Arthur D. Craig - US grants

This research project is based on the concept that lamina I of the dorsal horn is an integral component of the central representation of pain and temperature sensibility. Only lamina I receives small-diameter primary afferent input from all tissues of the body and contains a specific concentration of nociceptive and thermoreceptive neurons. Lamina I neurons form half of the spinothalamic tract (STT), and their ascending axons are concentrated in the critical location for pain and temperature sensation. The functional and anatomical organization of lamina I projections needs to be elucidated. In the preceding grant period, we identified with anatomical, immunohistochemical and physiological methods a specific lamina I STT thalamic relay nucleus (VMpo) for pain and temperature sensation in macaques and humans, and we demonstrated lamina I propriospinal and brainstem projections that could provide the basis for noxious and thermal somato-autonomic reflexes. In cats, we also identified three major physiological/morphological classes of lamina I STT cells, showed that they have selective thalamic projections, and demonstrated that a simple model of thalamo-cortical integration of the activity of these lamina I STT sensory channels can explain the thermal grill illusion and the burn of cold pain. The proposed experiments address the functional anatomy of the lamina I spino-thalamo-cortical pathway in primates. We will (i) determine whether the cortical projections of the major lamina I STT projection targets that we have identified (VMpo, VPI, and MDvc) in the monkey correspond with the major cortical areas activated by pain in human PET studies, (2) determine whether the different morphological classes of lamina I STT neurons identified in the cat are distinguishable in the monkey and project differentially, (3) examine whether VMpo and neighboring structures in monkey and human thalamus are chemoarchitectonically organized, (4) quantitatively determine whether physiologically distinct classes of lamina I STT cells exist in the monkey like those identified in the cat, and (5) quantitatively determine whether VMpo neurons maintain the physiological specificity of lamina I response classes or show evidence of integrative convergence of lamina I STT inputs. The thermal grill and the itch-inducing agent histamine will also be used to examine whether the postulated integration of lamina I STT activity occurs in VMpo. These experiments test specific hypotheses that delineate integral components of the central substrates of specific pain and temperature sensibility in monkeys and humans. We believe that the postulated model of thalamo-cortical integration of lamina I sensory channels could provide a basis for the cold allodynia and burning dysesthetic pain that characterize the classic (central post-stroke) thalamic pain syndrome.

DESCRIPTION (Adapted from the Investigator's Abstract): Our understanding of the functional organization of central pathways for pain and temperature sensations depends on anatomical elucidation of the substrates involved. We recently identified a thalamic nucleus in monkeys and humans (VMpo) that we believe is a sensory relay nucleus for specific pain and temperature activity. We will test this hypothesis by examining the synaptic organization of the lamina I trigemino- and spino-thalamic (TSTT) terminations in VMpo in order to determine whether they provide the basis for secure synaptic relay of sensory information. We will build volume-rendered three-dimensional models of single anterogradely-labeled TSTT terminals from serial ultrathin sections, using a methodology that we have applied successfully in the cat thalamus. Though technically difficult and time consuming, this is the only means of fully characterizing the relationships of these terminals with adjacent ultrastructural elements. In reconstructing their synaptology, we will seek to identify triadic arrangements with presynaptic dendrites, which are characteristic of thalamic sensory relay nuclei but which others say are not associated with TSTT terminals in the primate. Further, we will examine the possibilities that the terminations of nociceptive and thermoreceptive lamina I TSTT fibers may be different, as suggested by prior work in the cat, and that lamina I terminations in the ventral posterior n. (VP) may differ from those in VMpo. These studies will improve our understanding of the thalamic substrates underlying the processing of pain and temperature information, and they will offer insight into the synaptic basis for the sensations of pain and cold that can be produced by stimulation of this region in the human thalamus.

Ascending inputs to the brainstem from the spinal cord are critical for the control of homeostatic (pre-autonomic) functions, such as cardiovascular and respiratory responses to noxious or thermal stimuli that challenge the stable physiological condition of the body. It has been recognized for over 30 years that small-diameter (A-delta and C-fiber) afferent inputs generate powerful somato-autonomic reflexes in the brainstem, but there is still very little information available regarding the spinobulbar neurons that carry such activity to homeostatic brainstem integration sites. Neurons in lamina I of the superficial dorsal horn that receive direct - delta and C-fiber are the major source of spinal input to the brainstem. We have shown in prior work that lamina I neurons project to the homeostatic regions of the brainstem. New evidence suggests that lamina I spinobulbar neurons are unique population of lamina I neurons that has never been studied before. The goal of this project is to discriminate lamina I spinobulbar neurons anatomically and physiologically. In anatomic studies, we will (Aim 1) use retrograde labeling to identify lamina I and other spinal neurons that project to particular homeostatic sites in the brainstem and to verify that lamina I and other spinal neurons that project to particular homeostatic sites in the brainstem and to verify that lamina I spinobulbar an spinothalamic neurons are distinct (using double-labeling). In physiologic studies, we will (Aim 2) record and characterize single lamina I spinobulbar neurons, using antidromic activation and natural cutaneous and deep somatic stimulation, and differentiate them from spinothalamic neurons. In addition, we will (Aim 3) stimulate the anterior hypothalamus and the periacqueductal gray, two pre-autonomic control sites that drive sympathetic vasoconstrictor output, in order to determine whether descending homeostatic controls differentially modulate the activity of spinobulbar and spinothalamic lamina I neurons. Using protocols that we have refined in experiments in cats (which nonetheless have fundamental neuroanatomical differences from primates), these experiments will obtain data in macaque monkeys that will be directly relevant to human physiology. Preliminary evidence strongly indicates that these experiments will confirm the central hypotheses that lamina I spinobulbar neurons are a distinct population of neurons. These experiments will differentiate and characterize for the first time the ascending modality-selective spinal neurons that carry small- diameter A-delta and C-fiber afferent inputs to homeostatic and pre- autonomic integration mechanisms in the brain stem. The fundamental knowledge will provide new opportunities for explaining maladaptive homeostatic responses to somatic physiological changes, including such human pathological conditions as fibromyalgia.

