Stella Koutsikou

Spontaneous pain, both neuropathic and inflammatory, is related to frequency of spontaneous firing in intact C-fiber nociceptors

Spontaneous pain, a poorly understood aspect of human neuropathic pain, is indicated in animals by spontaneous foot lifting (SFL). To determine whether SFL is caused by spontaneous firing in nociceptive neurons, we studied the following groups of rats: (1) untreated; (2) spinal nerve axotomy (SNA), L5 SNA 1 week earlier; (3) mSNA (modified SNA), SNA plus loose ligation of the adjacent L4 spinal nerve with inflammation-inducing chromic gut; and (4) CFA (complete Freund’s adjuvant), intradermal complete Freund’s adjuvant-induced hindlimb inflammation 1 and 4 d earlier. In all groups, recordings of SFL and of spontaneous activity (SA) in ipsilateral dorsal root ganglion (DRG) neurons (intracellularly) were made. Evoked pain behaviors were measured in nerve injury (SNA/mSNA) groups. Percentages of nociceptive-type C-fiber neurons (C-nociceptors) with SA increased in intact L4 but not axotomized L5 DRGs in SNA and mSNA (to 35%), and in L4/L5 DRGs 1–4 d after CFA (to 38–25%). SFL occurred in mSNA but not SNA rats. It was not correlated with mechanical allodynia, extent of L4 fiber damage [ATF3 (activation transcription factor 3) immunostaining], or percentage of L4 C-nociceptors with SA. However, L4 C-nociceptors with SA fired faster after mSNA (1.8 Hz) than SNA (0.02 Hz); estimated L4 total firing rates were ∼5.0 and ∼0.6 kHz, respectively. Similarly, after CFA, faster L4 C-nociceptor SA after 1 d was associated with SFL, whereas slower SA after 4 d was not. Thus, inflammation causes L4 C-nociceptor SA and SFL. Overall, SFL was related to SA rate in intact C-nociceptors. Both L5 degeneration and chromic gut cause inflammation. Therefore, both SA and SFL/spontaneous pain after nerve injury (mSNA) may result from cumulative neuroinflammation.

Partial nerve injury induces electrophysiological changes in conducting (uninjured) nociceptive and nonnociceptive DRG neurons: Possible relationships to aspects of peripheral neuropathic pain and paresthesias

Summary After partial nerve injury, changes in uninjured nociceptors and nonnociceptors (spontaneous firing, decreased electrical thresholds, hyperpolarisation) may account for the different aspects of neuropathic and paresthesias. Abstract Partial nerve injury leads to peripheral neuropathic pain. This injury results in conducting/uninterrupted (also called uninjured) sensory fibres, conducting through the damaged nerve alongside axotomised/degenerating fibres. In rats seven days after L5 spinal nerve axotomy (SNA) or modified‐SNA (added loose‐ligation of L4 spinal nerve with neuroinflammation‐inducing chromic‐gut), we investigated a) neuropathic pain behaviours and b) electrophysiological changes in conducting/uninterrupted L4 dorsal root ganglion (DRG) neurons with receptive fields (called: L4‐receptive‐field‐neurons). Compared to pretreatment, modified‐SNA rats showed highly significant increases in spontaneous‐foot‐lifting duration, mechanical‐hypersensitivity/allodynia, and heat‐hypersensitivity/hyperalgesia, that were significantly greater than after SNA, especially spontaneous‐foot‐lifting. We recorded intracellularly in vivo from normal L4/L5 DRG neurons and ipsilateral L4‐receptive‐field‐neurons. After SNA or modified‐SNA, L4‐receptive‐field‐neurons showed the following: a) increased percentages of C‐, Ad‐, and Ab‐nociceptors and cutaneous Aa/b‐low‐threshold mechanoreceptors with ongoing/spontaneous firing; b) spontaneous firing in C‐nociceptors that originated peripherally; this was at a faster rate in modified‐SNA than SNA; c) decreased electrical thresholds in A‐nociceptors after SNA; d) hyperpolarised membrane potentials in A‐nociceptors and Aa/b‐low‐threshold‐mechanoreceptors after SNA, but not C‐nociceptors; e) decreased somatic action potential rise times in C‐ and A‐nociceptors, not Aa/b‐low‐threshold‐mechanoreceptors. We suggest that these changes in subtypes of conducting/uninterrupted neurons after partial nerve injury contribute to the different aspects of neuropathic pain as follows: spontaneous firing in nociceptors to ongoing/spontaneous pain; spontaneous firing in Aa/b‐low‐threshold‐mechanoreceptors to dysesthesias/paresthesias; and lowered A‐nociceptor electrical thresholds to A‐nociceptor sensitization, and greater evoked pain.

