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The Journal of Neuroscience, January 28, 2004, 24(4):836-842; doi:10.1523/JNEUROSCI.4221-03.2004
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Cellular/Molecular
Morphological and Physiological Features of a Set of Spinal Substantia Gelatinosa Neurons Defined by Green Fluorescent Protein Expression
Adam W. Hantman,1 Anthony N. van den Pol,2 andEdward R. Perl1 1Department of Cell and Molecular Physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, and 2Department of Neurosurgery, Yale University School of Medicine, New Haven, Connecticut 06520
Abstract
Top
Abstract
Introduction
The spinal substantia gelatinosa (SG) is known to be involved in the manipulation of nociceptive and thermal primary afferent input; however, the interrelationships of its neuronal components are poorly understood. As a step toward expanding understanding, we took a relatively unique approach by concentrating on a set of SG neurons selectively labeled by green fluorescent protein (GFP) in a transgenic mouse. These GFP-expressing SG neurons prove to have homogenous morphological and electrophysiological properties, are systematically spaced in the SG, contain GABA, receive C-fiber primary afferent input, and upregulate c-Fos protein in response to noxious stimuli. Together, the properties established for these GFP-labeled neurons are consistent with a modular SG organization in which afferent activity related to nociception or other C-fiber signaling are subject to integration/modulation by repeating, similar circuits of neurons.
Key words: C-fiber; dorsal horn; GABA; nociception; spinal; substantia gelatinosa; pain
Introduction
Establishing the functional architecture of the spinal substantia gelatinosa (SG) (lamina II) is challenging. Developmental mapping has shown that the neuronal composition and synaptic arrangements of the SG originate from multiple lineages of neuronal progenitor cells (Lee and Jessell, 1999; Müller et al., 2002
Despite limitations in our knowledge of SG architecture, there is compelling evidence that the SG is involved in mechanisms related to pain, itch, and temperature senses. Thinly myelinated (A
) and unmyelinated (C) primary afferent fibers concentrate their central projections in the SG (Light and Perl, 1979a
Progress in knowledge of the relationship between SG intrinsic neurons and somatosensory mechanisms would be advanced by systematic characterizations of particular SG neuronal categories and their relationships to dorsal root input. The absence of obvious anatomical patterning in the arrangement of SG neuronal subtypes makes it difficult to collect electrophysiological and morphological observations on a given category of neuron. Immunohistochemical studies frequently do not provide adequate information on either the classes of neurons expressing particular antigens or the cellular function of the antigens. To circumvent these difficulties, we targeted a specific population of SG neurons for study. The transgenic mouse [prion promotor (Prp)–GFP] used for this set of experiments expresses green fluorescent protein (GFP) in a limited set of SG cells under the control of the prion promoter (van den Pol et al., 2002
Materials and Methods
Animals
All of the procedures were approved by the Institutional Animal Care and Use Committee of the University of North Carolina at Chapel Hill and Yale University. The adult White Swiss-Webster transgenic mice used in this study were designed to express enhanced, red-shifted, mammalian codon-corrected GFP under control of the mouse prion promoter (van den Pol et al., 2002Immunocytochemistry
Spinal tissue was fixed either by immersion (slices) or perfusion with 4% paraformaldehyde. For use of the Somogyi and Sigma (St. Louis, MO) GABA antibodies, the perfusion fixative was 2.5% glutaraldehyde and 1% paraformaldehyde. After a minimum of 8 hr of immersion fixation or 2 hr of postfixation for perfused tissue, the tissue was cryoprotected using a 30% sucrose phosphate buffer solution. Cryostat-cut spinal cord sections were incubated with primary antibodies selective for substance P (1:1000; DiaSorin, Stillwater, MN), neuronal nuclei (NeuN) (1:100; Chemicon, Temecula, CA), neuron-specific enolase (1:100; Chemicon), GABA (1:250; Chemicon), GABA (1:3000; Somogyi; gift from P. Somogyi, University of Oxford, Medical Research Council-Anatomical Neuropharmacology Unit, Oxford, UK), GABA (1:6000; Sigma), calcitonin gene-related peptide (1:1000; Peninsula, San Carlos, CA), and c-Fos (1: 200; Genosys, The Woodlands, TX). Fluorophore-conjugated secondary antibodies raised against the appropriate species were then incubated with the tissue. Isolectin B4 (IB4) binding was detected by sequential incubation of tissue sections with biotinylated Griffonia simplicifolia-IB4 (IB4; Vector Laboratories, Burlingame, CA) and streptavidin-Cy5 (Zymed, San Francisco, CA). For GABA preabsorption controls, equal volumes of GABA–BSA conjugate (2.5 mg/ml) and the GABA antibody were combined before the primary incubation step and applied to the tissue.Biocytin immunocytochemistry. After a minimum of 20 min in the whole-cell, tight-seal patch-clamp configuration, the biocytin-filled electrodes were withdrawn from the targeted neuron, and the slices were immersed in 4% paraformaldehyde. After 24–48 hr of fixation, the tissue was cryoprotected by immersion in a 30% sucrose phosphate buffer. To detect the biocytin, the tissue was washed and incubated with Texas Red streptavidin (1:800; Vector Laboratories).
