Due to the complex circuitry and plethora of cell types involved in somatosensation, it is becoming increasingly important to be able to observe cellular activity at the population level. In addition, since cells rely on an intricate variety of extracellular factors, it is important to strive to maintain the physiological environment. Many electrophysiological techniques require the implementation of artificially-produced physiological environments and it can be difficult to assess the activity of many cells simultaneously. Moreover, imaging Ca transients using Ca-sensitive dyes often requires in vitro preparations or in vivo injections, which can lead to variable expression levels. With the development of more sensitive genetically-encoded Ca indicators (GECIs) it is now possible to observe changes in Ca transients in large populations of cells at the same time. Recently, groups have used a GECI called GCaMP to address fundamental questions in somatosensation. Researchers can now induce GCaMP expression in the mouse genome using viral or gene knock-in approaches and observe the activity of populations of cells in the pain pathway such as dorsal root ganglia (DRG), spinal neurons, or glia. This approach can be used in vivo and thus maintains the organism's biological integrity. The implementation of GCaMP imaging has led to many advances in our understanding of somatosensation. Here, we review the current findings in pain research using GCaMP imaging as well as discussing potential methodological considerations.
Afferent Pathways ; physiology ; Animals ; Calcium ; metabolism ; Calcium Signaling ; drug effects ; genetics ; Ganglia, Spinal ; metabolism ; Humans ; Pain ; metabolism ; pathology

Natural toxic substances have a bitter taste and their ingestion sends signals to the brain leading to aversive oral sensations. In the present study, we investigated chronological changes in c-Fos immunoreactivity in the nucleus tractus solitarius (NTS) to study the bitter taste reaction time of neurons in the NTS. Equal volumes (0.5 mL) of denatonium benzoate (DB), a bitter tastant, or its vehicle (distilled water) were administered to rats intragastrically. The rats were sacrificed at 0, 0.5, 1, 2, 4, 8, or 16 h after treatment. In the vehicle-treated group, the number of c-Fos-positive nuclei started to increase 0.5 h after treatment and peaked 2 h after gavage. In contrast, the number of c-Fos-positive nuclei in the DB-treated group significantly increased 1 h after gavage. Thereafter, the number of c-Fos immunoreactive nuclei decreased over time. The number of c-Fos immunoreactive nuclei in the NTS was also increased in a dose-dependent manner 1 h after gavage. Subdiaphragmatic vagotomy significantly decreased DB-induced neuronal activation in the NTS. These results suggest that intragastric DB increases neuronal c-Fos expression in the NTS 1 h after gavage and this effect is mediated by vagal afferent fibers.
Adjuvants, Immunologic/pharmacology ; Afferent Pathways/physiology ; Animals ; Injections/veterinary ; Ligands ; Male ; Proto-Oncogene Proteins c-fos/*metabolism ; Quaternary Ammonium Compounds/*pharmacology ; Rats ; Rats, Sprague-Dawley ; Receptors, G-Protein-Coupled/*metabolism ; Solitary Nucleus/*physiology ; Vagus Nerve/*drug effects/*physiology

The rostral ventromedial medulla (RVM) is a prominent component of the descending modulatory system involved in the control of spinal nociceptive transmission. In the current study, we investigated melanocortin-4 receptor (MC4R) expression in the RVM, where the neurons involved in modulation of nociception reside. Using a line of mice expressing green fluorescent protein (GFP) under the control of the MC4R promoter, we found a large number of GFP-positive neurons in the RVM [nucleus raphe magnus (NRM) and nucleus gigantocellularis pars α (NGCα)]. Fluorescence immunohistochemistry revealed that approximately 10% of MC4R-GFP-positive neurons coexpressed tyrosine hydroxylase, indicating that they were catecholaminergic, whereas 50%-75% of those coexpressed tryptophan hydroxylase, indicating that they were serotonergic. Our findings support the hypothesis that MC4R signaling in RVM may modulate the activity of serotonergic sympathetic outflow sensitive to nociceptive signals, and that MC4R signaling in RVM may contribute to the descending modulation of nociceptive transmission.
Animals ; Female ; Male ; Medulla Oblongata ; cytology ; metabolism ; Mice ; Mice, Transgenic ; Neural Pathways ; cytology ; metabolism ; Neurons, Afferent ; cytology ; metabolism ; Nociception ; physiology ; Receptor, Melanocortin, Type 4 ; genetics ; metabolism ; Serotonergic Neurons ; metabolism ; Tyrosine 3-Monooxygenase ; metabolism

