Simon McMullan

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.

trkA Is Expressed in Nociceptive Neurons and Influences Electrophysiological Properties via Nav1.8 Expression in Rapidly Conducting Nociceptors

To test the hypothesis that trkA (the high-affinity NGF receptor) is selectively expressed in nociceptive dorsal root ganglion (DRG) neurons, we examined the intensity of trkA immunoreactivity in single dye-injected rat DRG neurons, the sensory receptor properties of which were identified in vivo with mechanical and thermal stimuli. We provide the first evidence in single identified neurons that strong trkA expression in DRGs is restricted to nociceptive neurons, probably accounting for the profound influence of NGF on these neurons. Furthermore, we demonstrate that trkA expression is as high in rapidly conducting (Aα/β) as in more slowly conducting (Aδ and C) nociceptors. All Aα/β low-threshold mechanoreceptors (LTMs) are trkA negative, although weak but detectable trkA is present in some C and Aδ LTMs. NGF can influence electrophysiological properties of DRG neurons, probably by binding to trkA. We found positive correlations for single identified Aα/β (but not C or Aδ) nociceptors between trkA immunocytochemical intensity and electrophysiological properties typical of nociceptors, namely long action potential and afterhyperpolarization durations and large action potential amplitudes. Furthermore, for Aα/β (notCorAδ) nociceptors, trkA intensity is inversely correlated with conduction velocity. Similar relationships, again only in Aα/β nociceptors, between electrophysiological properties and trkA expression exist for sodium channel Nav1.8 but not Nav1.9 immunoreactivities. These findings suggest that in Aα/β nociceptors, influences of NGF on expression levels of Nav1.8 are related to, and perhaps limited by, expression levels of trkA. This view is supported by a positive correlation between immuno-intensities of trkA and Nav1.8 in A-fiber, but not C-fiber, nociceptors.

Differential regulation of the central neural cardiorespiratory system by metabotropic neurotransmitters

Central neurons in the brainstem and spinal cord are essential for the maintenance of sympathetic tone, the integration of responses to the activation of reflexes and central commands, and the generation of an appropriate respiratory motor output. Here, we will discuss work that aims to understand the role that metabotropic neurotransmitter systems play in central cardiorespiratory mechanisms. It is well known that blockade of glutamatergic, gamma-aminobutyric acidergic and glycinergic pathways causes major or even complete disruption of cardiorespiratory systems, whereas antagonism of other neurotransmitter systems barely affects circulation or ventilation. Despite the lack of an ‘all-or-none’ role for metabotropic neurotransmitters, they are nevertheless significant in modulating the effects of central command and peripheral adaptive reflexes. Finally, we propose that a likely explanation for the plethora of neurotransmitters and their receptors on cardiorespiratory neurons is to enable differential regulation of outputs in response to reflex inputs, while at the same time maintaining a tonic level of sympathetic activity that supports those organs that significantly autoregulate their blood supply, such as the heart, brain, retina and kidney. Such an explanation of the data now available enables the generation of many new testable hypotheses.

HCN1 and HCN2 in Rat DRG Neurons: Levels in Nociceptors and Non-Nociceptors, NT3-Dependence and Influence of CFA-Induced Skin Inflammation on HCN2 and NT3 Expression