Spinal Processing of Noxious and Innocuous Cold Information: Differential Modulation by the Periaqueductal Gray

In addition to cold being an important behavioral drive, altered cold sensation frequently accompanies pathological pain states. However, in contrast to peripheral mechanisms, central processing of cold sensory input has received relatively little attention. The present study characterized spinal responses to noxious and innocuous intensities of cold stimulation in vivo and established the extent to which they are modulated by descending control originating from the periaqueductal gray (PAG), a major determinant of acute and chronic pain. In lightly anesthetized rats, hindpaw cooling with ethyl chloride, but not acetone, was sufficiently noxious to evoke withdrawal reflexes, which were powerfully inhibited by ventrolateral (VL)-PAG stimulation. In a second series of experiments, subsets of spinal dorsal horn neurons were found to respond to innocuous and/or noxious cold. Descending control from the VL-PAG distinguished between activity in nociceptive versus non-nociceptive spinal circuits in that innocuous cold information transmitted by non-nociceptive class 1 and wide-dynamic-range class 2 neurons remained unaltered. In contrast, noxious cold information transmitted by class 2 neurons and all cold-evoked activity in nociceptive-specific class 3 neurons was significantly depressed. We therefore demonstrate that spinal responses to cold can be powerfully modulated by descending control systems originating in the PAG, and that this control selectively modulates transmission of noxious versus innocuous information. This has important implications for central processing of cold somatosensation and, given that chronic pain states are dependent on dynamic alterations in descending control, will help elucidate mechanisms underlying aberrant cold sensations that accompany pathological pain states.

Neural substrates underlying fear-evoked freezing: the periaqueductal grey-cerebellar link.

At the heart of the brain circuitry underlying fear behaviour is the periaqueductal grey (PAG). We address an important gap in understanding regarding the neural pathways and mechanisms that link the PAG to distinct patterns of motor response associated with survival behaviours. We identify a highly localised part of the cerebellum (lateral vermal lobule VIII, pyramis) as a key supraspinal node within a chain of connections that links the PAG to the spinal cord to elicit fear‐evoked freezing behaviour. Expression of fear‐evoked freezing behaviour, both conditioned and innate, is dependent on cerebellar pyramis neural input–output pathways. We also address an important controversy in the literature, namely whether or not ventrolateral PAG (vlPAG) increases muscle tone. We provide evidence that activation of the vlPAG causes an increase in α‐motoneurone excitability, consistent with a role in generating muscle tone associated with fear‐evoked freezing.

Neural substrates underlying fear-evoked freezing

At the heart of the brain circuitry underlying fear behaviour is the periaqueductal grey (PAG). We address an important gap in understanding regarding the neural pathways and mechanisms that link the PAG to distinct patterns of motor response associated with survival behaviours. We identify a highly localised part of the cerebellum (lateral vermal lobule VIII, pyramis) as a key supraspinal node within a chain of connections that links the PAG to the spinal cord to elicit fear‐evoked freezing behaviour. Expression of fear‐evoked freezing behaviour, both conditioned and innate, is dependent on cerebellar pyramis neural input–output pathways. We also address an important controversy in the literature, namely whether or not ventrolateral PAG (vlPAG) increases muscle tone. We provide evidence that activation of the vlPAG causes an increase in α‐motoneurone excitability, consistent with a role in generating muscle tone associated with fear‐evoked freezing.

