use-vivo-single-fiber-recording-intact-dorsal-root-ganglion-with

Use of In Vivo Single-fiber Recording and Intact Dorsal Root Ganglion with Attached Sciatic Nerve to Examine the Mechanism of Conduction Failure

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for ...

Fourth Military Medical University

In this study, we compared the sensitivity of non-periodically and periodically active neurons in chronically compressed dorsal root ganglion in rats to norepinephrine and sympathetic stimulation. Forty-nine of 58 (84.5%) neurons with non-periodic activity showed responses to norepinephrine, whereas only five of 48 (10.4%) neurons with periodic activity displayed any response. The dose-response relationship of norepinephrine to the irregular burst pattern neurons shifted towards the left significantly compared to that of the periodic activity neurons. Responses to norepinephrine became apparent in eight neurons after their periodic firing activity was transformed into the non-periodic firing activity through the increase in Ca(2+). Changes in the time-response curves indicate a higher sensitivity of irregular burst pattern neurons to sympathetic stimulation than the periodic activity neurons. Finally, deterministic dynamics contained within the interburst interval series for non-periodic activity were identified. From these results, we suggest that the non-periodic activity neurons have a higher adrenergic sensitivity than those displaying periodic activity, and that this sensitivity may depend on the deterministic chaos within its firing dynamic system.

Adrenergic sensitivity of neurons with non-periodic firing activity in rat injured dorsal root ganglion.

Our recent data revealed adrenergic sensitivity in chronically compressed dorsal root ganglion (DRG) of rats. As neuropeptide Y (NPY) is a common sympathetic co-transmitter, we investigated the effect of NPY on injured DRG neurons. The expression of NPY Y1 and Y2 receptors and the effect of NPY on chronically compressed DRG neurons were studied using in situ hybridization and extracellular single fiber recording in vitro, respectively. After DRG compression, the expression of Y1 receptor was distinctly increased in large and medium-sized DRG neurons, while Y2 receptor was increased in small DRG neurons. NPY inhibited both the spontaneous activity and the excitatory effect of norepinephrine in injured DRG A-neurons. The results suggest a possibility that NPY may inhibit the hyperexcitability of injured DRG A-neurons via increased Y1 receptor following chronic compression.

Neuropeptide Y inhibits the hyperexcitability of type A neurons in chronically compressed dorsal root ganglion of the rat.

Our previous works suggested that sensitivity of neurons with chaotic firing patterns to stimuli is significantly greater than that in neurons with periodic firing patterns, which shows that responsiveness of neurons may depend on the complexity of the firing series. This study was performed to determine the relation between responsiveness of the hippocampal CA1 neurons with epileptiform activity (EA) to neurotransmitters and their complexity of firing series.

Relation between responsiveness to neurotransmitters and complexity of epileptiform activity in rat hippocampal CA1 neurons.

Neurons can make different responses to identical inputs. According to the emerging frequency of repetitive firing, neurons are classified into two types: type 1 and type 2 excitability. Though in mathematical simulations, minor modifications of parameters describing ionic currents can result in transitions between these two excitabilities, empirical evidence to support these theoretical possibilities is scarce. Here we report a joint theoretical and experimental study to test the hypothesis that changes in parameters describing ionic currents cause predictable transitions between the two excitabilities in mesencephalic V (Mes V) neurons. We developed a simple mathematical model of Mes V neurons. Using bifurcation analysis and model simulation, we then predicted that changes in conductance of two low-threshold currents would result in transitions between type 1 and type 2. Finally, by applying specific channel blockers, we observed the transition between two excitabilities forecast by the mathematical model.

Transition between two excitabilities in mesencephalic V neurons.

