All methods and procedures were approved in advance by an institutional animal care and use committee (IACUC) and are in full compliance with National Institutes of Health policy on animal welfare.
Subjects, Anesthesia, Surgical Procedures, Euthanasia
Subjects were adult male and female squirrel monkeys (Saimiri sciureus and peruviensus; n = 10). The subject was placed in a light-tight enclosure and general anesthesia induced with a gas mix (4% halothane in a 50/50 mixture of oxygen and N2O). Tubing connecting a veterinary anesthesia machine to an external port on the enclosure enabled delivery and effective confinement of the anesthetic gas mix to the interior of the enclosure. After induction of anesthesia the subject was removed from the enclosure and placed on a surgical table. The trachea was intubated and connected via tubing to the anesthesia machine. Methylprednisolone sodium succinate (20 mg/kg) and gentamicin sulfate (2.5 mg/kg) were injected intramuscularly to protect against cerebral edema and bacterial septicemia, respectively.
The composition of the anesthetic gas mix was adjusted (1.5–3.0% halothane in 50/50 N2O/oxygen) to ensure maintenance of adequate and stable general anesthesia throughout performance of the following procedures. A valved polyethylene cannula filled with saline was inserted into the femoral vein of the subject's right hindlimb to enable administration of drugs, glucose (5%), and electrolytes (0.9% saline). A 1–1.5 cm2 opening was made in the skull overlying the central sulcus in the right hemisphere, and a cylindrical Lexan recording chamber placed over the opening and attached to the surrounding skull with dental acrylic. The dura was incised and removed, and the recording chamber filled with artificial cerebrospinal fluid and sealed with a glass plate to prevent cortical dehydration. Sensory input from tissues within the surgical fields on the head (in the vicinity of the recording chamber) and right hindlimb was minimized by topical application of local anesthetic in oil (Cetacaine), and skin wounds were closed with silk sutures.
After completion of the surgical procedures neuromuscular blockade was achieved by intravenous administration of Norcuron (loading dose: 0.25–0.5 mg/kg; maintenance dose: 0.025–0.05 mg/kg/h). For the remainder of the experiment a 50/50 mix of N2O and oxygen was provided via a positive pressure ventilator, and the halothane concentration adjusted (typically between 0.5% and 1.0%) to maintain heart rate, arterial blood pressure (recorded noninvasively using pressure plethysmography—blood pressure monitoring system BP-3Plus, VetSpecs, Canton, GA), and electroencephalography slow-wave content at values consistent with general anesthesia. Rate and depth of ventilation were monitored and adjusted to maintain end-tidal CO2 between 3.0% and 4.5%. Rectal temperature was maintained at 37.5 °C with a heating pad. Euthanasia was achieved by injection of pentobarbital (50 mg/kg; i.v.), followed by intracardial perfusion with 0.9% NaCl, and subsequently with fixative (10% formalin in saline).
Neurophysiological Recording Methods
Extracellular recordings of the spike discharge activity of area 3a neurons and small neuron groupings were obtained using glass-insulated tungsten wires (impedance 300–500 kΩ at a test frequency of 10 kHz). Micropositioners enabled placement of the tip of recording microelectrode at any site within the hydraulically sealed chamber. A microdrive was used to advance (through an “O-ring” in the glass coverplate) the microelectrode tip from a point above the cortical surface to intracortical position(s) at which neuronal spike discharge activity was detected. At the maximal depth of a penetration, and/or at a site where recordings of particular interest were obtained, an electrolytic lesion was created by passing 5–10 μA of DC current through the microelectrode. Such a lesion typically allowed postexperimental identification of the laminar location of the recording site ().
Figure 1. Microelectrode penetrations of area 3a. (A) Photograph of surface of squirrel monkey right hemisphere. Dots located anterior to central sulcus (CS) indicate entry point of each of the 21 microelectrode penetrations performed in the 10 subjects that yielded (more ...)
Thermotactile Stimulator, Stimulation Protocols
The stimulator used in all experiments (CS-540, Cantek Enterprises, Canonsburg, PA) enabled simultaneous delivery of precisely controlled thermal and mechanical stimulation to a preselected skin site. The stimulator made contact with the site via a cylindrical copper probe (5 mm diameter; flattened at the tip). Probe temperature could be varied from one contact to the next, or maintained at a temperature (25–56 °C; accuracy ±0.1 °C) over a series of successive contacts. The stimulator's control system allowed probe temperature to be modified only when the probe was not in contact with the skin. When the probe attained the desired temperature (investigator-specified via software) it was rapidly advanced (20 mm/s) from its “rest” position (~5 mm above the skin site targeted for stimulation) until the tip of the probe indented the skin by ~1 mm. The probe then remained stationary for an investigator-specified interval (1–7 s) after which the stimulator's control system abruptly (20 mm/s) retracted the probe to the rest position. Controlling software permitted precise specification of probe temperature, duration of probe contact with the skin, number of successive contacts delivered at a given probe temperature, time interval between successively applied same-temperature contacts, and the time interval between successive contacts applied at different temperatures.