Laminar organization of spinal dorsal horn neurones activated by C‐ vs. A‐heat nociceptors and their descending control from the periaqueductal grey in the rat

The periaqueductal grey can differentially control A‐ vs. C‐nociceptor‐evoked spinal reflexes and deep spinal dorsal horn neuronal responses. However, little is known about the control of A‐ vs. C‐fibre inputs to lamina I and the lateral spinal nucleus, and how this correlates with the control of deeper laminae. To address this, the laminar distributions of neurones expressing Fos‐like immunoreactivity were determined following preferential activation of A‐ or C‐heat nociceptors, using fast or slow rates of skin heating, respectively, in the absence or presence of descending control evoked from the periaqueductal grey. In lamina I, numbers of Fos‐positive neurones following both fast and slow rates of skin heating were reduced significantly following activation in the ventrolateral and dorsolateral/lateral periaqueductal grey. In contrast, in the deep dorsal horn (laminae III–VI), activation in both the ventrolateral and dorsolateral/lateral periaqueductal grey significantly reduced the numbers of Fos‐positive neurones evoked by C‐ but not A‐nociceptor stimulation. C‐ but not A‐heat nociceptor activation evoked Fos bilaterally in the lateral spinal nucleus. Stimulation in the ventrolateral but not the dorsolateral/lateral periaqueductal grey significantly increased the numbers of Fos‐positive neurones evoked by A‐ and C‐nociceptor stimulation bilaterally in the lateral spinal nucleus. These data have demonstrated differences in the descending control of the superficial vs. the deep dorsal horn and lateral spinal nucleus with respect to the processing of A‐ and C‐fibre‐evoked events. The data are discussed in relation to the roles of A‐ and C‐nociceptors in acute and chronic pain.

Separation of A- versus C-nociceptive inputs into spinal-brainstem circuits.

This study was designed to determine the organization of nociceptive inputs with different behavioral significance into spinal-brainstem circuits in the rat. Induction of Fos protein was used to localize spinal dorsal horn and hypothalamic neurons activated by noxious heating of the hind paw dorsum at rates known to preferentially activate C- or A-heat nociceptors. This was combined with retrograde transport of cholera toxin subunit B from the dorsolateral/lateral- (DL/L-) or the ventrolateral- (VL-) periaqueductal gray (PAG) in order to map the organization of A- and C-fiber input to spinal-brainstem circuits. The majority of dorsal horn heat-activated neurons were located in laminae I and II. A significantly larger proportion of C-fiber-activated neurons projected to the VL-PAG (P<0.05) compared with its DL/L-sector. In contrast, there was no columnar separation in the projections of A-fiber-activated neurons. However, a significantly greater proportion of A-fiber-activated neurons (P<0.05) were retrogradely labeled from the DL/L-PAG, when compared with C-fiber-activated neurons. A large proportion (25-50%) of A- and C-fiber-activated neurons in the lateral spinal nucleus projected to the PAG. A-fiber-activated neurons were found throughout the rostral hypothalamus but those projecting to the PAG were focused in the lateral area of the anterior hypothalamus (LAAH), from where approximately 20% projected to the VL-PAG, which was significantly more than to the DL/L PAG (P<0.05). We hypothesize that the organization of A- versus C-fiber inputs to the PAG enables the coordination of coping strategies appropriate to meet the demands imposed by these different noxious stimuli. Hypothalamic-PAG projections activated by A-fiber inputs did not reflect this level of organization and we suggest that this may relate to their role in thermoregulation as opposed to autonomic responses to particular nociceptive inputs.

The olivo-cerebellar system and its relationship to survival circuits

How does the cerebellum, the brain’s largest sensorimotor structure, contribute to complex behaviors essential to survival? While we know much about the role of limbic and closely associated brainstem structures in relation to a variety of emotional, sensory, or motivational stimuli, we know very little about how these circuits interact with the cerebellum to generate appropriate patterns of behavioral response. Here we focus on evidence suggesting that the olivo-cerebellar system may link to survival networks via interactions with the midbrain periaqueductal gray, a structure with a well known role in expression of survival responses. As a result of this interaction we argue that, in addition to important roles in motor control, the inferior olive, and related olivo-cortico-nuclear circuits, should be considered part of a larger network of brain structures involved in coordinating survival behavior through the selective relaying of “teaching signals” arising from higher centers associated with emotional behaviors.

