Morphological study of myelinated and unmyelinated fibres in the sacrococcygeal dorsal roots of the rat.

BACKGROUND: The number and calibre of myelinated and unmyelinated fibres of the sacrococcygeal dorsal roots innervating the tail of rats were studied by means of light and electron microscopy. MATERIALS AND METHODS: There were an estimated total of 12,500 myelinated and 25,500 unmyelinated dorsal root fibres innervating the tail of a rat. RESULTS: The results showed that from the second sacral (S2) to the fourth sacral (S4) segment, the fibre diameter spectrum of myelinated fibres within each dorsal root was bimodal with two peaks at 5 microns and 10 microns, respectively. The first sacral (S1) segment was composed of numerous smaller-size myelinated fibres, thus forming a right-skewed distribution. The coccygeal (Co) segments showed a unimodal distribution peaking at 10 microns for the first (Co1) segment and gradually shifting to 7 microns for the third (Co3) segment. Overall, there was a continuous relative increase of the larger vs. the smaller myelinated fibres from the sacral to coccygeal segments. The fibre diameter of unmyelinated fibres of all these roots was unimodal with a single peak at 0.5 microns. The ratio of unmy- elinated to myelinated fibre numbers was on average 2.83 for the S1-S2 roots, 1.66 for the S3-S4 roots, and 1.24 for the coccygeal roots. CONCLUSIONS: The comparison of the left- and right-side nerve fibres show that there was no significant difference, thus implying a symmetrical sensory innervation of the rat's tail.

A simple method for the construction of a recording-injection microelectrode with glass-insulated microwire.

A rapid method for the production of a glass-insulated microwire electrode is described. A microwire was threaded into a glass capillary which was then pulled on a vertical pipette puller. A conical tip of the microwire was formed when the strongly heated glass capillary broke together with the wire in it. A tight seal of the glass-insulated microwire electrode between the glass and the metal was accomplished with silicone glue. The manufactured electrode performed consistently at different immersion depths, and yielded stable recordings of single units in the cerebral cortex and the medulla of rats. The strength and low impedance characteristics of the glass-insulated microwire electrode may make it useful for the recording of single units in deep brain structures. Furthermore, the electrode can be easily combined with another glass micropipette to form a dual recording-injection microelectrode unit.

A modified "triangular pulse" stimulator for C-fibers stimulation.

A low-cost, battery-powered stimulator is described. This device generates asymmetric current pulse with fast rising phase and slower exponential decay. The current intensity and the time constant of the exponential decay can be independently and continuously varied. An example of using this stimulator to selectively activate C-fibers is demonstrated. In this case the total charge injected in one stimulation is only 67 nanocolumb, which is much smaller than that injected by conventional DC polarization technique. Detailed information about the circuit design is described.

Effect of conduction distance on amplitude and area of compound action potentials of A fiber and C fiber.

We used 24 sacral dorsal roots of the rat to analyze amplitude and area changes of biphasic and monophasic compound action potentials (CAPs) at 4 conduction distances. Both the CAPs of the A-fiber and C-fiber were analyzed. The changes were examined with the paired t test and linear regression. All the variables decreased linearly with increasing conduction distance except area of monophasic CAP, which remained constant throughout. CAP data were also compared between the S1 and S2 roots by using the pooled t test. Only area of monophasic CAP showed good correspondence with the anatomical data. Therefore, area of monophasic CAP is suggested as the best parameter for representing the functional state of a nerve.

A multichannel system for recording and analysis of cortical field potentials in freely moving rats.

A system has been developed to record and analyze the cortical electrical activity from 16 different sites in freely moving rats. The hardware includes a 16-channel amplifier system whose high input impedance, low noise, small size, light weight and shielded multistrand connecting cable allow high quality multichannel recording of field potentials. The software developed for this system consists of data acquisition, data analysis and topographic mapping of cortical-evoked potentials as well as electroencephalograms. Cortical field potentials evoked by CO2-laser stimulation were compared between wakeful and pentobarbital-treated conditions. To investigate the background interference produced by sleep spindle, three kinds of reference-free methods (the Wilson, local average and weighted average methods) were utilized to compare the coherence between field potentials obtained from two cerebral hemispheres using monopolar vs. reference-free recordings.

Algorithmic complexity as an index of cortical function in awake and pentobarbital-anesthetized rats.

This study introduces algorithmic complexity to measure characteristics of brain functions. The EEG of the rat was recorded with implanted electrodes. The normalized complexity value was relatively independent of data length, and it showed a simpler and easier calculation characteristic than other non-linear indexes. The complexity index revealed significant differences among awake, asleep, and anesthetized states. It may be useful in tracking short-term and long-term changes in brain functions, such as anesthetized depth, drug effects, or sleep-wakefulness.

Interactive program for spectral and area analysis of compound action potentials of A-fiber and C-fiber.

