figure 7 Electron micrograph showing: in A, a P2X3-IR bouton Figure 6 Quantification of IB4-positive boutons in direct (arrow) forming an axo-somatic contact onto a lamina I cell apposition to the cell body and primary dendrites of NK-1r-IR body (CB), which is also postsynaptic to unlabeled axonal lamina I spinoparabrachial neurons. Results are expressed in boutons (V); in B, a P2X3-IR central bouton of a type Ia number of appositions from boutons per 100 μ of membrane glomerulus (CI P2X3+) presynaptic to 3 lamina I dendrites (D) length of NK-1r-IR cell. N = 6 animals. and apposed to an axonal bouton (V2). Scale bar = 0.5 μm. Saeed and Ribeiro-da-Silva Molecular Pain 2012, 8:64 Page 6 of 10 http://www.molecularpain.com/content/8/1/64 Figure 8 Electron micrographs showing axo-dendritic contacts between P2X3-positive boutons (arrows) and NK-1r-positive dendrites (D) (A and B). Note the silver-intensified gold particles along the plasma membrane of the dendrites, representing NK-1r-IR sites. B, unlabeled axonal bouton. Scale bar = 0.5 μm. examined at the ultrastructural level ultrathin sections NK-1r) because of photobleaching during the perfor- recut from thicker sections previously examined by con- mace of the Z-stacks. However, we examined enough focal microscopy. Naim et al.'s study showed that immu- cells using the quadruple labeling to ensure that the pro- noreactive varicosities seen in close apposition to the portion of axonal boutons colocalizing IB4 binding and membrane of NK-1r-IR under the confocal microscope CGRP immunoreactivity in apposition to lamina I neu- actually formed synapses when viewed under the elec- rons was low. An example of the quadruple labeling is tron microscope. Therefore, we are confident that the given on Figure 4. IB4+ varicosities in apposition to NK-1r-IR lamina I pro- jection neurons should establish synapses in a high pro- Innervation of lamina I projection neurons by non- portion of cases. peptidergic C fibers It is known that in rat there is a small proportion of The main objective of this study was to investigate the peptidergic sensory fibers in lamina I and II that coloca- issue of the innervation of NK-1r-IR lamina I projection lize CGRP and somatostatin, do not respond to NGF neurons by non-peptidergic C fibers because in our and bind IB4 [19,20]. This obliged us to investigate the knowledge a systematic study combining labeling of co-localization of IB4 binding and P2X3 immunoreactiv- these sensory fibers with labeling of these lamina I neu- ity, our markers of non-peptidergic nociceptive C fibers, rons had never been done. This is an important issue for with CGRP immunoreactivity. Indeed, as predicted, we the reasons given below. Previous work by Lu and Perl found a limited level of co-localization of either marker [14], using simultaneous whole-cell recordings from of non-peptidergic afferents with CGRP immunoreactiv- pairs of neurons, provided some evidence that input ity. However, most varicosities that we observed in lam- from primary afferent C fibers terminating in inner lam- ina I which were IB4+ or P2X3-IR did not co-localize ina II may reach lamina I projection neurons via inter- CGRP immunoreactivity, what reassured us regarding posed interneurons. Since the great majority of C fibers the validity of our findings regarding the overall innerv- innervating inner lamina II are non-peptidergic, it has ation of lamina I by non-peptidergic afferents. But it was been later suggested that the above pathway may be im- still possible that a subpopulation of these afferents in- portant for pain-related information conveyed by non- nervating NK-1r-IR lamina I projection neurons would peptidergic C fibers to reach lamina I projection neurons represent exactly this minor subpopulation that is simul- which then project to supraspinal levels. If this is true, taneously CGRP-IR and IB4+. Therefore, we carried out lamina I nociceptive projection neurons would receive a quadruple labeling in which we detected CTb (the direct input from peptidergic afferents and polysynaptic retrograde label), NK-1r, CGRP and IB4 binding. Unfor- input from the non-peptidergic afferents [24]. tunately, because of antibody incompatibilities, we could Alternatively, it has been proposed that the non- not perform P2X3 staining and had to use the lectin IB4 peptidergic afferents are part of a distinct and parallel conjugated to a fluorochrome. Although our confocal pathway from that of their peptidergic counterpart. This microscope can identify 4 separate signals reliably using view obtained some support from a study by Braz et al. the multi-track approach, it was technically impossible [16] in a transgenic mouse, which demonstrated that the to perform a quantitative analysis of the innervation of termination of non-peptidergic afferents on lamina II lamina I cells using more than 3 signals (IB4, CTb and would be on excitatory interneurons, which in turn Saeed and Ribeiro-da-Silva Molecular Pain 2012, 8:64 Page 7 of 10 http://www.molecularpain.com/content/8/1/64 would synapse on deep lamina V projection neurons to give the reader an idea of the order of magnitude of with ascending connections to the amygdala, hypothal- the two types of innervation. Indeed, Todd at al. [13] amus and bed nucleus of stria terminalis. As they found have detected a density of peptidergic innervation of minimal connection with lamina I neurons expressing 16.2 contacts/100 μm of NK-1r-IR lamina I projection the NK-1r, the above group proposed the involvement of neuron membrane. Therefore, the non-peptidergic in- the non-peptidergic afferents in the affective/emotional nervation is of lamina I NK-1R-IR neurons is substantial, component of pain. Alternatively, other studies also in although less abundant than the peptidergic. Although the mouse, proposed that distinct subsets of primary the focus of this study was mainly on NK-1r-IR projec- sensory afferents selectively mediate responses to differ- tion neurons in lamina I, there is also a population of ent stimulus modalities. These studies provided some NK-1r-positive neurons in deep laminae III-V, which evidence suggesting that, in the mouse, non-peptidergic also project supraspinally and send processes to laminae afferents play a particular role in transmitting mechan- I-II [25]. These neurons have been shown to receive on ical pain, as opposed to the peptidergic which would be average 18.8 contacts per 100 μm of dendrite length involved in conveying thermal pain [26,27]. This idea of from substance P-IR boutons in these superficial layers divergent pain pathways can be criticized since all the [25]. Another study has reported that these deeper neu- studies supporting it were performed in mice, which rons receive contacts from IB4-binding non-peptidergic have been shown to demonstrate an explicit dichotomy afferents in laminae I-II, although in much lower density between the C fiber populations that does not apply to (just 2 appositions per 100 μm of length of NK-1r-IR rats and higher order species such as primates. In par- dendrite membrane) than for the peptidergic afferents ticular, the vanilloid TRPV1 receptors are localized only [34]. These values of non-peptidergic innervation of in the peptidergic fibers in mice [28,29] whereas they are laminae III/IV neurons are considerably lower than present in both peptidergic and non-peptidergic affer- those we found in the current study. ents in the rat and higher species [30,31]. Our data provides evidence that non-peptidergic pri- Conclusion mary afferents establish direct connections with NK-1r- These results expand our previous knowledge concern- IR lamina I projections neurons, in addition to the pos- ing the termination in dorsal horn of non-pepdergic pri- sible indirect connections via an interposed interneuron mary afferents by providing evidence that they terminate suggested in previous studies [14]. Although the non- directly on lamina I neurons expressing the NK-1r. peptidergic primary afferent termination in lamina I may seem as a minor contribution to the lamina I synaptic Materials & methods circuitry when compared to their termination in inner The guidelines of the Canadian Council on Animal Care lamina II, they may prove to have a significant role in for the care and use of experimental animals