It has been confirmed that the voltage-gated persistent sodium currents mediate the generation of subthreshold membrane potential oscillations (SMPOs) and contribute to shaping repetitive firing. Our previous study indicated that gabapentin (GBP) administration induced a dose-dependent inhibition of SMPO in chronically compressed dorsal root ganglion (CCD) neurons. To investigate the mechanisms and possible site(s) of action of GBP, the persistent sodium currents (I(NaP)) were measured and the effects of GBP on I(NaP) were examined in CCD neurons electrophysiologically in vitro. DRG neurons possess slow TTX-sensitive inactivating sodium currents that significantly contribute to the generation of membrane oscillations by amplifying the resonance behavior. GBP reduced the resonant amplitude of DRG neurons as well as inhibiting the firing and SMPO induced by injection current, which was strongly due to the inhibitory effect on persistent sodium currents. Furthermore, we found that GBP (1-20 microM) administration inhibited the persistent sodium currents in dose-dependent manner, while the changes of K(+) and Ca(2+) current minimally contributed to the effect of GBP on oscillation and resonant behavior of DRG neurons. In contrast, the amplitude and voltage-dependence of transient sodium current were unchanged by GBP. The results suggest that GBP decreased the amplitude of resonance and abolished the SMPO of A-type DRG neurons through the inhibition of I(NaP), and thus inhibited the SMPO dependent repetitive and bursting firings.

Gabapentin selectively reduces persistent sodium current in injured type-A dorsal root ganglion neurons.

Respiratory behavior expresses diverse forms of plasticity by altering breathing patterns. Failure of respiratory neuroplasticity often leads to malfunctions. Long-term facilitation (LTF), the most frequently studied model induced by episodic hypoxia to produce long-lasting enhancement of phrenic motor output, is thought to be serotonin 2A (5-HT(2A)) receptor-dependent. Previous studies have described 5-HT-induced prompt apnea in intact animals. However, the role of exogenous 5-HT in mediating respiratory neuroplasticity is less attended in vivo study. We hypothesized that an in vivo 5-HT challenge contributes to respiratory neuroplasticity. Here, we found that systemic bolus administration of 5-HT exerted an initial transient inhibition followed by marked facilitation, forming a biphasic pattern of phrenic nerve activity in artificially ventilated, midcervically vagotomized, and anesthetized adult rats. The facilitatory phase corresponded to the enhanced phrenic nerve activity that lasted for at least one hour after drug exposure, characterized as phrenic LTF (pLTF). The 5-HT-induced biphasic pattern and pLTF were 5-HT(2A) receptor-dependent and coupled to protein kinase C (PKC) activation. The initial inhibition of phrenic nerve activity was found to be nodose ganglion-associated, whereas the subsequent facilitation was carotid body-associated, establishing a peripheral inhibitory-facilitatory afferent balance. Immunoreactive expressions of 5-HT/5-HT(2A) receptors and phospho-PKC isoforms/PKC substrate provide morphological evidence of existence of a 5-HT/5-HT(2A) receptor/PKC mechanism in the nodose ganglion and the carotid body. We speculate that 5-HT challenge in vivo may contribute to respiratory neuroplasticity, to yield pLTF or augmented pLTF in animals with reduced or absent peripheral inhibitory inputs.

5-HT induces enhanced phrenic nerve activity via 5-HT(2A) receptor/PKC mechanism in anesthetized rats.

Methylcobalamin (MeCbl), the activated form of vitamin B12, has been used to treat some nutritional diseases and other diseases in clinic, such as Alzheimer's disease and rheumatoid arthritis. As an auxiliary agent, it exerts neuronal protection by promoting regeneration of injured nerves and antagonizing glutamate-induced neurotoxicity. Recently several lines of evidence demonstrated that MeCbl may have potential analgesic effects in experimental and clinical studies. For example, MeCbl alleviated pain behaviors in diabetic neuropathy, low back pain and neuralgia. MeCbl improved nerve conduction, promoted the regeneration of injured nerves, and inhibited ectopic spontaneous discharges of injured primary sensory neurons. This review aims to summarize the analgesic effect and mechanisms of MeCbl at the present. 

Methylcobalamin: a potential vitamin of pain killer.

Intrinsic resonant frequency properties of neurons in the brain provide a basis for different behavioral states and determine the responding modality (i.e. excitability) of different neurons. Previously, three types of excitability behaviors in rat Mesencephalic V neurons were found. To investigate the different resonant frequency and ionic current mechanisms underlying different resonances among three types of excitability, we performed whole-cell patch recordings and applied ZAP current on Mesencephalic V cells (N=70) in rat slices. The results show that: Class 2 neurons (30/30) have two resonances with U-shaped voltage dependence. One is a high frequency resonance (75.4 ± 2.11 Hz) significant at depolarized potentials (about -50 mV) and the other is a low frequency resonance (5.46 ± 0.31 Hz) significant at hyperpolarized potentials (about -70 mV). Voltage clamp experiments reveal two non-inactivating currents operating in the subthreshold voltage range: (1) 4-aminopyridine sensitive K(+) current, which activates at membrane potentials positive than -60 mV and was blocked by 4-AP (50 µM), was underlying the high frequency resonance of Class 2 neuron; (2) h-current, which activates negative than -60mV and was blocked by the hyperpolarization-activated cyclic nucleotide-gated channel blocker ZD7288 (10 µM), was underlying the low frequency resonance. Class 1 neurons do not show voltage-dependence resonant behaviors (10/10). Class 3 neurons (23/30) have two resonances, which are similar with Class 2 in both resonant frequency and currents. Our results provide clear evidence for the existence of multiple kinds of frequency resonances and how the relationship between the resonant frequency and the ionic current is based in Mesencephalic V neurons.

