Mingchen Jiang

Participation of central descending nociceptive facilitatory systems in secondary hyperalgesia produced by mustard oil

The present series of experiments were designed to examine a potential role for central descending pain facilitatory systems in mediating secondary hyperalgesia produced by topical application of mustard oil and measuring the nociceptive tail-flick reflex in awake rats. Topical application of mustard oil (100%) to the lateral surface of the hind leg produced a facilitation of the tail-flick reflex that was significantly reduced in spinal transected animals. Mustard oil hyperalgesia was also inhibited in animals that had received electrolytic lesions in the rostral ventromedial medulla (RVM). Intrathecal (i.t.) administration of the non-selective cholecystokinin (CCK) receptor antagonist proglumide (10 micrograms) prior to mustard oil application completely blocked both the lesser and greater hyperalgesic responses observed in spinal transected and normal animals, respectively, and produced an inhibition of the tail-flick reflex in normal animals. Administration of the selective CCKB receptor antagonist L-365260 i.t. dose-dependently inhibited mustard oil hyperalgesia (ID50 = 364 ng) at doses approximately 5-fold less than the CCKA receptor antagonist devazepide (ID50 = 1760 ng). Similar to spinal proglumide, microinjection of the neurotensin antagonist SR48692 (3.5 micrograms) into the RVM blocked mustard oil hyperalgesia and inhibited the tail-flick reflex. These data suggest that secondary hyperalgesia produced by mustard oil is mediated largely by a central, centrifugal descending pain facilitatory system which involves neurotensin in the RVM and spinal CCK (via CCKB receptors). The inhibition of the tail-flick reflex produced by mustard oil following spinal or supraspinal administration of receptor antagonists suggests concurrent activation of central descending facilitatory and inhibitory systems.

Parvalbumin in respiratory neurons of the ventrolateral medulla of the adult rat

A column of parvalbumin immunoreactive neurons is closely associated with the location of respiratory neurons in the ventrolateral medulla of the rat. The majority (66%) of bulbospinal neurons in the medullary ventral respiratory column (VRC) that were retrogradely labeled by tracer injections in the phrenic nucleus were also positive for parvalbumin. In contrast, only 18.8% of VRC neurons retrogradely labeled after a tracer injection in the VRC, also expressed parvalbumin. The average cross-sectional area of VRC neurons retrogradely labeled after VRC injections was 193.8 μm2 ± 6.6 SE. These were significantly smaller than VRC parvalbumin neurons (271.9 μm2 ± 12.3 SE). Parvalbumin neurons were found in the Bötzinger Complex, the rostral ventral respiratory group (VRG), and the caudal VRG, areas which all contribute to the bulbospinal projection. In contrast, parvalbumin neurons were sparse or absent in the preBötzinger Complex and in the vicinity of the retrotrapezoid nucleus, areas that have few bulbospinal projections. Parvalbumin was rarely colocalized within Neurokinin-1 receptor positive (NK1R) VRC neurons, which are found in the preBötzinger complex and in the anteroventral part of the rostral VRG. Parvalbumin neurons in the Bötzinger Complex and rostral VRG help define the rostrocaudal extent of these regions. The absence of parvalbumin neurons from the intervening preBötzinger complex also helps establish the boundaries of this region. Regional boundaries described in this manner are in good agreement with earlier physiological and anatomical studies. Taken together, the distributions of parvalbumin, NK1R and bulbospinal neurons suggest that the rostral VRG may be subdivided into distinct, anterodorsal, anteroventral, and posterior subdivisions.