and of the the transmission of nociceptive signals. Indeed, the International Association for the Study of Pain were fol- terminals of these fibers in inner lamina II represent the lowed in all the experiments. Furthermore, all studies central bouton of type Ia glomeruli, and are involved in were previously approved by the McGill University complex modulatory circuits involving GABAergic pre- Faculty of Medicine Animal Care Committee. synaptic inhibition [24]. A direct termination of non- A total of 26 male Sprague Dawley rats (Charles River, peptidergic afferents on lamina I cells would bypass such Quebec, Canada) weighing 225-250 g were used for the modulation. Since non-peptidergic afferents have a more experiments. The number of animals used and their suf- extensive distribution in the epidermis than peptidergic fering were kept to the minimum necessary for the con- afferents [32], show larger and more sustained responses duction of the study. Animals were exposed to 12 hr to capsaicin than peptidergic afferents [33] and signal light/dark cycle and given food and water ad libitum. mainly via glutamate without the presence of neuropep- The cages were fitted with soft bedding and a plastic tide co-transmitters, the activation of NK-1r-IR lamina I tube for an enriched environment and the animals were projection neurons by these non-peptidergic afferents housed in cages of four. would result in a different signal transmitted than when activated by the peptidergic afferents. Animal preparation Our results revealed that, on average, there were 6.7 Injection of tracers appositions from non-peptidergic boutons per 100 μm For the immunohistochemistry experiments requiring of length of NK-1r-IR lamina I projection neuron mem- retrograde tracing, six animals were anesthetized using brane. How does this compare to the peptidergic innerv- 5% isoflurane with oxygen and placed in a stereotaxic ation of these cells as shown in other studies? Although apparatus (David Kopf Instruments, Tujunga, CA) and such comparison is not fully legitimate because of meth- stabilized with non-perforating ear bars. The coordinates odological differences with our study, we report it here for the parabrachial nucleus (rostral/caudal: -9.12; Saeed and Ribeiro-da-Silva Molecular Pain 2012, 8:64 Page 8 of 10 http://www.molecularpain.com/content/8/1/64 medial/lateral: -2.1; dorsal/ventral: -6.3) were calculated different species), or IB4, in PBS + T containing 5% NDS. from the Paxinos & Watson Rat Brain Atlas [35] with Next, the sections were washed in PBS + T and then incu- Bregma as the reference point. A small hole was bored bated in species-specific secondary antibodies that were through the skull at the target point, exposing the dura raised in donkey and conjugated to either AlexaFluor 488, mater and a glass micropipette (Wiretrol II, Drummond AlexaFluor 405, Rhodamine RedX or biotin. The sections Scientific Company, Broomall, PA) was lowered to the were incubated in 3 different cocktails: #1) rabbit anti- stereotaxic position of the parabrachial nucleus. Two μl CGRP at a 1:200 dilution (Sigma, St Louis, MO) and lectin of 1.0% solution of CTb (List, Campbell, CA) was slowly IB4 conjugated to AlexaFluor 568 at a 1:200 dilution (Mo- injected into the parabrachial nucleus over a period of lecular Probes); #2) rabbit anti-CGRP and guinea pig anti- 20 minutes, followed by a waiting period of 10 minute P2X3 at a 1:25,000 dilution (Neuromics, Edina, MN); #3) before the micropipette was retracted from its position goat anti-CTb at a 1:5000 dilution (List Biological), rabbit to minimize leakage of the tracer. CTb was injected anti-NK-1r at a 1:10000 dilution (Sigma, St Louis, MO), seven days prior to sacrificing the animals. guinea pig anti-CGRP at a 1:8000 dilution (Peninsula, San Carlos, CA) and lectin IB4 conjugated to AlexaFluor 647 Animal perfusion at a 1:200 dilution (Molecular Probes). All the sections For immunohistochemistry, eighteen animals were were washed with PBS + T and then (for #1) incubated for deeply anesthetized with 0.3 ml/100 g of body weight of 2 hours at room temperature with donkey anti-rabbit Equithesin (6.5 