Imaging. The fluorescent signals of the spinal cord sections were collected as z-series images using a confocal microscope [Leica (Nussloch, Germany) DM IRBE, DM RXE; Leica TCSNT]. Signal bleed-through was always checked and, when present, was eliminated by sequential capture of the fluorescent signals. Photoshop 6.0 (Adobe Systems, Mountain View, CA), Leica confocal software, Slicer 1.1 (Fortner Research, Sterling, VA), and Volocity 2.0 (Improvision, Lexington, MA) were used to analyze the confocal images. Laminar boundaries of the SG were determined from cytoarchitecture and, when possible, immunohistochemical markers. Positive immunoreactivity was defined as fluorescent signals with intensities at least 20% greater than background. Colocalization was judged on the basis of three-dimensional alignment of positive immunoreactivity of two fluorescent signals.
Electrophysiology
Electrophysiological experiments were performed as described in Grudt and Perl (20026 ml/min: pharmacological compounds were added to ACSF. A fluorescent upright microscope (Leica DMLFS) and a charge-coupled device camera system (Cohu, San Diego, CA) were used to identify fluorescence during electrophysiological recording. Infrared imagery was used to direct a recording pipette, backfilled with 0.5% biocytin (Sigma), to SG neurons. SG–GFP neurons were targeted by directing the recording pipette to an infrared image of an SG neuron that had a corresponding fluorescent signal. The internal pipette solution contained (in mM): 130 potassium gluconate, 5 NaCl, 1 CaCl2, 1 MgCl2, 10 HEPES, and 4 Na2ATP. After acquiring gigaohm seals, tight-seal, whole-cell recordings were obtained. An Axopatch 1D (Axon Instruments, Foster City, CA) and pClamp6 software (Axon Instruments) were used to acquire data. Dorsal root volleys were initiated by pulses from a constant-voltage pulse generator applied through the suction electrode. Successive dorsal root-evoked responses with latencies showing a coefficient of variation <2% were considered to be monosynaptic (Li and Perl, 1994
, >5 m/sec; A
c-Fos experiments
Similar volumes of 5% formalin (n = 6) or 0.9% NaCl (n = 4) were injected subcutaneously into the dorsal surface of the right hindpaw of 4- to 6-week-old awake male mice. Behavior of the animals was monitored for 2 hr after the injections. At 2 hr postinjection, the animals were decapitated, and the spinal cords were removed, as described for the electrophysiological experiments, and immersed in 4% paraformaldehyde. Confocal microscopy was used to assess colocalization as described above. The number of SG–GFP neurons that expressed c-Fos protein was counted for the ispilateral and contralateral sides of 20 transverse spinal cord sections (30 µm) from two animals for each condition: no injection, a saline injection, and a formalin injection. Chance differences in values were evaluated by statistical tests mentioned in Results (p < 0.05 was taken as significant).