Development of mustard oil-induced hyperalgesia in rats

Abstract Age‐dependent changes in nociceptive responses were investigated using either the electromyogram (EMG) recorded from the hamstring muscle in response to electrical stimulation of the hind foot in spinal transected rats or measurement of the tail‐flick (TF) reflex latency in intact rats. The development of hyperalgesia produced by topical application of mustard oil was subsequently studied. In experiments involving EMG recordings, rats were tested from day 2 to day 34 after birth (4‐day interval) and as adults. In experiments involving measurement of the TF reflex, rats were tested from day 5 to day 30 after birth (5‐day interval) and as adults. It was found that the latency and the duration of an early component of the EMG decreased with an increase in animal age, and was similar to adult animals at approximately 18 days after birth. The thermal tail withdraw threshold was lower in pups in comparison with older rats, and took more than 30 postnatal days to become similar to that of adult rats. Although nociceptive behaviors such as biting, body movement, and vocalization could be produced in intact rats by mustard oil in rats as young as 5 days old, the intensities of these responses were subjectively less than those of adult rats. Mustard oil application enhanced significantly the EMG response to electrical stimulation and the effect increased with increasing age. Similarly, mustard oil applied to a hind leg facilitated the TF reflex (decreased response latency). In both experiments, it took approximately 34–40 postnatal days for mustard oil‐produced hyperalgesia to become similar to that of adult rats. These data confirm that nociceptive processing is not mature in the young animal and that a developmental period after birth is required for hyperalgesia‐related mechanisms to mature.

Progressive Changes in Synaptic Inputs to Motoneurons in Adult Sacral Spinal Cord of a Mouse Model of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is characterized by progressive degeneration of motoneurons. One potential mechanism is excitotoxicity. We studied the behaviors of spinal neurons using an in vitro preparation of the sacral cord from the G93A SOD1 mouse model of ALS. Measurements were conducted at presymptomatic [approximately postnatal day 50 (∼P50)], early (∼P90), and late (>P120) stages of the disease. Short-latency reflexes (SRs) in ventral roots, presumably monosynaptic, were evoked by electrical stimulation of a dorsal root. The fraction of motoneurons capable of responding to this activation was evaluated by measuring the compound action potential [total motor activity (TMA)] evoked by antidromic stimulation of the distal ventral root. In mutant SOD1 (mSOD1) mice, both the SR and the TMA decreased with age compared with nontransgenic littermates, ruling out the SR as a source of increasing excitotoxicity. Spinal interneuron activity was assessed using the synchronized ventral root bursts generated by both bath application of blockers of inhibitory neurotransmitters (glycine, GABAA) and agonists of glutamate receptors (especially NMDA receptors). After symptom onset, a higher percentage of preparations from mSOD1 mice exhibited bursting, and these bursts exhibited more sub-bursts and a more disorganized pattern. In mSOD1 mice with clear muscle tremor, the ventral roots exhibited spontaneous synchronized bursts, which were highly sensitive to the blockade of NMDA receptors. These data suggest that although short-latency sensory input does not increase as symptoms develop, interneuron activity does increase and may contribute to excitotoxicity.

Parabrachial-lateral pontine neurons link nociception and breathing.

We investigated the role of the parabrachial complex in cutaneous nociceptor-induced respiratory stimulation in chloralose-urethane anesthetized, vagotomized rats. Noxious stimulation (mustard oil, MO) applied topically to a forelimb or hindlimb enhanced the peak amplitude of the integrated phrenic nerve discharge and, with forelimb application, increased phrenic nerve burst frequency. Bilateral inactivation of neural activity in the parabrachial complex with injection of the GABA agonist muscimol (3nl) markedly attenuated the response to MO application. Injection of the retrograde tracer FluoroGold within the medullary ventral respiratory column labeled neurons in dorsolateral pontine regions known to receive nociceptive inputs (i.e., Kolliker-Fuse, lateral crescent, and superior lateral subnuclei of the parabrachial complex). Extracellular recordings of 65 dorsolateral parabrachial neurons revealed about 15% responded to a noxious cutaneous pinch with either an increase or a decrease in discharge and approximately 40% of these exhibited a phasic respiratory-related component to their discharge. In conclusion, parabrachial pontine neurons contribute to cutaneous nociceptor-induced increases in breathing.