mg chloral hydrate and 3 mg sodium AlexaFluor 488; (for #2) incubated for 90 minutes in a bio- pentobarbital i.p.). They were then perfused through the tin conjugated donkey anti-guinea pig IgG (Jackson left cardiac ventricle with perfusion buffer (for compos- Immunoresearch, West Grove, PA, 1:200). Further ampli- ition see [36]) for one minute, followed by 4% parafor- fication of the P2X3 signal was achieved by treating the maldehyde (PFA) in 0.1 M phosphate buffer (PB), pH sections with 1 hour incubation in an avidin-biotin (A + 7.4, for 30 minutes. The spinal cord segments L4-L5, as B) complex (Vectastain Elite ABC kit, Vector Laborator- well as the brains from animals injected with the retro- ies) followed by tyramide (Perkin-Elmer, Norwalk, CT, grade tracer, were extracted and post-fixed for 1 or 1:75) for 7 minutes. Sections were then incubated in strep- 2 hours, respectively, in the same fixative. tavidin conjugated to AlexaFluor 568 (Molecular Probes, For examination under the electron microscope, eight Eugene, OR, 1:200) and donkey anti-rabbit AlexaFluor animals were anesthetized as mentioned above, and then 488. For #3, sections wereincubated for 2 hours at room perfused with perfusion buffer for one minute, followed temperature with secondary antibodies: donkey anti-goat by 30 minutes perfusion with a mixture of 4% PFA and Rhodamine Red X, donkey anti-rabbit AlexaFluor 488, 15% picric acid in 0.1 M PB. The spinal cord segments and donkey anti-guinea pig AlexaFluor 405. Finally, all L4-L5 were taken out and post-fixed for one hour in the sections were washed with PBS, mounted on gelatin- same fixative. subbed slides and coverslipped with an anti-fading All specimens were immersed overnight in 30% su- mounting medium (Aqua Polymount; Polysciences, crose in PB at 4°C for cryoprotection. Warrington, PA). Slides were stored at −4°C pending further processing. Tissue processing To evaluate the injection site in the parabrachial nu- Confocal microscopy cleus, brainstem sections were incubated with anti-CTb The injection site at the level of the parabrachial nucleus antibody followed by a biotinylated donkey anti-goat IgG was examined by cutting serial, 100 μm-thick coronal sec- and streptavidin conjugated to AlexaFluor 568. They tions of the relevant brain region. The dorsal aspect of the were then mounted and coverslipped as described above. L4-L5 spinal cord segment was cut into serial, 50 μm- thick horizontal sections (n = 10), 50 μm-thick parasagittal Electron microscopy sections (n = 4) or 50 μm-thick transverse sections (n = 4). Spinal cord specimens from the L4-L5 region were All sections were cut using a freezing sledge microtome freeze-thawed in liquid nitrogen for 30 seconds, then cut (Leica, Richmond Hill, Ontario) and collected as free- into 50 μm-thick transverse sections on a Vibratome floating in phosphate-buffered saline (PBS) with 0.2% (TPI, St. Louis, MO, USA) and collected as free-floating Triton-X 100 (PBS + T). To block unspecific staining, all in PBS. To increase further antibody penetration, sec- spinal cord sections were incubated, for one hour, in 10% tions were incubated for 15 minutes in PBS + T, but all normal donkey serum (NDS) (Jackson, West Grove, PA) further incubations were carried out in PBS without Tri- in PBS + T at room temperature. Subsequently, the sec- ton. Following a short PBS wash, the sections were incu- tions were placed in primary antibodies (or conjugated bated with 50% ethanol for 30 minutes. They were then lectin IB4 - see below) for 48 hours at 4°C. We used a incubated for 30 minutes in 0.5% BSA (bovine serum al- mixture of 2 or 4 primary antibodies (each raised in a bumin) and subsequently incubated for one hour in 10% Saeed and Ribeiro-da-Silva Molecular Pain 2012, 8:64 Page 9 of 10 http://www.molecularpain.com/content/8/1/64 NDS. Sections were incubated for 48 hours, at 4°C, ei- separate detections of AlexaFluor 488, streptavidin con- ther with a primary antibody mixture of guinea pig anti- jugated to AlexaFluor 568 or Rhodamine Red X, IB4- P2X3 at dilution 1:200,000 and rabbit anti-NK-1r at dilu- conjugated to AlexaFluor 647 and/or AlexaFluor 