Results
Distribution of GFP-expressing spinal cord cells
On cytoarchitectural grounds, the majority of GFP-expressing cells were located in the outer part of Rexed's (1952
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Figure 1. GFP-expressing cells in the spinal cord are predominately located in the SG. a, SG–GFP cells (green) are concentrated in lamina IIo and are evenly distributed in the mediallateral axis of the spinal cord. b, The majority of SG–GFP neurons are in SG regions delineated by IB4 binding (blue signal). c, SG–GFP cells are neurons. Red represents immunoreactivity to a NeuN antibody. Boundary indicated between lamina IIi and IIo (line) is approximate. Arrows demark SG–GFP somata. Dorsal is shown at top. d, Higher magnification view of SG–GFP cells in a horizontal section showing their similar morphologies. Medial is shown at the top, and rostral is shown to the left. Scale bars, 25 µm.
A rare GFP cell was noted ventral to the SG in the deep dorsal horn (supplemental Fig. 1; available at http://www.med.unc.edu/physiology/fac_hantman.htm). In addition, ependymal cells lining the central canal throughout the length of the spinal cord expressed GFP.Neuronal identity of the GFP-expressing spinal cord cells
The neuronal nature of the GFP-expressing cells was evaluated by examining for the NeuN protein (Mullen et al., 1992Classification of mouse SG neurons
The distribution of GFP-labeled neurons and the morphological similarity of the individual GFP cells (Fig. 1d) raised the possibility that the SG–GFP neurons represented a single subtype. To match the SG–GFP neurons to an existing SG neuronal classification scheme, we examined the mouse SG for analogy to that of other species. To this end, detailed morphological and electrophysiological features of individual SG neurons were determined by performing tight-seal, whole-cell electrophysiological studies with biocytin-filled recording electrodes. Histochemical detection of intracellular biocytin was used to visualize neuronal morphology. Mouse SG neurons not labeled by GFP (n = 41) were morphologically and electrophysiologically diverse, representing examples (Fig. 2) of categories equivalent to those described for hamster SG neurons (Grudt and Perl, 2002
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Figure 2. Examples of the morphology of neurons recorded from the center of the SG that did not express GFP. Maximum projection confocal images of biocytin-labeled neurons. a, Vertical cell in a sagittal spinal section (represented by icon at bottom right). b, Medial-lateral cell in a transverse spinal section. c, Radial cell in a transverse spinal section (represented by icon at bottom right). d, Central cell in a sagittal spinal section. Insets are merged images of GFP signal (green) and biocytin signal (red); absence of overlap indicates that the cell did not contain GFP. Dorsal is shown at the top. Scale bar, 25 µm.
Morphological profile of individual SG–GFP neurons
The somata of SG–GFP neurons labeled by biocytin (n = 21) were oval in shape with the longest dimension (13 ± 3 µm) in the rostrocaudal axis (Fig. 3). Rostrocaudal dendrites averaged 194 ± 91 µm in sagittal sections (Fig. 3a). Dendrites extending in the medial and lateral direction were rare and, when present, were short (44 ± 24 µm) in transverse sections (Fig. 3b). The mean extent of dorsoventral processes was 92 ± 43 µm (Fig. 3). Biocytin-labeled axons of SG–GFP neurons did not appear to project outside the SG (a three-dimensional reconstruction of an SG–GFP neuron appears in supplemental Fig. 2, available at http://www.med.unc.edu/physiology/fac_hantman.htm). On the basis of the morphological characteristics of the SG neuronal subtypes defined in the hamster and rat (Grudt and Perl, 2002
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Figure 3. Morphology of biocytin-filled SG–GFP neurons expressing GFP. a, Maximum projection confocal image of a representative neuron in a sagittal spinal section. b, Maximum projection confocal image of a representative neuron in a transverse section. Insets are merged images of GFP signal (green) and biocytin signal (red). Orange-yellow indicates biocytin labeling of a GFP-expressing neuron. SG–GFP neurons are morphologically equivalent to central neurons. Dorsal is shown at the top. Scale bar, 25µm.