Throughout each experiment probe temperature was continuously displayed and recorded as part of the permanent experimental record. To minimize sensitization of a skin site: 1) “control” skin contact stimuli were delivered with the probe at a temperature selected from the range 25–38 °C; 2) a series of successive skin contacts was used (typically 6 when contact duration was 5–7 s; 10 contacts were used when contact duration was 1 s) to characterize the area 3a neuron response to probe temperatures ≥49 °C; 3) the probe did not contact the skin before, after, or between successive stimuli; and 4) at a probe temperature ≥49 °C a substantial no-stimulus period (never less than 30 s) separated successive contacts when contact duration was >1 s.
Stimulation Protocols (n = 3)
The initial experiments made extensive use of a 3-phase protocol (Protocol #1) to study the effects of 5- to 7-s heated skin contact on the spike discharge activity of area 3a neurons. First, a series of contacts was applied with the probe preheated to a temperature selected from the range 25–38 °C (“Control”). Next, a second series of contacts (“Test”) was applied to the same site. The contacts in this series were identical in all respects except one to those delivered during the initial series—that is, throughout this second series the probe was maintained at a temperature selected from the range 47–51 °C. And finally, a third series of contacts (“Recovery”) was reapplied to the same skin site, once again with the probe at the temperature used in the initial series. All contacts were 5–7 s in duration. A 15-s interstimulus interval (ISI) was allowed between successive contacts in the control and recovery phases of the protocol; an ISI of 30-s separated successive contacts in each phase. A 3-min no-stimulus delay separated the control, test, and recovery phases.
A different protocol (Protocol #2) was used to assess the temperature-dependency of area 3a neurons. In this protocol the response of a neuron was recorded to 6 series of contacts delivered to the same skin site (each series consisted of 4 contacts; total contacts = 24). Each series included one contact with the probe at 38 °C, 49 °C, 50 °C, and 51 °C, respectively; order of the different-temperature contacts within a series was varied from one series to the next. A 30-s ISI separated successive contacts in the same series. A 3-min no-stimulus delay separated the last contact of one series and the first contact of the next.
The third protocol (Protocol #3; repetitive 1/3 s, 0.8- to 1.0-s contact with a 5 mm diameter skin site by a probe maintained at a temperature between 49–56 °C) that was used was especially well-suited for study of the cerebral cortical mechanisms relevant to second pain. Previous studies have shown this protocol to 1) be uniquely effective for evoking second (“slow”) pain in a conscious subject (Vierck et al. 1997
); 2) enable clear demonstration of the slow temporal summation characteristic of second pain in humans (Vierck et al. 1997
); and 3) evoke a temporal profile of spike discharge activity from lamina I neurons in the spinal cord dorsal horn (the initial stage of central nervous system [CNS] processing of the input from C-nociceptors) that closely resembles the temporal profile of second pain in humans (Andrew and Craig 2002
The range of probe temperatures chosen for study was based on the very different perceptual experiences that result when a site on the thenar eminence is contacted with the probe at a temperature selected from within the range 25–56 °C (Vierck et al. 1997
; Li et al. 2000
). The investigators rated a 5-s exposure to static contact of the thenar with the probe at 32–38 °C as “thermoneutral/nonpainful,” “warm/marginally painful” at 47 °C, “marginally/moderately painful” at 48 °C, and as “moderately-strongly painful” at a temperature between 49.5 °C and 51 °C. Also relevant is the published observation (Vierck et al. 1997
) that contact for 1 s by a probe maintained at ≥50 °C evokes a delayed second (“slow”) pain percept that is maximal in intensity at 1–2 s after the probe is withdrawn from the skin, and grows progressively stronger in intensity when the probe contacts the skin repetitively at a frequency ≥1/3 s (exhibits prominent slow temporal summation). At no probe temperature used in the experiments described in this paper did a single or repetitive contact stimulus elicit escape when it was applied to the skin of the investigators. Nor did any of the conditions/protocols that were used result in either visually apparent skin damage or skin sensitization.
Neural Data Collection
Area 3a neuron spike discharge activity occurring before, during, and after each contact stimulus was collected, digitized (sampled at 20 kHz), and stored as an electronic file. The activity attributable to a single unit (SUR) or to small neuronal groupings composed of 2–3 neurons (MUR) was amplitude-discriminated using nonoverlapping voltage windows (2–3 units per neuronal groupings could reliably be distinguished in this way at a single recording locus). The electronic file generated for each voltage window recorded at a cortical depth registered the time of occurrence (accuracy ±100 μs) of each action potential whose peak voltage fell within that window, as well as the times of each stimulator event of interest (e.g., onset of probe contact with the skin; onset of probe withdrawal from the skin).