The periaqueductal gray orchestrates sensory and motor circuits at multiple levels of the neuraxis

The periaqueductal gray (PAG) coordinates behaviors essential to survival, including striking changes in movement and posture (e.g., escape behaviors in response to noxious stimuli vs freezing in response to fear-evoking stimuli). However, the neural circuits underlying the expression of these behaviors remain poorly understood. We demonstrate in vivo in rats that activation of the ventrolateral PAG (vlPAG) affects motor systems at multiple levels of the neuraxis through the following: (1) differential control of spinal neurons that forward sensory information to the cerebellum via spino-olivo-cerebellar pathways (nociceptive signals are reduced while proprioceptive signals are enhanced); (2) alterations in cerebellar nuclear output as revealed by changes in expression of Fos-like immunoreactivity; and (3) regulation of spinal reflex circuits, as shown by an increase in α-motoneuron excitability. The capacity to coordinate sensory and motor functions is demonstrated in awake, behaving rats, in which natural activation of the vlPAG in fear-conditioned animals reduced transmission in spino-olivo-cerebellar pathways during periods of freezing that were associated with increased muscle tone and thus motor outflow. The increase in spinal motor reflex excitability and reduction in transmission of ascending sensory signals via spino-olivo-cerebellar pathways occurred simultaneously. We suggest that the interactions revealed in the present study between the vlPAG and sensorimotor circuits could form the neural substrate for survival behaviors associated with vlPAG activation. SIGNIFICANCE STATEMENT Neural circuits that coordinate survival behaviors remain poorly understood. We demonstrate in rats that the periaqueductal gray (PAG) affects motor systems at the following multiple levels of the neuraxis: (1) through altering transmission in spino-olivary pathways that forward sensory signals to the cerebellum, reducing and enhancing transmission of nociceptive and proprioceptive information, respectively; (2) by alterations in cerebellar output; and (3) through enhancement of spinal motor reflex pathways. The sensory and motor effects occurred at the same time and were present in both anesthetized animals and behavioral experiments in which fear conditioning naturally activated the PAG. The results provide insights into the neural circuits that enable an animal to be ready and able to react to danger, thus assisting in survival.

The periaqueductal grey modulates sensory input to the cerebellum: a role in coping behaviour?

The paths that link the periaqueductal grey (PAG) to hindbrain motor circuits underlying changes in behavioural responsiveness to external stimuli are unknown. A major candidate structure for mediating these effects is the cerebellum. The present experiments test this directly by monitoring changes in size of cerebellar responses evoked by peripheral stimuli following activation of the PAG. In 22 anaesthetized adult Wistar rats, climbing fibre field potentials were recorded from the C1 zone in the paramedian lobule and the copula pyramidis of the cerebellar cortex evoked, respectively, by electrical stimulation of the ipsilateral fore‐ and hindlimb. An initial and a late response were attributable to activation of Aβ and Aδ peripheral afferents respectively (hindlimb onset latencies 16.9 and 23.8 ms). Chemical stimulation at physiologically‐identified sites in the ventrolateral PAG (a region known to be associated with hyporeactive immobility) resulted in a significant reduction in size of both the Aβ and Aδ evoked field potentials (mean reduction relative to control ± SEM, 59 ± 7.5 and 66 ± 11.9% respectively). Responses evoked by electrical stimulation of the dorsal or ventral funiculus of the spinal cord were also reduced by PAG stimulation, suggesting that part of the modulation may occur at supraspinal sites (including at the level of the inferior olive). Overall, the results provide novel evidence of descending control into motor control centres, and provide the basis for future studies into the role of the PAG in regulating motor activity in different behavioural states and in chronic pain.