Bai-Chuang Shyu

A fMRI study of brain activations during non-noxious and noxious electrical stimulation of the sciatic nerve of rats.

An acute pain animal model for fMRI study would provide useful spatial and temporal information for studying the supraspinal nociceptive neuronal responses. The aim of the present study was to investigate whether the nociceptive responses in different brain areas can be differentiated by using functional magnetic resonance imaging (fMRI) in anesthetized rats. Functional changes in brain regions activated by noxious or non-noxious stimuli of the sciatic nerve were investigated using fMRI in a 4.7 T MR system in alpha-chloralose anaesthetized rats. To determine the electrical intensity for noxious and non-noxious stimuli, compound action potential recording was employed to reveal the type of fibers activated by graded electrical stimulation of sciatic nerve. It showed that innocuous A-beta fibers were excited by two times the muscle twitch threshold and nociceptive A-delta and C fibers were recruited and excited by 10 and 20 times threshold, respectively. A series of four-slice gradient echo images were acquired during innocuous (two times threshold) and noxious (10 and 20 times threshold) stimuli in a 4.7 T MR system. Contralateral somatosensory cortex was the most prominent brain area activated by innocuous stimuli. Both signal intensity and activated areas were significantly increased in the somatosensory cortex, cingulate cortex, medial thalamus and hypothalamus during noxious stimuli. These four brain areas activated by noxious stimuli were significantly suppressed by prior intravenous injection of morphine (5 mg/kg). The present findings demonstrated that the difference of the innocuous and nociceptive responses in the brain could be detected and localized by an in vivo spatial map using fMRI. Results suggest that fMRI may be an invaluable tool for studying pain in anesthetized animals.

A New Scenario for Negative Functional Magnetic Resonance Imaging Signals: Endogenous Neurotransmission

Functional magnetic resonance imaging (fMRI) has revolutionized investigations of brain functions. Increases in fMRI signals are usually correlated with neuronal activation, but diverse explanations have been proposed for negative fMRI responses, including decreases in neuronal activity, the vascular-steal effect, and large increases in oxygen consumption. These possible scenarios, although encompassing a wide range of potential neurovascular responses, cannot yet be used to interpret certain types of negative fMRI signals. Recent studies have found that intravenous injection of dopamine D2 receptor (D2DR) agonist reduced the hemodynamic responses in the caudate–putamen (CPu); however, whether endogenous dopaminergic neurotransmission contributes to fMRI signals remains obscure. Since it has been suggested that the D2DR is involved in pain modulation, and the CPu shows equivocal fMRI signals during noxious stimulation, the present study established an animal model based on graded electrical stimulation to elicit different levels of nociception, and aimed to determine whether nociception-induced endogenous dopaminergic neurotransmission is sufficient to generate negative fMRI responses. Our results from cerebral blood volume (CBV)-weighted fMRI, Fos immunohistochemistry, and electrophysiological recording demonstrated a salient bilateral CBV decreases associated with heightened neuronal activity in the CPu induced by unilateral noxious electrical stimulation. In addition, preinjection of D2DR antagonist reduced the observed CBV decreases. Our findings reveal the role of the D2DR in regulating striatal vascular responses and suggest that endogenous neurotransmission-induced CBV decreases underlie negative fMRI signals. Hence, the influence of endogenous neurotransmission should be considered when interpreting fMRI data, especially in an area involved in strong vasoactive neurotransmission.

Short-term synaptic plasticity in the nociceptive thalamic-anterior cingulate pathway.

BackgroundAlthough the mechanisms of short- and long-term potentiation of nociceptive-evoked responses are well known in the spinal cord, including central sensitization, there has been a growing body of information on such events in the cerebral cortex. In view of the importance of anterior cingulate cortex (ACC) in chronic pain conditions, this review considers neuronal plasticities in the thalamocingulate pathway that may be the earliest changes associated with such syndromes.ResultsA single nociceptive electrical stimulus to the sciatic nerve induced a prominent sink current in the layer II/III of the ACC in vivo, while high frequency stimulation potentiated the response of this current. Paired-pulse facilitation by electrical stimulation of midline, mediodorsal and intralaminar thalamic nuclei (MITN) suggesting that the MITN projection to ACC mediates the nociceptive short-term plasticity. The short-term synaptic plasticities were evaluated for different inputs in vitro where the medial thalamic and contralateral corpus callosum afferents were compared. Stimulation of the mediodorsal afferent evoked a stronger short-term synaptic plasticity and effectively transferred the bursting thalamic activity to cingulate cortex that was not true for contralateral stimulation. This short-term enhancement of synaptic transmission was mediated by polysynaptic pathways and NMDA receptors. Layer II/III neurons of the ACC express a short-term plasticity that involves glutamate and presynaptic calcium influx and is an important mechanism of the short-term plasticity.ConclusionThe potentiation of ACC neuronal activity induced by thalamic bursting suggest that short-term synaptic plasticities enable the processing of nociceptive information from the medial thalamus and this temporal response variability is particularly important in pain because temporal maintenance of the response supports cortical integration and memory formation related to noxious events. Moreover, these modifications of cingulate synapses appear to regulate afferent signals that may be important to the transition from acute to chronic pain conditions associated with persistent peripheral noxious stimulation. Enhanced and maintained nociceptive activities in cingulate cortex, therefore, can become adverse and it will be important to learn how to regulate such changes in thalamic firing patterns that transmit nociceptive information to ACC in early stages of chronic pain.

