Longitudinal FDG-PET scan study of brain changes in mice with cancer-induced bone pain and after morphine analgesia.

Morphine is the most commonly used drug for treating physical and psychological suffering caused by advanced cancer. Although morphine is known to elicit multiple supraspinal analgesic effects, its behavioral correlates with respect to the whole-brain metabolic activity during cancer-induced bone pain have not been elucidated. We injected 4T1 mouse breast cancer cells into the left femur bone marrow cavity of BALB/c mice. All mice developed limb use deficits, mechanical allodynia, and hypersensitivity to cold, which were effectively suppressed with morphine. Serial 18F-fluorodeoxyglucose positron emission tomography (FDG-PET) was performed for each mouse before cancer induction (0 day), after cancer-induced bone pain was established (14 days), and during effective morphine treatment (16 days). The longitudinal FDG-PET imaging analysis demonstrated that cancer-induced bone pain increased glucose uptake in the insular cortex and hypothalamus and decreased the activity of the retrosplenial cortex. Morphine reversed the activation of the insular cortex and hypothalamus. Furthermore, morphine activated the amygdala and rostral ventromedial medulla and suppressed the activity of anterior cingulate cortex. Our findings of hypothalamic and insular cortical activation support the hypothesis that cancer-induced bone pain has strong inflammatory and affective components in freely moving animals. Morphine may provide descending inhibitory and facilitatory actions in the treatment of cancer-induced bone pain in a clinical setting.

Differential Expression of Phosphorylated ERK and c-Fos of Limbic Cortices Activities in Response to Tactile Allodynia of Neuropathic Rats.

Neuropathic pain is due to lesion or dysfunction of the somatosensory system. Treating patients with neuropathic pain is difficult because the underlying mechanisms are understood limitedly, especially at the supraspinal level. In this study, we used two kinds of molecular markers to investigate the neuronal activity changes in the anterior cingulate cortex, insular cortex (IC), and medial prefrontal cortex (mPFC) of the neuropathic rats under tactile allodynia. We used spared nerve injury of the sciatic nerve (SNI) as the neuropathic pain model. Two weeks after SNI surgery, we applied repetitive allodynic stimulation to the conscious rats. After stimulation, the rats were sacrificed, and the immunohistochemistry of phosphorylated extracellular signal-regulated kinase (pERK) and c-Fos was performed. Quantification of immunoreactive cells was carried out by stereological method. For pERK study, the expression of pERK was significantly increased in the mPFC and IC of the SNI rats. For c-Fos study, only mPFC had elevated expression of c-Fos in the SNI rats. The analgesic, gabapentin, reversed the mechanical hyper-sensitivity and the augmented expression of limbic pERK and c-Fos in the SNI rats. Immunofluorescent staining revealed the expression of pERK or c-Fos was restricted to neurons, not glia cells. Our results demonstrated that tactile allodynia represented differential expression of pERK and c-Fos in the limbic cortices of the neuropathic rats.

Granger causality-based synaptic weights estimation for analyzing neuronal networks.

Granger causality (GC) analysis has emerged as a powerful analytical method for estimating the causal relationship among various types of neural activity data. However, two problems remain not very clear and further researches are needed: (1) The GC measure is designed to be nonnegative in its original form, lacking of the trait for differentiating the effects of excitations and inhibitions between neurons. (2) How is the estimated causality related to the underlying synaptic weights? Based on the GC, we propose a computational algorithm under a best linear predictor assumption for analyzing neuronal networks by estimating the synaptic weights among them. Under this assumption, the GC analysis can be extended to measure both excitatory and inhibitory effects between neurons. The method was examined by three sorts of simulated networks: those with linear, almost linear, and nonlinear network structures. The method was also illustrated to analyze real spike train data from the anterior cingulate cortex (ACC) and the striatum (STR). The results showed, under the quinpirole administration, the significant existence of excitatory effects inside the ACC, excitatory effects from the ACC to the STR, and inhibitory effects inside the STR.

Spatiotemporal Changes of Neuronal Responses in the Primary Somatosensory Cortex to Noxious Tail Stimulation in Awake and Pentobarbital-Anesthetized Rats.