O-Antigen Modulates Infection-Induced Pain States

The molecular initiators of infection-associated pain are not understood. We recently found that uropathogenic E. coli (UPEC) elicited acute pelvic pain in murine urinary tract infection (UTI). UTI pain was due to E. coli lipopolysaccharide (LPS) and its receptor, TLR4, but pain was not correlated with inflammation. LPS is known to drive inflammation by interactions between the acylated lipid A component and TLR4, but the function of the O-antigen polysaccharide in host responses is unknown. Here, we examined the role of O-antigen in pain using cutaneous hypersensitivity (allodynia) to quantify pelvic pain behavior and using sacral spinal cord excitability to quantify central nervous system manifestations in murine UTI. A UPEC mutant defective for O-antigen biosynthesis induced chronic allodynia that persisted long after clearance of transient infections, but wild type UPEC evoked only acute pain. E. coli strains lacking O-antigen gene clusters had a chronic pain phenotype, and expressing cloned O-antigen gene clusters altered the pain phenotype in a predictable manner. Chronic allodynia was abrogated in TLR4-deficient mice, but inflammatory responses in wild type mice were similar among E. coli strains spanning a wide range of pain phenotypes, suggesting that O-antigen modulates pain independent of inflammation. Spinal cords of mice with chronic allodynia exhibited increased spontaneous firing and compromised short-term depression, consistent with centralized pain. Taken together, these findings suggest that O-antigen functions as a rheostat to modulate LPS-associated pain. These observations have implications for an infectious etiology of chronic pain and evolutionary modification of pathogens to alter host behaviors.

In vitro sacral cord preparation and motoneuron recording from adult mice

We report the development of an intracellular recording technique for adult mouse motoneurons in sacral spinal cord. Based on a similar preparation for adult rat, we modified the cord preparation solution and filled the sharp electrode with a solution that has physiological osmolarity and pH. The viability of the preparation was examined by recording root reflexes. Short-latency reflexes mediated through monosynaptic transmission between S1 and S3 ventral root were reliably produced by dorsal root electrical stimuli and were stably recorded for more than eight hours. Long-lasting potentiation of the root reflex was observed by bath application of methoxamine, a noradrenergic alpha1 receptor agonist. Bath application of strychnine and picrotoxin, antagonists for glycine and GABA(A) receptors respectively, unmasked long-lasting reflexes that may contain polysynaptic components. In addition, on the background of strychnine and picrotoxin, adding methoxamine induced spontaneous ventral root activity. For intracellular recording, the motoneurons could be reliably penetrated and held for up to 30 min. In all 16 motoneurons recorded, resting membrane potential, input resistance, action potentials and repetitive firing were comparable to those of rat motoneurons. Thus, this preparation is viable and provides a new method for combined electrophysiological and genetic studies of the adult mouse spinal cord.

Cerebellar input to magnocellular neurons in the red nucleus of the mouse: synaptic analysis in horizontal brain slices incorporating cerebello-rubral pathways.

We studied the synaptic input from the nucleus interpositus of the cerebellum to the magnocellular division of the red nucleus (RNm) in the mouse using combined electrophysiological and neuroanatomical methods. Whole-cell patch-clamp recordings were made from brain slices (125-150 microm) cut in a horizontal plane oriented to pass through both red nucleus and nucleus interpositus. Large cells that were visually selected and patched were injected with Lucifer Yellow and identified as RNm neurons. Using anterograde tracing from nucleus interpositus in vitro, we examined the course of interposito-rubral axons which are dispersed in the superior cerebellar peduncle. In vitro monosynaptic responses in RNm were elicited by an electrode array placed contralaterally in this pathway but near the midline. Mixed excitatory post-synaptic potentials (EPSPs)/inhibitory post-synaptic potentials (IPSPs) were observed in 48 RNm neurons. Excitatory components of the evoked potentials were studied after blocking inhibitory components with picrotoxin (100 microM) and strychnine (5 microM). All RNm neurons examined continued to show monosynaptic EPSPs after non-N-methyl-D-aspartate (NMDA) glutamate receptor components were blocked with 10 microM 6,7-dinitroquinoxaline-2,3-dione or 5 microM 2,3-dihydro-6-nitro-7-sulfamoyl-benzo(f)-quinoxaline (NBQX; n=12). The residual potentials were identified as NMDA receptor components since they (i) were blocked by the addition of the NMDA receptor antagonist, D,L-2-amino-5-phosphonovaleric acid (APV), (ii) were voltage-dependent, and (iii) were enhanced by Mg(2+) removal. Inhibitory components of the evoked potentials were studied after blocking excitatory components with NBQX and APV. Under these conditions, all RNm neurons studied continued to show IPSPs. Blockade of GABA(A) receptors reduced but did not eliminate the IPSPs. These were eliminated when GABA(A) receptor blockade was combined with strychnine to eliminate glycine components of the IPSPs. Thus, IPSPs evoked by midline stimulation of the superior cerebellar peduncle, while blocking alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and NMDA receptors, raise the possibility of direct inhibitory inputs to RNm from the cerebellum. In summary we propose that the special properties of the NMDA receptor components are considered important for the generation of RNm motor commands: their slow time course will contribute a steady driving force for sustained discharge and their voltage dependency will facilitate abrupt transitions from a resting state of quiescence to an active state of intense motor command generation.