Resonance characteristic and its ionic basis of rat mesencephalic trigeminal neurons.

Recently several lines of evidence demonstrated that methylcobalamin (MeCbl) might have potential analgesic effect in experimental and clinical studies. However, it was reported that MeCbl had no effect on treating lumbar spinal stenosis induced pain. Thus, the effects of short-term and long-term administration of MeCbl were examined in the chronic compression of dorsal root ganglion (CCD) model. We found that mechanical allodynia was significantly inhibited by a continuous application of high dose and a single treatment of a super high dose of MeCbl. Little is known about mechanisms underlying the analgesia of MeCbl. We examined the effect of MeCbl on the spontaneous activity (SA), the excitability, and hyperpolarization-activated nonselective cation ion current in compressed medium-sized dorsal root ganglion (DRG) neurons using extracellular single fiber recording in vivo and whole-cell patch clamp in vitro. We found that MeCbl significantly inhibited the SA of A-type sensory neurons in a dose-dependent manner and inhibited the excitability of medium-sized DRG neurons. In addition, MeCbl also decreased I h current density in injured medium-sized DRG neurons. Our results proved that MeCbl might exert an analgesic effect through the inhibition I h current and then might inhibit the hyperexcitability of primary sensory neurons under neuropathic pain state. 

Inhibition of Hyperpolarization-Activated Cation Current in Medium-Sized DRG Neurons Contributed to the Antiallodynic Effect of Methylcobalamin in the Rat of a Chronic Compression of the DRG.

Postoperative cognitive dysfunction (POCD), mainly characterized by short-term decline of learning and memory, occurs after operations under anesthesia. However, the underlying mechanisms are poorly understood. The μ-opioid receptors (MOR) are highly expressed in interneurons of hippocampus, and is believed to be critical for the dysfunction of synaptic plasticity between hippocampal neurons. Therefore, we investigated the effect of fentanyl, a strong agonist of MOR and often used for anesthesia and analgesia in clinical settings, on hippocampal synaptic plasticity in the Schaffer-collateral CA1 pathway during acute exposure and washout in vitro. Our results revealed that acute fentanyl exposure (0.01, 0.1, 1 μM) dose-dependently increased the field excitatory postsynaptic potentials (fEPSPs), which was prevented by pre-administration of picrotoxin (50 μM) or MOR antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Phe-Thr-NH2 (CTOP, 10 μM). While fentanyl exposure-increased fEPSPs amplitude was prevented by picrotoxin [an inhibitor of γ-aminobutyric acid receptor (GABAR)] treatment or fentanyl washout, pretreatment of picrotoxin failed to prevent the fentanyl-impaired long-term potentiation (LTP) of synaptic strength as well as the fentanyl-enhanced long-term depression (LTD). These results demonstrated that fentanyl acute exposure and washout increases hippocampal excitability in the Schaffer-collateral CA1 pathway, depending on disinhibiting interneurons after MOR activation. In addition, fentanyl acute exposure and washout modulated synaptic plasticity, but the inhibitory activation was not critical. Elucidating the detailed mechanisms for synaptic dysfunction after fentanyl exposure and washout may provide insights into POCD generation after fentanyl anesthesia. 

Effect of acute fentanyl treatment on synaptic plasticity in the hippocampal CA1 region in rats.