Primary somatosensory cortex (SI) is a key area in the processing of nociceptor inputs to our consciousness. To clarify the columnar and laminar organization of SI for pain processing, we compared spatiotemporal changes in neuronal activities of the primary sensorimotor cortex (SmI) of the rat in response to noxious laser heat stimulation applied to the mid-tail. Longitudinal and vertical array microelectrodes were chronically implanted in the cerebral cortex. Evoked neuronal activities, including intracortical local field potentials (LFP) and ensemble single-unit activity (SU) around SmI were simultaneously recorded. The effect of pentobarbital on the neuronal responses was evaluated in comparison with the neuronal responses in conscious animals to explore the potential substrate of nociceptive processing in the conscious state. The results from the experiment with longitudinal microelectrode arrays indicated that noxious stimulation induced a neuronal response which was spread widely around the SmI of the conscious rat, and the range of neuronal responses was limited to the tail region of the SmI under anesthesia. The results from the experiment with vertical microelectrode arrays showed the universal neuronal responses through all cortical layers of the SmI in conscious rats, and sodium pentobarbital suppressed these neuronal responses in the supragranular layers significantly relative to the deeper layers and basal activity. These results imply that a wider range of cortical activation, both in the horizontal or vertical dimension, might be important for nociceptive processing in the conscious state.

Gabapentin reverses central hypersensitivity and suppresses medial prefrontal cortical glucose metabolism in rats with neuropathic pain.

BACKGROUND: Gabapentin (GBP) is known to suppress neuropathic hypersensitivity of primary afferents and the spinal cord dorsal horn. However, its supra-spinal action sites are unclear. We identify the brain regions where GBP changes the brain glucose metabolic rate at the effective dose that alleviates mechanical allodynia using 18 F-fluorodeoxyglucose-positron emission tomography (FDG-PET) scanning. RESULTS: Comparing the PET imaging data before and after the GBP treatment, the spared nerve injury-induced increases of glucose metabolism in the thalamus and cerebellar vermis were reversed, and a significant decrease occurred in glucose metabolism in the medial prefrontal cortex (mPFC), including the anterior cingulate cortex. GBP treatment also reversed post-SNI connectivity increases between limbic cortices and thalamus. CONCLUSIONS: Our results indicate that GBP analgesic effect may be mediated by reversing central hypersensitivity, and suppressing mPFC, a crucial part of the cortical representation of pain, in the brain.

Collateral projections from vestibular nuclear and inferior olivary neurons to lobules I/II and IX/X of the rat cerebellar vermis: a double retrograde labeling study.

Axon collateral projections to various lobules of the cerebellar cortex are thought to contribute to the coordination of neuronal activities among different parts of the cerebellum. Even though lobules I/II and IX/X of the cerebellar vermis are located at the opposite poles in the anterior-posterior axis, they have been shown to receive dense vestibular mossy fiber projections. For climbing fibers, there is also a mirror-image-like organisation in their axonal collaterals between the anterior and posterior cerebellar cortex. However, the detailed organisation of mossy and climbing fiber collateral afferents to lobules I/II and IX/X is still unclear. Here, we carried out a double-labeling study with two retrograde tracers (FluoroGold and MicroRuby) in lobules I/II and IX/X. We examined labeled cells in the vestibular nuclei and inferior olive. We found a low percentage of double-labeled neurons in the vestibular nuclei (2.1 ± 0.9% of tracer-labeled neurons in this brain region), and a higher percentage of double-labeled neurons in the inferior olive (6.5 ± 1.9%), especially in its four small nuclei (18.5 ± 8.0%; including the ß nucleus, dorsal cap of Kooy, ventrolateral outgrowth, and dorsomedial cell column), which are relevant for vestibular function. These results provide strong anatomical evidence for coordinated information processing in lobules I/II and IX/X for vestibular control.

Cingulate seizure-like activity reveals neuronal avalanche regulated by network excitability and thalamic inputs.