Differential projections from the mediodorsal and centrolateral thalamic nuclei to the frontal cortex in rats

The aim of the present study was to investigate afferent projections from the medial thalamic nuclei (MT) to the frontal cortical areas using a single small iontophoretic injection of biotinylated dextran amine (BDA) and analysis of the anterogradely labeled fibers and varicosities. Projections from the mediodorsal (MD) nuclei were found primarily and extensively in the anterior cingulate cortex (ACC), whereas those from the centrolateral (CL) thalamic nucleus were found in the frontal motor cortex. The density of terminals in the ACC was high in layers II and III and sparse in layer I. The majority of projected fibers from the CL were found at a high density in layer V, with a moderate density in the superficial layers. The differential projection patterns were topographically organized in the medial prefrontal cortex and sensory motor cortex. These findings support the results of our previous electrophysiological studies suggesting that neurons in the medial thalamic nuclei relay nociceptive information to the limbic or sensory motor cortical areas. The present results agree with the current notion that the medial thalamo-frontal cortical network circuitry plays an important role in processing the emotional aspect of nociception.

Contribution of the anterior cingulate cortex to laser-pain conditioning in rats

The emotional component of nociception is seldom distinguished from pain behavioral testing. The aim of the present study was to develop a behavioral test that indicates the emotional pain responses using the classical conditioning paradigm. The role of the anterior cingulate cortex (ACC) in the process of this pain conditioning response was also evaluated. In laser-pain conditioning, free moving rats were trained to associate a tone (conditioned stimulus, CS) and short CO(2) laser pulsation (unconditioned stimulus, US). Monotonous tone (800 Hz, 0.6 s) was delivered through a loud-speaker as CS. CO(2) laser pulses (5 W at 50 or 100 ms in duration) applied to the hind paw was adopted as US. The CS-US interval was 0.5 s. Laser-pain conditioning was developed during 40 CS-US pairings. CS and US pairing with 100-ms laser pulse stimuli was more effective in establishing conditioning responses than that of 50-ms stimuli. The conditioning responses remained, tested by presenting CS alone, immediate to and 24 h subsequent to training. The performance of laser-pain conditioning was significantly reduced after bilateral lesioning of the ACC. Similar results were also obtained by bilateral lesions of the amygdala. The conditioning responses were also diminished following morphine treatment. The association between a neutral stimulus and a noxious stimulus could be demonstrated in a Pavlovian conditioning test in free moving rats. Thus, the conditioned response may be employed as a measure of the emotional component of the nociception. It is also suggested that the ACC may play an important role in mediating this conditioning effect.

Anxiety- and depressive-like responses and c-fos activity in preproenkephalin knockout mice: oversensitivity hypothesis of enkephalin deficit-induced posttraumatic stress disorder.

The present study used the preproenkephalin knockout (ppENK) mice to test whether the endogenous enkephalins deficit could facilitate the anxiety- and depressive-like symptoms of posttraumatic stress disorder (PTSD). On Day 1, sixteen wildtype (WT) and sixteen ppENK male mice were given a 3 mA or no footshock treatment for 10 seconds in the footshock apparatus, respectively. On Days 2, 7, and 13, all mice were given situational reminders for 1 min per trial, and the freezing response was assessed. On Day 14, all mice were tested in the open field test, elevated plus maze, light/dark avoidance test, and forced swim test. Two hours after the last test, brain tissues were stained to examine c-fos expression in specific brain areas. The present results showed that the conditioned freezing response was significant for different genotypes (ppENK vs WT). The conditioned freezing effect of the ppENK mice was stronger than those of the WT mice. On Day 14, the ppENK mice showed more anxiety- and depressive-like responses than WT mice. The magnitude of Fos immunolabeling was also significantly greater in the primary motor cortex, bed nucleus of the stria terminalis-lateral division, bed nucleus of the stria terminalis-supracapsular division, paraventricular hypothalamic nucleus-lateral magnocellular part, central nucleus of the amygdala, and basolateral nucleus of the amygdala in ppENK mice compared with WT mice. In summary, animals with an endogenous deficit in enkephalins might be more sensitive to PTSD-like aversive stimuli and elicit stronger anxiety and depressive PTSD symptoms, suggesting an oversensitivity hypothesis of enkephalin deficit-induced PTSD.