Although conduction failure has been observed in nociceptive C-fibers, little is known regarding its significance or therapeutic potential. In a previous study, we demonstrated that C-fiber conduction failure, which is regarded as an intrinsic self-inhibition mechanism, was reduced in circumstances of painful diabetic neuropathy. In this study, we extend this finding in the complete Freund's adjuvant model of inflammatory pain and validate that the degree of conduction failure decreased and led to a greater amount of pain signals conveyed to the central nervous system. In complete Freund's adjuvant-injected animals, conduction failure occurred in a C-fiber-selective, activity-dependent manner and was associated with an increase in the rising slope of the C-fiber after-hyperpolarization potential. To target conduction failure in a therapeutic modality, we used ZD7288, an antagonist of hyperpolarization-activated, cyclic nucleotide-modulated channels which are activated by hyperpolarization and play a pivotal role in both inflammatory and neuropathic pain. ZD7288 promoted conduction failure by suppressing Ih as a mechanism to reduce the rising slope of the after-hyperpolarization potential. Moreover, perineuronal injection of ZD7288 inhibited abnormal mechanical allodynia and thermal hyperalgesia without affecting motor function or heart rate. Our data highlight the analgesic potential of local ZD7288 application and identify conduction failure as a novel target for analgesic therapeutic development.

A novel intrinsic analgesic mechanism: the enhancement of the conduction failure along polymodal nociceptive C-fibers.

Sensory neuron types have been distinguished by distinct morphological and transcriptional characteristics. Excitability is the most fundamental functional feature of neurons. Mathematical models described by Hodgkin have revealed three types of neuronal excitability based on the relationship between firing frequency and applied current intensity. However, whether natural sensory neurons display different functional characteristics in terms of excitability and whether this excitability type undergoes plastic changes under pathological pain states have remained elusive. Here, by utilizing whole-cell patch clamp recordings, behavioral and pharmacological assays, we demonstrated that large dorsal root ganglion (DRG) neurons can be classified into three classes and four subclasses based on their excitability patterns, which is similar to mathematical models raised by Hodgkin. Analysis of hyperpolarization-activated cation current () revealed different magnitude of  in different excitability types of large DRG neurons, with higher  in Class 2-1 than that in Class 1, 2-2 and 3. This indicates a crucial role of  in the determination of excitability type of large DRG neurons. More importantly, this pattern of excitability displays plastic changes and transition under pathological pain states caused by peripheral nerve injury. This study sheds new light on the functional characteristics of large DRG neurons and extends functional classification of large DRG neurons by integration of transcriptomic and morphological characteristics.

Article	Year
Adrenergic sensitivity of neurons with non-periodic firing activity in rat injured dorsal root ganglion. Neuroscience\| PubMed ID: 11113317	2000
Neuropeptide Y inhibits the hyperexcitability of type A neurons in chronically compressed dorsal root ganglion of the rat. Neuroscience letters\| PubMed ID: 11911992	2002
Relation between responsiveness to neurotransmitters and complexity of epileptiform activity in rat hippocampal CA1 neurons. Epilepsia\| PubMed ID: 12423382	2002
Transition between two excitabilities in mesencephalic V neurons. Journal of computational neuroscience\| PubMed ID: 17643188	2008
Gabapentin selectively reduces persistent sodium current in injured type-A dorsal root ganglion neurons. Pain\| PubMed ID: 19269740	2009
5-HT induces enhanced phrenic nerve activity via 5-HT(2A) receptor/PKC mechanism in anesthetized rats. European journal of pharmacology\| PubMed ID: 21296069	2011
Methylcobalamin: a potential vitamin of pain killer. Neural plasticity\| PubMed ID: 24455309	2013
Resonance characteristic and its ionic basis of rat mesencephalic trigeminal neurons. Brain research\| PubMed ID: 25449887	2015
Inhibition of Hyperpolarization-Activated Cation Current in Medium-Sized DRG Neurons Contributed to the Antiallodynic Effect of Methylcobalamin in the Rat of a Chronic Compression of the DRG... Neural plasticity\| PubMed ID: 26101670	2015
Effect of acute fentanyl treatment on synaptic plasticity in the hippocampal CA1 region in rats. Frontiers in pharmacology\| PubMed ID: 26578961	2015
A novel intrinsic analgesic mechanism: the enhancement of the conduction failure along polymodal nociceptive C-fibers. Pain\| PubMed ID: 27583680	2016
Characterization of Different Types of Excitability in Large Somatosensory Neurons and Its Plastic Changes in Pathological Pain States. International journal of molecular sciences\| PubMed ID: 29303989	2018

Sanjue Hu

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