OBJECTIVE: To evaluate the influence of inferior vestibular and part of mandibular branch of trigeminal neurotomy on sound-induced masseter reflex potentials. METHOD: Twenty guinea pigs were randomly divided into four groups, including 5 normal control guinea pigs, 5 received unilateral inferior vestibular neurotomy, 5 received unilateral inferior alveolar nerve neurotomy, and 5 received auriculotemporal nerve neurotomy. Click sound-induced masseter reflex potentials were recorded in four groups, respectively. RESULT: The thresholds of negative peak (NP) of click sound-induced masseter reflex potentials in normal control group were (90.00 +/- 8.16) dBnHL. The response rates of the NP of the masseter reflex potentials using 100, 90, 80 and 70 dBnHL monaural acoustic stimulation with unilateral recording were 100%, 70%, 40% and 0, respectively. The mean latencies of the NP were (6.55 +/- 0.25) ms, (6.61 +/- 0.16) ms and (6.70 +/- 0.13) ms, when elicited by 100,90 and 80 dBnHL acoustic stimulation respectively. There was no significant difference between the stimulus intensity and the mean latency of the NP (P > 0.05). Negative peak of click sound-induced masseter reflex potentials was not observed in the inferior vestibular neurotomy group. The NP was preserved in the inferior alveolar nerve and auriculotemporal nerve neurotomy groups. There were no significant difference of the mean thresholds and latencies of NP between normal control group and inferior alveolar nerve and auriculotemporal nerve neurotomy group (P > 0.05). CONCLUSION Click sound-induced masseter reflex potentials originates from vestibular afferents. Afferent of inferior alveolar nerve and auriculotemporal nerve can not influence the vestibular evoked masseter reflex potentials.
Afferent Pathways ; surgery ; Animals ; Guinea Pigs ; Masseter Muscle ; physiology ; Reflex, Acoustic ; Trigeminal Nerve ; surgery ; Vestibular Nerve ; surgery

AIMTo explore the influence of GABAergic neurotransmitters and GABAA receptors on the auditory afferent impulses recorded in the brainstem evoked by electro-stimulation.

METHODSBrainstem slices were prepared using ddy/ddy mice of postnatal 0-5th days. The brainstem slices were stained with a voltage-sensitive dye(NK3041). The cut end of the vestibulocochlear nerve (nVIIIth) connected with slices was stimulated by a tungsten electrode, a 16 x 16 pixels silicon photodiode array apparatus was used to record the optical mapping from auditory brainstem slices. The data were analyzed by ARGUS-50/PDA software.

RESULTSThe spatial-temporal patterns of the excitatory propagation from the vestibulocochlear nerve (nVIIIth) to cochlear nucleus and vestibular nucleus were displayed with multiple-sites optical recording. The optical signal coming from one pixel consisted of a fast spike-like response and a following slow response. Inhibitory neurotransmitter GABA decreased the fast spike-like response and following slow response of evoked optical signals, while an antagonist BMI against GABAA receptors increased the both responses.

CONCLUSIONA 16 x 16 pixel silicon photodiode array apparatus can be used to record multiple-sites optical mapping evoked by electro-stimulation to the cut end of the vestibulocochlear nerve. The every optical signal consists of both presynaptic and postsynaptic elements. Inhibitory neurotransmitter GABA and an antagonist BMI of GABAA receptors can modulate the excitatory propagation of evoked optical signals.

Animals ; Animals, Newborn ; Auditory Pathways ; physiology ; Brain Stem ; physiology ; Evoked Potentials, Auditory, Brain Stem ; physiology ; In Vitro Techniques ; Mice ; Neurons, Afferent ; physiology ; Optics and Photonics ; Photic Stimulation ; Receptors, GABA-A ; physiology ; gamma-Aminobutyric Acid ; physiology

OBJECTIVETo investigate the role of nitric oxide in the inhibitory effect of somatic afferent input on the pressor response caused by electrical stimulation of the paraventricular nucleus of the hypothalamus (PVN).