The essential and downstream common proteins of amyotrophic lateral sclerosis: A protein-protein interaction network analysis

Amyotrophic Lateral Sclerosis (ALS) is a devastative neurodegenerative disease characterized by selective loss of motoneurons. While several breakthroughs have been made in identifying ALS genetic defects, the detailed molecular mechanisms are still unclear. These genetic defects involve in numerous biological processes, which converge to a common destiny: motoneuron degeneration. In addition, the common comorbid Frontotemporal Dementia (FTD) further complicates the investigation of ALS etiology. In this study, we aimed to explore the protein-protein interaction network built on known ALS-causative genes to identify essential proteins and common downstream proteins between classical ALS and ALS+FTD (classical ALS + ALS/FTD) groups. The results suggest that classical ALS and ALS+FTD share similar essential protein set (VCP, FUS, TDP-43 and hnRNPA1) but have distinctive functional enrichment profiles. Thus, disruptions to these essential proteins might cause motoneuron susceptible to cellular stresses and eventually vulnerable to proteinopathies. Moreover, we identified a common downstream protein, ubiquitin-C, extensively interconnected with ALS-causative proteins (22 out of 24) which was not linked to ALS previously. Our in silico approach provides the computational background for identifying ALS therapeutic targets, and points out the potential downstream common ground of ALS-causative mutations.

Hyperexcitability in synaptic and firing activities of spinal motoneurons in an adult mouse model of amyotrophic lateral sclerosis

Hyperexcitability is hypothesized to contribute to the degeneration of spinal motoneurons (MNs) in amyotrophic lateral sclerosis (ALS). Studies, thus far, have not linked hyperexcitability to the intrinsic properties of MNs in the adult ALS mouse model with the G93A-mutated SOD1 protein (mSOD1G93A). In this study, we obtained two types of measurements: ventral root recordings to assess motor output and intracellular recordings to assess synaptic properties of individual MNs. All studies were carried out in an in vitro preparation of the sacral spinal cords of mSOD1G93A mice and their non-transgenic (NT) littermates, both in the age range of 50-90days. Ventral root recordings revealed that maximum compound action potentials (coAPs) evoked by a short-train stimulation of corresponding dorsal roots were similar between the two types of mice. Although the progressive depression of coAPs was present during the train stimulation in all recordings, the coAP depression in mSOD1G93A mice was to a lesser extent, which suggests an increased firing tendency in mSOD1G93A MNs. Intracellular recordings showed no changes in fast excitatory postsynaptic potentials (EPSPs) in mSOD1G93A MNs. However, recording did show that oscillating EPSPs (oEPSPs) were induced by poly-EPSPs at a higher frequency and by less-intense electrical stimulation in mSOD1G93A MNs. These oEPSPs were dependent upon the activities of spinal network and N-methyl-d-aspartate receptors (NMDARs), and were subjected to riluzole modulation. Taken together, these findings revealed abnormal electrophysiology in mSOD1G93A MNs that could underlie ALS excitotoxicity.