405, re- tion 1:4000, or the guinea-pig anti-P2X3 antibody (at the spectively. Images used for quantification represent ser- same dilution) alone, in 5% NDS and 0.1% BSA. ial optical sections obtained along the z-axis (z-stacks), Sections were then washed several times with PBS and using a 63X plan-apochromatic oil-immersion objective. then incubated for 90 minutes in a biotinylated donkey Furthermore, for an unbiased representation of the anti-guinea pig IgG antibody at dilution of 1:1000 in PBS images taken, all the parameters of laser power, pinhole and processed for tyramide amplification as described size and image detection were kept constant for all sam- above. Afterwards, the sections were incubated, for one ples. The images obtained were converted to TIFF files. hour, in A + B enzyme complex. Labeling, for the P2X3 Our criteria of identification and quantification of lam- antibodies, was revealed using following the incubation of ina I neurons have been described extensively in previ- the section with intensified DAB (3’,3’diaminobenzidine ous publications from our laboratory [18] (see also with 1% cobalt chloride and 1% nickel ammonium sulfate) Results section). To calculate the density of IB4-positive to which 1% hydrogen peroxide was added [37]. The reac- boutons per unit length of neuronal membrane, the tion was stopped by two washes in PBS. Sections pro- length of membrane in the soma and proximal dendrites cessed for P2X3 staining were then osmicated (see below). of NK-1r-IR neurons was measured with the help of an Sections previously incubated in two primary antibodies MCID Elite Image Analysis System (Imaging Research, (anti-P2X3 and anti-NK-1r) were placed for 10 minutes in St.Catharines, ON, Canada) in each optical section and washing buffer (0.8% BSA and 0.5% fish gelatin in PBS; the number of appositions from IB4-positive boutons pH 7.4) and then incubated overnight at 4°C with anti- onto it was counted. This was done on alternate optical rabbit IgG conjugated to gold particles (1.4 nm diameter, sections from the z-stack, to avoid counting the same Nanoprobes, Yaphank, NY) in washing buffer, at dilution boutons twice. of 1:200. The following day, all sections are washed for Competing interests 3 minutes in washing buffer, rinsed for 3 minutes in PBS, The authors declare they have no competing interests. and then immersed for 10 minutes in 1% glutaraldehyde in PBS. The sections were washed with de-ionized water Authors’ contributions for 2 minutes and then incubated for 8 minutes with an AWS designed and performed all experimental protocols described in this HQ silver enhancement reagent following manufacturer's manuscript as well as the writing of the initial draft of the manuscript. ARS provided supervision for data analysis, study direction, image acquisition, instructions (Nanoprobes, Yaphank, NY). The sections manuscript design and revisions. Both authors have read and approved the were then thoroughly rinsed with de-ionized water and final draft of this manuscript. then washed with 0.1 M PB. The sections were then post-fixed in 1% osmium tet- Acknowledgements roxide in PB for 1 hour at room temperature and, subse- Supported by Canadian Institute of Health Research (CIHR) grant MOP-79411 (to A.R.-da-S). The authors would like to thank Manon St-Louis for laboratory quently, dehydrated in ascending concentrations of expertise, Jacynthe Laliberté for confocal microscopy assistance and Johanne ethanol and propylene oxide, followed by flat-embedding Ouellette for electron microscopy assistance. the sections in Epon [37]. The Epon-embedded sections Author details were observed at low power with a light microscope and Department of Pharmacology and Therapeutics, McGill University, Montreal, selected areas were trimmed and re-embedded in Epon 2 Quebec H3G 1Y6, Canada. Alan Edwards Centre for Research on Pain, McGill blocks. The Epon blocks were then trimmed, cut into University, Montreal, Quebec H3A 2B2, Canada. 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Journal

Molecular Pain – Springer Journals

Published: Sep 10, 2012

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