BACKGROUND: Cortical neurons display network-level dynamics with unique spatiotemporal patterns that construct the backbone of processing information signals and contribute to higher functions. Recent years have seen a wealth of research on the characteristics of neuronal networks that are sufficient conditions to activate or cease network functions. Local field potentials (LFPs) exhibit a scale-free and unique event size distribution (i.e., a neuronal avalanche) that has been proven in the cortex across species, including mice, rats, and humans, and may be used as an index of cortical excitability. In the present study, we induced seizure activity in the anterior cingulate cortex (ACC) with medial thalamic inputs and evaluated the impact of cortical excitability and thalamic inputs on network-level dynamics. We measured LFPs from multi-electrode recordings in mouse cortical slices and isoflurane-anesthetized rats. RESULTS: The ACC activity exhibited a neuronal avalanche with regard to avalanche size distribution, and the slope of the power-law distribution of the neuronal avalanche reflected network excitability in vitro and in vivo. We found that the slope of the neuronal avalanche in seizure-like activity significantly correlated with cortical excitability induced by γ-aminobutyric acid system manipulation. The thalamic inputs desynchronized cingulate seizures and affected the level of cortical excitability, the modulation of which could be determined by the slope of the avalanche size. CONCLUSIONS: We propose that the neuronal avalanche may be a tool for analyzing cortical activity through LFPs to determine alterations in network dynamics.

Discontinuity third harmonic generation microscopy for label-free imaging and quantification of intraepidermal nerve fibers.

Label-free imaging methodologies for nerve fibers rely on spatial signal continuity to identify fibers and fail to image free intraepidermal nerve endings (FINEs). Here, we present an imaging methodology-called discontinuity third harmonic generation (THG) microscopy (dTHGM)-that detects three-dimensional discontinuities in THG signals as the contrast. We describe the mechanism and design of dTHGM and apply it to reveal the bead-string characteristics of unmyelinated FINEs. We confirmed the label-free capability of dTHGM through a comparison study with the PGP9.5 immunohistochemical staining slides and a longitudinal spared nerve injury study. An intraepidermal nerve fiber (IENF) index based on a discontinuous-dot-connecting algorithm was developed to facilitate clinical applications of dTHGM. A preliminary clinical study confirmed that the IENF index was highly correlated with skin-biopsy-based IENF density (Pearson's correlation coefficient R = 0.98) and could achieve differential identification of small-fiber neuropathy (p = 0.0102) in patients with diabetic peripheral neuropathy.

Long-term changes in trigeminal ganglionic and thalamic neuronal activities following inferior alveolar nerve transection in behaving rats.

The transection of the inferior alveolar nerve (IANx) produces allodynia in the whisker pad (V2 division) of rats. Ectopic discharges from injured trigeminal ganglion (TG) neurons and thalamocortical reorganization are possible contributors to the sensitization of uninjured V2 primary and CNS neurons. To test which factor is more important, TG and ventroposterior medial nucleus (VPM) neurons were longitudinally followed before, during, and after IANx for up to 80 d. Spontaneous discharges and mechanical stimulation-evoked responses were recorded in conscious and in anesthetized states. Results show (1) a sequential increase in spontaneous activities, first in the injured TG neurons of the IAN (2-30 d), followed by uninjured V2 ganglion neurons (6-30 d), and then VPM V2 neurons (7-30 d) after IANx; (2) ectopic discharges included burst and regular firing patterns in the IAN and V2 branches of the TG neurons; and (3) the receptive field expanded, the modality shifted, and long-lasting after-discharges occurred only in VPM V2 neurons. All of these changes appeared in the late or maintenance phase (7-30 d) and disappeared during the recovery phase (40-60 d). These observations suggest that ectopic barrages in the injured IAN contribute more to the development of sensitization, whereas the modality shift and evoked after-discharges in the VPM thalamic neurons contribute more to the maintenance phase of allodynia by redirecting tactile information to the cortex as nociceptive.

Affective mirror and anti-mirror neurons relate to prosocial help in rats.

Although empathic emotion is closely related to prosocial behavior, neuronal substrate that accounts for empathy-associated prosocial action remains poorly understood. We recorded neurons in the anterior cingulate cortex (ACC) and insular cortex (InC) in rats when they observed another rat in pain. We discovered neurons with anti-mirror properties in the ACC and InC, in addition to those with mirror properties. ACC neurons show higher coupling between activation of self-in-pain and others-in-pain, whereas the InC has a higher ratio of neurons with anti-mirror properties. During others-in-pain, ACC neurons activated more when actively nose-poking toward others and InC neurons activated more when freezing. To further illustrate prosocial function, we examined neuronal activities in the helping behavior test. Both ACC and InC neurons showed specific activation to rat rescuing which is contributed by mirror, but not anti-mirror neurons. Our work indicates the functional involvement of mirror neuron system in prosocial behaviors.