Use of OIS Imaging
In 7 of the 10 squirrel monkey subjects the method of OIS imaging (Tommerdahl et al. 1996
) was used initially. Images obtained with this method were used in the subsequent (neurophysiological recording) component of the experiment to guide the placement of microelectrode penetrations. Availability of images of the cortical optical response to the same conditions of noxious skin heating used to study the response of individual neurons ensured that extracellular recordings of neuronal spike discharge activity were obtained from the same region of anterior parietal cortex that developed a prominent (typically the maximal) OIS in response to noxious skin-heating stimulation.
The OIS imaging method was not used in the final 3 experiments because by that point it was evident that noxious heating stimulation of either the glabrous skin of the hand or foot evokes vigorous spike discharge activity from neurons within a highly consistent location in contralateral anterior parietal cortex. In each of the 7 subjects that provided OIS imaging results, the region of the primary somatosensory cortex (SI) that responded maximally to ≥49 °C contact with a site on the hand occupied a part of area 3a located 1–4 mm more medially than the region (in area 3b) that responded maximally to same-site 25–38 °C skin contact or 25 Hz flutter; whereas the sector of area 3a that developed the maximal optical response to contact of a site on the volar foot with a probe ≥49 °C was located ~1–2 mm lateral to the region (in area 3b) that responded maximally to same-site 25–38 °C skin contact or 25 Hz flutter.
Area 3a Neuron Sample/Approach to Placement of Microelectrode Penetrations
Ten squirrel monkeys were studied. The spike discharge activity of 103 single neurons (SURs) or small neuronal groupings (typically 2–5; MURs) was recorded during 21 microelectrode penetrations performed in area 3a () Fourteen of the 21 penetrations traversed the area 3a region that in the same subject developed the maximal OIS in response to noxious heating of the same skin site used to evoke area 3a neuron spike discharge activity. The region of area 3a targeted by the remaining 7 penetrations was determined using a different strategy—that is, each of these penetrations was performed subsequent to determining (using extracellular recordings of neuronal spike discharge activity) the anterior parietal locus of neurons highly responsive to gentle mechanical stimulation of the volar surface of the radial hand. Once that region was identified, the position of the recording electrode then was shifted anteriorly (by 1–2 mm) and medially (by 1–4 mm)—in accord with the relative locations of the distinctly different SI regions that develop a maximal OIS in response to same-site tactile versus noxious skin-heating stimulation (Tommerdahl et al. 1996
Intradermal Injection of Algogen
The spike discharge activity of 29 area 3a neurons was recorded before, during, and for an extended period (typically >1 h) following intradermal algogen injection (capsaicin—21 neurons; α, β methylene ATP—8 neurons). Recordings were obtained from each neuron in the absence of intentional skin stimulation (“spontaneous activity”), and also during and following the delivery of precisely controlled thermomechanical skin stimulation. Capsaicin or α, β methylene ATP was injected intradermally at a single site located 4–10 mm adjacent to the site contacted by the probe of the thermomechanical stimulator. For 3 of the 21 neurons whose activity was recorded before, during and after intradermal capsaicin injection the contribution of ongoing C-nociceptor afferent drive to the altered area 3a neuron response to 38 °C skin contact was assessed by intradermal injection of 25–50 μL of 10 mg/mL lidocaine HCl (1% xylocaine; AstraZeneca, London, UK). The local anesthetic was injected at 1–2 sites between the capsaicin injection site and the site contacted by the stimulator probe.
Histological Procedures, Identification of Cytoarchitectural Boundaries
In each subject the region of SI traversed by microelectrode penetrations was removed, fixed in 10% formalin, placed in a cryostat, and serially sectioned either in the sagittal or coronal planes. All sections were stained with cresyl fast violet and inspected microscopically to distinguish anterior parietal regions on the basis of established cytoarchitectonic criteria (Powell and Mountcastle 1959
; Jones and Porter 1980
; Friedman and Jones 1981; Sur et al. 1982
; see ). Boundaries between adjacent cytoarchitectonic areas were identified by scanning sections separated by no more than 300 μm. For each subject a 2-dimensional plot showing the locations of area 3a, 3b, 1, and 2 was generated using a microscope and drawing tube attachment. The sites at which recordings were made along each track were reconstructed using 1) micrometer readings of the depth at which recordings were obtained during each microelectrode penetration, and 2) depth at which a microlesion was placed in each penetration.