Electrophysiological characteristics of SG–GFP neurons
The input resistance of SG–GFP neurons averaged 158 ± 68 M(n = 28) and the mean resting membrane potential was –48 ± 7 mV (n = 28). Twenty-five of 28 SG–GFP neurons exhibited inward rectification (Fig. 4a1,2). Most SG–GFP neurons (20 of 28) expressed a hyperpolarization-activated inward current (Ih); however, none (n = 28) showed a hyperpolarization-activated outward tail current (IA). SG–GFP neurons initiated action potentials on depolarization without delay (27 of 28); all (28 of 28) fired tonically throughout a depolarizing pulse. Most SG–GFP cells (26 of 28) generated action potentials at relatively constant interspike intervals (Fig. 4a3).
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Figure 4. Electrophysiological properties of mouse neurons. Row 1, Current versus voltage traces. Row 2, Current versus voltage plots taken from the beginning and end of each voltage step. Row 3, Action potential generation by a prolonged (1 sec) depolarization. a, A representative SG–GFP neuron showing small inward rectification (changing slope of current–voltage plots), slight Ih-like currents (hyperpolarization-activated depolarizing currents), and a tonic action potential generation pattern in response to a maintained depolarization. b, An example of an SG neuron in the Prp–GFP mouse lacking GFP. Note the absence of Ih, the presence of IA (outward tail current), and the transient pattern of action potential generation in response to a maintained depolarization. Neurons were held at –60 mV during voltage-clamp recordings.
In contrast to SG–GFP neurons, electrophysiological properties of nearby, randomly selected SG neurons without GFP labeling exhibited a much broader range of features (Yoshimura and Jessell, 1989Dorsal root input to the SG–GFP neurons
Dorsal root stimulation evoked responses in SG–GFP neurons (Fig. 5a). In 9 of 10 SG–GFP neurons, consecutive dorsal root-evoked responses varied little in latency (coefficient of variation, <2%), indicative of a monosynaptic connection. The conduction velocity of the dorsal root fibers underlying this presumed monosynaptic input (n = 9) ranged from 0.10 to 0.25 m/sec (average, 0.15 ± 0.06 m/sec), a value consistent with C-fiber input. The dorsal root-evoked responses were reversibly blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (10 µM), suggesting mediation by AMPA-type glutamate receptors (Fig. 5a). Dorsal root stimulation failed to generate monosynaptic or polysynaptic responses at latencies that would be indicative of a myelinated fiber input to SG–GFP neurons. In contrast, responses to activation of dorsal root fibers conducting at A
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Figure 5. Responses of SG neurons in the Prp–GFP mouse to dorsal root stimulation. Each trace represents the average of 10 successive stimulations. Gray traces represent dorsal root responses during the application of 10 mM CNQX. Dashed traces represent dorsal root responses during wash period. a, The lack of variation in the long latency responses of SG–GFP neurons is consistent with a monosynaptic C-fiber input. The block of response by CNQX suggests mediation by AMPA-type glutamate channels. b, The dorsal root response of an SG neuron lacking GFP expression shows a briefer latency and a more complex form, indicating input from A
Noxious stimuli activate SG–GFP neurons
Substantial noxious stimulation of peripheral tissue can cause the upregulation of c-Fos protein in a population of SG neurons (Hunt et al., 1987
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Figure 6. c-Fos expression in the Prp–GFP mouse after formalin injection into the foot. The number of c-Fos-expressing SG–GFP neurons ispilateral (a1, a2) to the injection was much higher than on the contralateral side (b1, b2). a1, b1, GFP signal from a transverse section of lumbar spinal cord is shown. a2, b2, Depicted is immunoreactivity to c-Fos antibodies in the sections shown in a1 and b1, respectively. Somata of SG–GFP neurons are indicated by arrows. Dorsal is shown at the top. Scale bars, 25 µm. c, Mean numbers (±SEM) of SG–GFP neurons per section expressing c-Fos for saline and formalin injections. Paired two-tailed t test p-values are shown for difference between the sides ipsilateral and contralateral to the formalin injections.