The spinothalamic tract (STT) is an important ascending pathway for activity associated with pain, temperature and itch sensations in primates and humans. Prior studies in this laboratory indicate that modality- selective lamina I STT neurons that project in the lateral STT have a integral role in these sensations. Studies in other laboratories, hoever, report that the responses of 'wide dynamic range' (WDR) STT neurons in lamina V, which ascend in the anterior STT, correlate with pain sensation and with hyperalgesia. Indeed, neurons in both populations are activated by stimulate that evoke pain, temperature, and itch sensations, yet both comprise several subclasses of cells that are activated by particular stimuli. This project is based on the global hypothesis that subclasses of lamina I and lamina V STT neurons carry complementary information to the forebrain that must be integrated to produce the several aspects of these sensations This hypothesis is consistent with clinical data suggesting that multiple ascending pathways are involved in pain, and with imaging observations showing that multiple forebrain regions are activated during these sensations. Knowledge of the activity in different subclasses of laminae I and V STT neurons that project to different targets in the primate thalamus is inadequate, despite considerable prior work. For example, polymodal nociceptive (HPC) lamina I STT cells that project to Vmpo have never been characterized, and the role of lamina V STT cells that project to VPL in the thermal grill illusion of pain has never been examined. In order to test specific hypothesis that anticipate which subclasses of laminae I aNd V STT cells display responses corresponding to different sensations, we will functionally characterize single laminae I and V STT neurons using five noel quantitative stimulus paradigms that distinguish different aspects of these sensations: (Aim 1) repeated brief contact heat [,second pain']; (Aim 2) graded tonic pressure ['first pain']; (Aim 3) iontophoretic histamine [itch]; (Aim 4) the algogens capsaicin and mustard oil (burning pain and hyperalgesia]; and (Aim 5) the thermal grill [illusory cold pain]. Using protocols that hae been refined in experiments in cats (which have almost no WDR lamina I STT cells and comparatively few lamina V STT cells), the goal of these experiments will be to obtain data in macaque monkeys that can be directly compared (across primates) with published human psychophysical evidence obtained using the same stimulus paradigms. Preliminary findings verify the feasibility of these experiments and validate the discriminative value of these paradigms. This research will provide new insights into the synergy between lamina I and V STT neurons and into the integration underlying pain, temperature and itch sensations that is altered in clinical disorders.

DESCRIPTION (provided by applicant): In order to understand the organization of central pathways for pain in humans, we need to know the underlying anatomical substrates in the primate brain. During the past several years, we have characterized a thalamic nucleus in monkeys and humans (VMpo) that, we believe, serves as a specific thalamocortical relay nucleus for pain, temperature, itch and other sensations related to the physiological condition of the body. This hypothesis differs from the conventional view that the ventral posterior nuclei (VP) are crucial for pain sensation, but it is supported by growing evidence. The preceding cycle of this NIH grant enabled us to test this hypothesis by examining the synaptology of lamina I trigemino- and spino-thalamic (TSTT) terminations in VMpo. Using three-dimensional models built from serial ultra-thin sections through anterogradely-labeled lamina I TSTT terminal fibers, we showed that lamina I terminations in VMpo have distinct characteristics (multiple contacts on single post-synaptic dendrites in triadic arrangements with presynaptic dendrites) that provide the basis for secure, high-fidelity synaptic transmission, consistent with a relay function. This contrasts strongly with prior ultrastructural observations by reliable investigators indicating that TSTT terminals in the primate thalamus generate single, isolated boutons that do not provide the basis for secure, high-fidelity synaptic contacts. That prior study focused on VP, but it also examined the region of VMpo; yet, it did not differentiate lamina I from lamina V TSTT terminations, which may have entirely different functional roles. We propose to resolve this discrepancy by directly examining the synaptology of identified lamina I TSTT terminations in VP. If lamina I TSTT terminations in VP form secure, high-fidelity synaptic contacts like they do in VMpo, this would support the view that both these regions have an important role in pain. Alternatively, if lamina I TSTT terminations in VP form single contacts, as suggested by prior observations, this would indicate that the synaptic transfer of pain-related activity in VMpo and VP is qualitatively different, supporting our view that VMpo and VP have different roles in sensation, and underscoring the significance of VMpo as a lamina I sensory relay nucleus. These studies will advance our understanding of the anatomical substrates for pain sensation in the primate thalamus.