METHODSSD rats anesthetized by urethane were used in the study. Bipolar stainless stimulating electrode was inserted into PVN for electrical stimulation. Multi-barreled micropipettes were used for microinjection of L-NAME or normal saline into the lateral ventricle or amygdala. Deep peroneal nerve (DPN) was stimulated with electrical current pulses of 0.4 mA with duration of 0.5 ms at 4 Hz for 5 min. PVN was stimulated by electrical current pulses of 0.3 mA with duration of 0.5 ms at 80 Hz for 10 sec.

RESULTElectrical stimulation of PVN increased mean arterial pressure. Stimulation of DPN significantly inhibited the pressor response induced by stimulation of PVN (P<0.01), with the inhibitory percentage of 43.27%. Microinjection of L-NAME (0.5 mol/L,10 microl) into the lateral ventricle of brain attenuated the inhibitory effect of DPN. The inhibitory percentage decreased from 47.73% to 12.49% (P<0.05). Microinjection of L-NAME (2 mol/L,100 nl) into amygdala reduced the inhibitory effect of DPN. The inhibitory percentage of stimulating DPN on the pressor response decreased from 50.71% to 25.30% (P<0.05).

CONCLUSIONNitric oxide in the brain and amygdala are involved in the inhibitory effect of somatic afferent input on central pressor response.

Afferent Pathways ; drug effects ; physiology ; Amygdala ; drug effects ; physiology ; Animals ; Blood Pressure ; physiology ; Electric Stimulation ; Enzyme Inhibitors ; pharmacology ; NG-Nitroarginine Methyl Ester ; pharmacology ; Nitric Oxide ; antagonists & inhibitors ; physiology ; Nitric Oxide Synthase ; antagonists & inhibitors ; Paraventricular Hypothalamic Nucleus ; drug effects ; physiology ; Peroneal Nerve ; physiology ; Pressoreceptors ; drug effects ; physiology ; Rats ; Rats, Sprague-Dawley

OBJECTIVEEarly researches found that different heartbeat perceivers have different heartbeat evoked potential (HEP) waves. Two tasks were considered in our experiments to get more details about the differences between good and poor heartbeat perceivers at attention and resting state.

METHODSThirty channels of electroencephalogram (EEG) were recorded in 22 subjects, who had been subdivided into good and poor heartbeat perceivers by mental tracking task. Principal component analysis (PCA) was applied to remove cardiac field artifact (CFA) from the HEP.

RESULTS(1) The good heart-beat perceivers showed difference between attention and resting state in the windows from 250 ms to 450 ms after R wave at C3 location and from 100 ms to 300 ms after R wave at C4 location; (2) The difference waveforms between good and poor heartbeat perceivers was a positive waveform at FZ from 220 ms to 340 ms after R wave, which was more significant in attention state.

CONCLUSIONAttention state had more effect on the HEPs of good heartbeat perceivers than that of poor heartbeat perceivers; and perception ability influenced HEPs more strongly in the attention state than in the resting state.

Adult ; Afferent Pathways ; physiology ; Attention ; physiology ; Awareness ; physiology ; Biofeedback, Psychology ; physiology ; Brain ; physiology ; Electroencephalography ; Evoked Potentials ; physiology ; Heart ; physiology ; Heart Rate ; physiology ; Humans ; Male ; Perception ; physiology ; Reference Values ; Sensation ; physiology

AIMTo investigate the inhibitory effect of the deep peroneal nerve (DPN) on the cardiovascular responses induced by excitation of the paraventricular nucleus of hypothalamus (PVN) and the role of central nucleus of amygdala (CeA) in this effect.

METHODSCeA was injected by L-glutamate or Kainic acid (KA). The femoral arterial pressure, mean arterial pressure (MAP), electrocardiogram (ECG) and heart rate (HR) of SD rats were recorded while PVN or DPN was electrically stimulated.

RESULTSIt showed that MAP increased when PVN was activated by electrical stimulation. Stimulating contralateral DPN inhibited this pressor response. Ten minutes after microinjection of KA(0.02 mol/L, 100 nl) into ipsilateral CeA, MAP increased for (13.8 +/- 3.2) mmHg when PVN was stimulated. Microinjection of KA into CeA could not only reduce the pressor response elicited by stimulation of PVN for (6.6 +/- 1.6) mmHg (P < 0.05), but also the inhibitory effect of DPN from 51.5% to 32.0% .