An interactive program was described to correct the baseline wandering of the compound action potentials (CAPs) of C-fiber, to calculate the area and the peak amplitude of CAPs, and to analyze their spectral distribution. Using this program, we found the optimal bandpass of the filter for recording CAPs to be from 10 Hz to 3 kHz.

Intracellular staining of physiologically identified neurons and axons in the somatosensory thalamus of the cat.

Neurons and axons responding to somesthetic stimulation in the thalamic ventrobasal complex (VB) were characterized electrophysiologically by intracellular recording and then individually injected with horseradish peroxidase. Two types of thalamocortical relay neuron were identified, primarily on the basis of dendritic morphology and axon diameter. Types with cutaneous or deep receptive fields were found in each class. Neither type had collateral axons in VB but each gave branches to the thalamic reticular nucleus (RTN). Small putative interneurons in VB and RTN neurons with somatosensory receptive fields were also injected. The RTN neurons had profusely branched widely ramifying axons in VB and adjoining nuclei. Injected medial lemniscal axons in VB had a range of receptive field properties and conduction velocities and ended in elongated anteroposterior domains with one or more dense concentrations of terminal boutons of varying size and with varying numbers of boutons.

Dynamic changes of touch- and laser heat-evoked field potentials of primary somatosensory cortex in awake and pentobarbital-anesthetized rats.

In this investigation, changes of mechanical- (MEP) and laser-evoked potentials (LEP) in rat primary somatosensory cortex during the course of pentobarbital (PB) anesthesia were examined. Temporal analysis of changes in the magnitude and latency of MEP and LEP, EEG activity, gross motor behaviors, and the tail flick response following laser stimulation before, during, and after PB administration (50 mg/kg, i.p.) was performed and correlated in chronically implanted rats. During the wakeful condition, there were two major cortical components each following mechanical stimulation (MEP1 and MEP2, n=17) and laser stimulation (LEP1 and LEP2, n=10), respectively. After PB administration, the positive peak in MEP1 was enhanced, and all other components disappeared. These components returned with different time courses. Two hours after PB administration, when the rat had spontaneous movements and flexor reflexes, LEP2 showed reversed polarity. MEP2 returned gradually 3 h after PB administration when the rat regained its ability to execute coordinated movements. After 4 h, LEP1 began to reappear and LEP2 returned to its negative polarity. We found that PB facilitated Abeta fiber-related cortical evoked potential (MEP1), while differentially inhibited Adelta and C fiber-related components (MEP2, LEP1 and LEP2). Characterization of these anesthesia-induced changes in cortical output may be useful in studying the neural basis of tactile and pain sensations.

Distribution of thalamic nociceptive neurons activated from the tail of the rat.

The purpose of the present study was to map systematically in the thalamus the distribution of neurons processing nociceptive information from the tail of the rat. Pentobarbital-anesthetized and gallamine-paralyzed rats were used. Glass microelectrodes were used to record extracellularly from thalamic neurons. Noxious radiant heat stimuli were applied to the tail with a tail-flick apparatus, and the recorded neurons were localized with horseradish peroxidase deposits or by marking electrodes left in situ. A number of 121 neurons were tested of which 45 responded. Of these, 13 were located in the ventrobasal complex (VB), 17 were located in the central lateral nucleus and the parafascicular nucleus of the intralaminar nuclei (ILN). The rest of the responding neurons were located in the posterior group, the reticular thalamic nucleus, and the zona incerta. The nucleus submedius was not examined specifically. It is concluded that the VB and the ILN are two of the most important thalamic nuclei for processing nociceptive information from the tail of the rat.

Comparison of touch- and laser heat-evoked cortical field potentials in conscious rats.

Field potentials and multiunit activities from chronically implanted cortical electrodes were used to study tactile and nociceptive information processing from the tail of the rat. Fourteen stainless steel screws implanted in the skull were used as electrodes to record field potentials in different cortical areas. Electrical, mechanical, and laser pulses were applied to the tail to induce evoked cortical field potentials. Evoked responses were compared before and after sodium pentobarbital anesthesia (50 mg/kg, i.p.). In both electrical- and mechanical-evoked potential (EEP and MEP) studies, two major peaks were found in the conscious animal. The polarity of the late component was modified after pentobarbital anesthesia. In the laser-evoked potential (LEP) study, two distinct negative peaks were found. Both peaks were very sensitive to anesthesia. Following quantitative analysis, our data suggest that the first positive peak of EEP and MEP corresponded to the activation of the Abeta fiber, the second negative peak of MEP and the first peak of LEP corresponded to Adelta fiber activation, while the second peak of LEP corresponded to C fiber activation. The absolute magnitudes of all cortical components were positively related to the intensity of the stimulation. From spatial mapping analysis, a localized concentric source of field potential was observed in the primary somatosensory cortex (SI) only after activation of the Abeta fiber. Larger responsive cortical areas were found in response to Adelta and C fiber activation. In an intracortical recording experiment, both tactile and nociceptive stimulation evoked heightened unit activity changes at latencies corresponding to respective field potentials. We conclude that different cortical areas are involved in the processing of A and C fiber afferent inputs, and barbiturate anesthesia modifies their processing.