SG–GFP neurons are GABAergic
SG–GFP neurons exhibit immunoreactivity to three different antibodies established to stain neurons containing GABA as a synaptic agent. These antibodies labeled known GABAergic cells such as the Renshaw cells of the ventral horn. More than 80% (703 of 843) of SG–GFP neurons showed GABA-like immunoreactivity (Fig. 7a1,2). This proportion is probably an underestimate, because GABA antibodies have limited penetration into tissue. SG–GFP neurons labeled by GABA antibodies constituted
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Figure 7. The majority of SG–GFP neurons express immunoreactivity to GABA antibodies. This immunoreactivity was blocked by preabsorption with a GABA–BSA conjugate. a1, b1, GFP signal from transverse sections of lumbar spinal cord. a2, b2, Immunoreactivity to a GABA antibody in sections shown in a1 and b1, respectively. The tissue section in b1 and b2 was preabsorbed with a GABA–BSA conjugate (2.5 mg/ml). Somata of GFP neurons are indicated by arrows. Dorsal is shown at the top. Scale bars, 25 µm.
Discussion
SG–GFP neurons have features of tonic central cells
Ramon y Cajal (1909The morphological and electrophysiological features of the SG–GFP neurons suggest that they represent tonic central cells. Cytoarchitecture (size and density of neurons) and the relationship of the location of SG–GFP somata to IB4 binding and the ventral border of immunoreactivity to substance P locate these cells in the zone containing the central class of SG neurons. The cellular morphology of the SG–GFP neurons, including the local projection of their axons, also corresponds to features of central cells in other species. The small inward rectification, small Ih currents, and the tonic generation of action potentials by a suprathreshold depolarization fit features of tonic central cells. However, the apparent limitation of direct dorsal root input to C-fibers may indicate that the SG–GFP neurons are a subset of tonic central cells, because, in some other rodents, tonic central cells receive monosynaptic dorsal root input from A
C-fiber input to inhibitory neurons
Our observations provide clues to a circuit possibly containing the SG–GFP neurons. Neurons that upregulate c-Fos protein in response to a noxious stimuli are argued to participate in nociceptive pathways (Hunt et al., 1987The case is made here that SG–GFP neurons constitute a homogenous population of tonic central cells. Their spaced, repeated location and their coherent functional attributes suggest that they represent a neuronal type with a repeating SG function. Repetition of a functional unit along the spinal cord would be consistent with a modular framework in which interconnected neurons are arranged to deal with related somatosensory inputs. Related inputs could arise from similar afferent sensory fibers or from comparable receptive fields. In such an organization, nests (sets) of neurons connected in a special functional way would subserve particular integrative functions related to selective primary afferent projections. This kind of modular arrangement would be comparable in principle with the neuronal columns of the somatosensory cerebral cortex and its relationship to primary afferent input.
Finally, these studies emphasize the relative ease of establishing features of a particular class of interneurons marked by the expression of a fluorescent protein. There are apparent advantages to this approach in analyzing function of a complex neuropil.
Footnotes
Received Sep 15, 2003; revised November 7, 2003; accepted November 7, 2003.This work was supported by Research Grant NS-10321 from the National Institute of Neurological Disorders and Stroke of the National Institutes of Health. We thank Ms. Bonnie Taylor-Blake for excellent assistance with immunocytochemical studies and Mr. Kirk McNaughton for superior histological work. We are grateful to Mrs. Sherry Joseph for valuable help in preparation of this manuscript. Peter Somogyi graciously provided a GABA antibody for use in these experiments.
Correspondence should be addressed to Dr. Edward R. Perl, Department of Cell and Molecular Physiology, University of North Carolina at Chapel Hill, 5109 Neuroscience Research Building, Campus Box 7545, Chapel Hill, NC 27599. E-mail: erp{at}med.unc.edu.
DOI:10.1523/JNEURSCI.4221-03.2004
Copyright © 2004 Society for Neuroscience 0270-6474/04/240836-07$15.00/0
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