CONCLUSIONThe results suggest that central nucleus of amygdala partly mediate the central pressor response induced by stimulation of PVN. The neurons in central nucleus of amygdala are involved in the inhibitory effect of DPN on the above pressor response.

Afferent Pathways ; Amygdala ; physiology ; Animals ; Blood Pressure ; Central Nervous System ; physiology ; Hypothalamus ; physiology ; Paraventricular Hypothalamic Nucleus ; physiology ; Peroneal Nerve ; physiology ; Rats ; Rats, Sprague-Dawley

Intraganglionic laminar endings (IGLEs) have been supposed to be the mechanoreceptors in the gut by electrophysiological recording techniques. But the specialized morphology of IGLEs could not be displayed closely associated with this function and the mechanism that IGLEs act as the mechanotransduction sites in the gut is not yet well understood. In the present study, we used styryl dye FM1-43 combined with stretch stimulation in the guinea pig esophagus to test whether IGLEs acted as the mechano-sensitive receptors of the vagal afferent nerves. At the same time, the special structure of IGLEs displayed by FM1-43 was further confirmed by neurobiotin anterograde labeling technique. To further investigate the characteristics of IGLEs as mechanosensitive receptors, different drugs were used to block or stimulate IGLEs activation. Our results indicated that only in the stretched preparation could FM1-43 enter the IGLEs and completely display their specialized structure, which was consistent with that shown by neurobiotin. The amount of IGLEs shown by stretch-evoked FM1-43 staining was much more than that shown without stretch stimulation [(90.4 +/- 9.5) % vs (10.7 +/- 2.1) %, P<0.05]. Ca(2+), TTX (0.6 mumol/L), atropine (0.6 mumol/L), SKF (50 mumol/L), and gadolium (100 mumol/L) had no effect on the IGLEs activation. But for benzamil (100 mumol/L), an epithelial sodium channel blocker, activation of IGLEs by stretch stimulation was significantly blocked. The potent ATP analogue, alpha,beta-methylene ATP (100 mumol/L) could not activate FM1-43 staining without stretch. These results indicate that IGLEs are sensitive to mechanical stimulation. This could lead to the deduction that IGLEs act as the mechanoreceptors of vagal afferent nerve. IGLEs could transmit mechanical stimuli directly through ion channels, independent of neurotransmitter release and action potential propagation. The stretch-sensitive channels on IGLEs probably belong to the epithelial sodium channel family rather than voltage-gated sodium ion channels. Furthermore, styryl dye FM1-43 is a useful activity-dependent marker to demonstrate the structure and function of IGLEs in guinea pig esophagus.
Afferent Pathways ; physiology ; Animals ; Esophagus ; innervation ; Female ; Ganglia, Autonomic ; physiology ; Guinea Pigs ; Male ; Mechanoreceptors ; physiology ; Nerve Endings ; physiology ; Vagus Nerve ; physiology

OBJECTIVETo investigate the role of the dorsal column (DC) in the inhibitory effect of somatic afferent inputs on the central pressor response.

METHODSThe femoral arterial pressure, mean arterial pressure (MAP), electrocardiogram (ECG) and heart rate (HR) of the male SD rats were recorded when the hypothalamic paraventricular nucleus (PVN) was electrically stimulated with or without destruction of DC. The inhibitory effect of the deep peroneal nerve (DPN) on the pressor response induced by stimulation of PVN was observed 20 min or 5 d after ipsilateral DC destruction.

RESULTSStimulating DPN inhibited the pressor response elicited by electrical stimulation of PVN with an inhibitory rate of 43.29%. Twenty minutes after destroying the right DC, stimulation of the right or left DPN could inhibit the pressor response with an inhibitory rate of 38.64% and 39.97%, respectively (P>0.05); five days later the inhibitory rates remained as 33.87% and 36.86% respectively (P>0.05). The pain responses of both hindlimbs in the rats with the right DC destroyed showed no significant difference compared with the intact rats.

CONCLUSIONDC is not involved in the inhibitory effect of DPN on the pressor response induced by PVN stimulation.

Afferent Pathways ; physiology ; Animals ; Blood Pressure ; physiology ; Electric Stimulation ; Male ; Paraventricular Hypothalamic Nucleus ; physiology ; Peroneal Nerve ; physiology ; Pressoreceptors ; physiology ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Spinal Cord ; physiology ; Spinothalamic Tracts ; physiology