Ih, which influences neuronal excitability, has recently been measured in vivo in sensory neuron subtypes in dorsal root ganglia (DRGs). However, expression levels of HCN (hyperpolarization-activated cyclic nucleotide-gated) channel proteins that underlie Ih were unknown. We therefore examined immunostaining of the most abundant isoforms in DRGs, HCN1 and HCN2 in these neuron subtypes. This immunostaining was cytoplasmic and membrane-associated (ring). Ring-staining for both isoforms was in neurofilament-rich A-fiber neurons, but not in small neurofilament-poor C-fiber neurons, although some C-neurons showed cytoplasmic HCN2 staining. We recorded intracellularly from DRG neurons in vivo, determined their sensory properties (nociceptive or low-threshold-mechanoreceptive, LTM) and conduction velocities (CVs). We then injected fluorescent dye enabling subsequent immunostaining. For each dye-injected neuron, ring- and cytoplasmic-immunointensities were determined relative to maximum ring-immunointensity. Both HCN1- and HCN2-ring-immunointensities were positively correlated with CV in both nociceptors and LTMs; they were high in Aβ-nociceptors and Aα/β-LTMs. High HCN1 and HCN2 levels in Aα/β-neurons may, via Ih, influence normal non-painful (e.g. touch and proprioceptive) sensations as well as nociception and pain. HCN2-, not HCN1-, ring-intensities were higher in muscle spindle afferents (MSAs) than in all other neurons. The previously reported very high Ih in MSAs may relate to their very high HCN2. In normal C-nociceptors, low HCN1 and HCN2 were consistent with their low/undetectable Ih. In some C-LTMs HCN2-intensities were higher than in C-nociceptors. Together, HCN1 and HCN2 expressions reflect previously reported Ih magnitudes and properties in neuronal subgroups, suggesting these isoforms underlie Ih in DRG neurons. Expression of both isoforms was NT3-dependent in cultured DRG neurons. HCN2-immunostaining in small neurons increased 1 day after cutaneous inflammation (CFA-induced) and recovered by 4 days. This could contribute to acute inflammatory pain. HCN2-immunostaining in large neurons decreased 4 days after CFA, when NT3 was decreased in the DRG. Thus HCN2-expression control differs between large and small neurons.

Midbrain control of spinal nociception discriminates between responses evoked by myelinated and unmyelinated heat nociceptors in the rat

Abstract Descending control of spinal nociception is a major determinant of normal and chronic pain. Myelinated (A‐fibre) and unmyelinated (C‐fibre) nociceptors convey different qualities of the pain signal (first and second pain, respectively), and they play different roles in the development and maintenance of chronic pain states. It is of considerable importance, therefore, to determine whether descending control has differential effects on the central processing of A‐ vs. C‐nociceptive input. In anaesthetised rats, biceps femoris EMG was recorded to monitor the thresholds and encoding properties of responses evoked by fast (7.5 °C s−1) or slow (2.5 °C s−1) rates of skin heating of the dorsal surface of a hindpaw to preferentially activate myelinated or unmyelinated heat nociceptors, respectively. Activation of neurones in the periaqueductal grey (PAG) by microinjection of dl‐homocysteic acid (DLH) or bicuculline (BIC) significantly increased response thresholds to slow rates of heating (P < 0.001), but not those to fast rates of heating (P > 0.05). The ability of the EMG to encode the stimulus intensity of fast rates of skin heating remained intact and unaltered (r2 = 0.99, P < 0.001) following BIC but not DLH injection. In contrast, encoding of the stimulus intensity of slow rates of skin heating was abolished following BIC and DLH injection. The functional significance of differential descending control of the central processing of C‐ and A‐nociceptive inputs is discussed with respect to role of the PAG in mediating antinociception as part of active coping strategies in emergency situations and the role of C‐ and A‐nociceptive inputs in animal models of chronic pain.

Somatostatin 2A Receptor-Expressing Presympathetic Neurons in the Rostral Ventrolateral Medulla Maintain Blood Pressure