Synaptic organization and input-specific short-term plasticity in anterior cingulate cortical neurons with intact thalamic inputs

The absence of a slice preparation with intact thalamocortical pathways has held back elucidation of the cellular and synaptic mechanisms by which thalamic signals are differentially transmitted to and processed in the anterior cingulate cortex (ACC). In this report we introduce an innovative mouse brain slice preparation in which it is possible to explore the electrophysiological properties of ACC neurons with intact long‐distance inputs from medial thalamic (MT) nuclei by intracellular recordings; this MT–ACC neuronal pathway plays an integral role in information transmission. Biocytin‐labeled fibers in a functional slice could be traced anterogradely or retrogradely from the MT via the reticular thalamic nuclei, striatum and corpus callosum to the cingulate cortical areas. Eighty‐seven cells downstream of the thalamic projections in 49 slices were recorded intracellularly. Intracellular recordings in the ACC showed that thalamocingulate transmission involves both α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA)/kainate and N‐methyl‐d‐aspartate (NMDA) subtypes of glutamate receptors. Thalamus‐evoked responses recorded extracellularly in the ACC were activated and progressed along a deep–superficial–deep trajectory loop across the ACC layers. We observed enhanced paired‐pulse facilitation and tetanic potentiation of thalamocingulate synapses, suggestive of input‐specific ACC plasticity and selective processing of information relayed by thalamocingulate pathways. Furthermore, we observed differential responses of ACC neurons to thalamic burst stimulation, which underscores the importance of MT afferents in relaying sensory information to the ACC. This new slice preparation enables the contribution of MT‐evoked ACC synaptic transmission to short‐term plasticity in the neuronal circuitry underlying sensory information processing to be examined in detail.

Intrathecally administered c-fos antisense oligodeoxynucleotide decreases formalin-induced nociceptive behavior in adult rats

c-fos antisense strategy was applied as a pharmacological approach to characterize its dose-dependent role and reversibility in the reduction of formalin-induced hyperalgesia. Nociceptive behavioral responses (weighted score, flinching response, licking/biting) following formalin (50 microl 5%) injection were assessed in adult Wistar rats receiving different doses (50 nM, 250 nM) of intrathecally administered c-fos antisense oligodeoxynucleotides at different times prior to formalin injections. The treatments dose dependently decreased both Fos immunoreactivity expression in dorsal horn of rat lumbar spinal cord and all nociceptive measures in the tonic phase of the formalin test. c-Fos correlated well with weighted pain score and/or flinching responses, but not with licking/biting behavior. With the exception of a 48-120 h period required for licking/biting behavior to be restored to its normal status, the suppressive effect on c-fos expression and other nociceptive behaviors disappeared 48 h following c-fos antisense oligodeoxynucleotide treatment. The results suggest a pharmacological potential of c-fos antisense oligodeoxynucleotides in the central nervous system to block immediate-early genes and their resulting physiological consequence following noxious stimulus.

Potentiation of local field potentials in the anterior cingulate cortex evoked by the stimulation of the medial thalamic nuclei in rats

The limbic thalamus and cingulate cortex are essential components in mediating the affective component of pain responses. In the present study, we examined the excitatory properties of medial thalamus (MT)-evoked field potentials in the anterior cingulated cortex (ACC). We also examined the effects of paired pulses and brief tetanic stimuli of the MT. The aim of this study was to determine whether nociceptive inputs to medial thalamic afferents cause plastic changes in the ACC. In alpha-chloralose (50 mg/kg, i.v.) anaesthetized rats, tungsten microelectrodes were used to stimulate the MT and to record field potentials in the ACC. The locations of MT were identified by searching and examining their responses to peripheral noxious stimuli. Early negative (about 4.7 ms latency) and late positive (about 11.7 ms) potentials could be evoked in the ACC by MT stimuli. The evoked field potentials were potentiated by prepulse stimulation. Maximal paired pulse facilitation (509+/-51%) was produced in 80-150 ms interpulse intervals. Evoked field potentials were also potentiated (28.8+/-6.3% and 29.6+/-5.9%, respectively) by low (10 Hz/10 s) and high (100 Hz/2s/2x) frequency tetanic stimulation of the MT, with a duration maintained for about 90 s and 120 min, respectively. The potentiation of MT-evoked ACC potentials provides a neural basis for synaptic plasticity, which may be essential for the establishment of pain-initiated conditioning behavior and affective responses to noxious stimuli.

A minimal stress model for the assessment of electroacupuncture analgesia in rats under halothane.

The use of anesthetics in acupuncture analgesia is controversial. We evaluate a steady‐state light anesthesia model to test whether minimal stress manipulation and reliable measurement of analgesia could be simultaneously achieved during electroacupuncture (EA) in animals.