Bulbospinal neurons in the rostral ventrolateral medulla (RVLM) are critical for the maintenance of sympathetic vasomotor tone and normal cardiovascular reflex function. So far, selectively eliminating/inhibiting distinct subpopulations of RVLM neurons has not significantly altered arterial pressure. Here we show that RVLM presympathetic neurons that express somatostatin 2A receptors are essential for maintaining and potentially generating sympathetic vasomotor tone. Combined immunocytochemistry and in situ hybridization were used to map the expression of somatostatin receptors 1, 2A, 2B, 3, and 4 (sst1 through 4, respectively) in the rat RVLM. sst1 and sst2B were absent; sst3 and sst4 were sparse. However, sst2A was found postsynaptically and detected in 35±5% of bulbospinal RVLM neurons a population that included 54±4% of catecholaminergic and 30±3% of enkephalinergic neurons. Bilateral microinjection into the RVLM of either somatostatin or the receptor-selective agonist lanreotide evoked dramatic, dose-dependent sympathoinhibition, hypotension, and bradycardia that were blocked by the sst2 receptor antagonist BIM-23627 in anesthetized rats. Bilateral RVLM microinjection of somatostatin also attenuated chemoreceptor and somatosympathetic reflex function. Somatostatin only eliminated the first sympathoexcitatory peak evoked by somatosympathetic reflex activation, whereas muscimol abolished both excitatory peaks providing functional evidence that the activity of only a subpopulation of RVLM presympathetic neurons is inhibited by somatostatin. We suggest that the subpopulation of bulbospinal RVLM neurons that expresses the sst2A receptor sets sympathetic vasomotor output. These neurons are essential for maintaining resting blood pressure under anesthesia and contribute to adaptive reflexes mediated through the RVLM.

Somatostatin selectively ablates post-inspiratory activity after injection into the Bötzinger complex

Somatostatin (SST) neurons in the ventral respiratory column (VRC) are essential for the generation of normal breathing. Little is known about the neuromodulatory role of SST on ventral respiratory neurons other than that local administration induces apnoea. Here, we describe the cardiorespiratory effects of microinjecting SST into the preBötzinger and Bötzinger complexes which together elaborate a normal inspiratory augmenting and expiratory respiratory pattern, and on spinally projecting respiratory subnuclei (rostral ventral respiratory group; rVRG). Microinjections (20-50 nl) of SST (0.15, 0.45, 1.5 mM) were made into respiratory subnuclei of urethane-anaesthetized, paralysed, vagotomized and artificially ventilated Sprague-Dawley rats (n=46). Unilateral microinjection of SST into the Bötzinger complex converted the augmenting activity of phrenic nerve discharge into a square-wave apneustic pattern associated with a lengthening of inspiratory period and shortening of expiratory time. Following bilateral microinjection the apneusis became pronounced and was associated with a dramatic variability in inspiratory duration. Microinjection of SST into the Bötzinger complex also abolished the post-inspiratory (post-I) motor activity normally observed in vagal and sympathetic nerves. In the preBötzinger complex SST caused bradypnoea and with increasing dose, apnoea. In the rVRG SST reduced phrenic nerve amplitude, eventually causing apnoea. In conclusion, SST powerfully inhibits respiratory neurons throughout the VRC. Of particular interest is the finding that chemical inhibition of the Bötzinger complex with SST ablates the post-I activity that is normally seen in respiratory activity and leads to apneusis. This loss of post-I activity is a unique feature of inhibition with SST and is not seen following inhibition with other agents such as galanin, GABA and endomorphin. The effect seen on post-I activity is similar to the effect of inhibiting the Kölliker-Fuse nucleus in the pons. The mechanism by which SST exerts this effect on Bötzinger neurons remains to be determined.

Circulating angiotensin II attenuates the sympathetic baroreflex by reducing the barosensitivity of medullary cardiovascular neurones in the rat

Chronic intravenous angiotensin II (Ang II) has been widely used to establish centrally mediated hypertension in experimental animals, and disruption of Ang II activity is a frontline treatment for hypertensive disease. However, the acute central actions of circulating Ang II are poorly understood. We examined the effects of intravenous pressor doses of Ang II on autonomic activity in anaesthetized rats under neuromuscular blockade, and compared baroinhibition evoked by Ang II pressor ramps to equipressor responses evoked by phenylephrine (PE). Baroinhibition of splanchnic sympathetic nerve activity was attenuated during Ang II trials compared with PE, and rats remained sensitive to electrical stimulation of the aortic depressor nerve at higher arterial pressures during Ang II trials. This was not due to a direct effect of Ang II on aortic nerve baroreceptors. In a separate series of experiments, we provide direct evidence that bulbospinal barosensitive neurones in the rostral ventrolateral medulla are differentially sensitive to pressure ramps evoked by Ang II or PE vasoconstriction. Nineteen out of 41 units were equally sensitive to increased arterial pressure evoked by Ang II or PE. In 17 of 41 units, barosensitivity was attenuated during Ang II trials, and in five of 41 cases units that had previously been barosensitive increased their firing rate during Ang II trials. These results show, for the first time, that circulating Ang II acutely modulates central cardiovascular control mechanisms. We suggest that this results from activation by Ang II of a central pathway originating at the circumventricular organs.

Spinal dorsal horn neuronal responses to myelinated versus unmyelinated heat nociceptors and their modulation by activation of the periaqueductal grey in the rat

The aim of this study was to further understand the central processing of inputs arising from unmyelinated and myelinated nociceptors by (i) determining the response characteristics of Class 2 dorsal horn neurones to preferential activation of C‐ and A‐fibre heat nociceptors, and (ii) investigating the control exerted by the dorsolateral/lateral region of the midbrain periaqueductal grey (DL/L‐PAG) on C‐ and A‐fibre‐evoked responses of these neurones. The use of different rates of skin heating to preferentially activate unmyelinated (C‐fibre; 2.5°C s−1) versus myelinated (A‐fibre; 7.5°C s−1) heat nociceptors revealed that, in response to C‐nociceptor activation, Class 2 neurones encode well only over the first 5°C above threshold, and that at higher temperatures responses decline. In contrast, responses to A‐nociceptor activation are linear and encode skin temperature over more than 10°C, and almost certainly into the tissue‐damaging range. PAG stimulation raised thresholds and decreased significantly the magnitude of responses to A‐ and C‐nociceptor activation. However, differences were revealed in the effects of descending control on the relationships between skin temperature and neuronal firing rate; the linear relationship that occurred over the first 5°C of slow rates of skin heating was no longer evident, whereas that to fast rates of skin heating was maintained over the entire range, albeit shifted to the right. These data indicate that the sensori‐discriminative information conveyed in A‐fibre nociceptors is maintained and that the information from C‐nociceptors is lost in the presence of descending control from the DL/L‐PAG. The data are discussed in relation to the role of the DL/L‐PAG in mediating active coping strategies.

Brain sources of inhibitory input to the rat rostral ventrolateral medulla

The rostral ventrolateral medulla (RVLM) contains neurons critical for cardiovascular, respiratory, metabolic, and motor control. The activity of these neurons is controlled by inputs from multiple identified brain regions; however, the neurochemistry of these inputs is largely unknown. Gamma‐aminobutyric acid (GABA) and enkephalin tonically inhibit neurons within the RVLM. The aim of this study was to identify all brain regions that provide GABAergic or enkephalinergic input to the rat RVLM. Neurons immunoreactive for cholera toxin B (CTB‐ir), retrogradely transported from the RVLM, were assessed for expression of glutamic acid decarboxylase (GAD67) or preproenkephalin (PPE) mRNA using in situ hybridization. GAD67 mRNA was expressed in CTB‐ir neurons in the following regions: the nucleus of the solitary tract (NTS, 6% of CTB‐ir neurons), area postrema (AP, 8%), caudal ventrolateral medulla (17%), midline raphe (40%), ventrolateral periaqueductal gray (VLPAG, 15%), lateral hypothalamic area (LHA, 25%), central nucleus of the amygdala (CeA, 77%), sublenticular extended amygdala (SLEA, 86%), interstitial nucleus of the posterior limb of the anterior commissure (IPAC, 56%), bed nucleus of the stria terminals (BNST, 59%), and medial preoptic area (MPA, 53%). PPE mRNA was expressed in CTB‐ir neurons in the following regions: the NTS (14% of CTB‐ir neurons), midline raphe (26%), LHA (22%), zona incerta (ZI, 15%), CeA (5%), paraventricular nucleus (PVN, 13%), SLEA (66%), and MPA (26%). Thus, limited brain regions contribute GABAergic and/or enkephalinergic input to the RVLM. Multiple neurochemically distinct pathways originate from these brain regions projecting to the RVLM. J. Comp. Neurol. 521:213–232, 2013.