Quantitative relationship between fluctuations of blood pressure and sympathetic nerve activity in pentobarbital anesthetized rats.

Transfer function analysis was used to examine the coupling between the sympathetic nerve activity (SNA) and the blood pressure (BP) fluctuations. In pentobarbital anesthetized Wistar rats, linear regression of the relationship between frequency (X-axis) and the logarithmic transfer magnitude, i.e. log (BP power density/renal SNA power density) (Y-axis), in the low frequency range (0.016-0.85 Hz) revealed an excellent fit (r = 0.97-0.98). Comparing the regression lines, rats under large dose of pentobarbital anesthesia (40 mg/kg, i.v. single dose) had significantly smaller intercept and slope values compared to rats under small dose of pentobarbital anesthesia (12.5 mg/kg). When intercept and slope values were compared between intact and acutely sinoaortic denervated rats, no significant difference were found. The results suggest that sympathetic modulation of vasomotor tone may be a major factor in generating BP fluctuations between 0.016 and 0.85 Hz in rats. Furthermore, these results support the possibility of using low frequency spectral power of BP to quantitatively estimate the fluctuations of SNA for rats under pentobarbital anesthesia if anesthetic depth is controlled.

Frequency dependent sympathetic modulation of vasomotor tone in the anesthetized rat.

The hypothesis that sympathetic nerve system modulates a specific frequency range of blood pressure fluctuation was tested by electrical stimulation of the medullary sympathetic excitatory sites in anesthetized, paralyzed, vagotomized, cardiac sympathetic blocked, baroreceptor transected and angiotensin II converting enzyme inhibited rats. The frequency tested ranged from 0.02 to 1.7 Hz. For blood pressure fluctuation within this range there was no specific sympathetic reactive zone. Instead, low frequency fluctuation of sympathetic flow produced blood pressure fluctuation of the same frequency. Transfer magnitude of renal sympathetic activity to blood pressure decrease logarithmically with the increase of stimulation frequency. The relationship between the sympathetic spectral power (P(SND), (microV.s)2/Hz) and the blood pressure spectral power (P(BP), mmHg2/Hz) was found as P(BP)=10(1.3) x P(SND) x 10(-4.7x(frequency)). This transfer function demonstrates that when blood pressure fluctuation is used to estimate the sympathetic activity, it should be frequency weighted.

Coexistence of autonomic and somatic mechanisms in the pressor areas of medulla in cats.

The effects of electrical stimulation and microinjection of sodium glutamate (0.5 M) in the sympathetic pressor areas of the dorsal medulla (DM), ventrolateral medulla (VLM), and parvocellular nucleus (PVC) on the knee jerk, crossed extension, and evoked potential of the L5 ventral root produced by intermittent electrical stimulation were studied in 98 adult cats anesthetized with chloralose and urethane. During electrical and glutamate stimulation of these pressor areas, in addition to the rise of systemic arterial blood pressure marked inhibition of the spinal reflex was produced, indicating presence of neuronal perikarya responsible for these actions. Mild to moderate augmentation of spinal reflexes was also observed during brain stimulation but only in a few cases. The magnitude of the somatic effects among the pressor areas of the VLM, DM, and PVC subsequent to glutamate activation was about the same. Induced spinal reflex inhibition, independent from the baroreceptor and vagal influence, remained essentially unaltered after acute midcollicular decerebration. The inhibition was also observed in cats decerebellated 8-10 days in advance. The inhibition was not affected after bilateral electrolytic- or kainic-acid-induced lesions in the paramedian reticular nucleus (PRN). On the contrary, PRN-induced spinal reflex inhibition was attenuated after bilateral lesions in the DM or VLM. Data suggest that there coexists neuronal subpopulations in the VLM, DM, and PVC that can affect both the sympathetic pressor systems and spinal reflexes.

Inhibition of spinal reflexes by paramedian reticular nucleus.

The inhibitory actions of the paramedian reticular nucleus (PRN), and its neighbouring structures, i.e., midline raphe nuclei (MRN) and dorsal medullary depressor area (DMD) on the knee jerk (KnJ) and crossed extension movement (CEM) induced by central sciatic stimulation and on the L5 ventral root response (EVRR) evoked by central tibial stimulation, were studied in cats under urethane (400 mg/kg) and alpha-chloralose (40 mg/kg) anesthesia alone, IP or further paralyzed with atracurium besylate (0.5 mg/kg/30 min), IV. Electrical stimulation of the above areas with rectangular pulses (80 Hz, 1.0 msec, 100-200 microA) decreased systemic arterial blood pressure (SAP) in an average value of: 36 +/- 3 mmHg for PRN; 19 +/- 2 mmHg for MRN; and 23 +/- 3 mmHg for DMD. The KnJ and CEM were almost completely suppressed by simultaneous PRN stimulation. The EVRR, including mono- and polysynaptic spinal reflexes with transmission velocity from 10 to 60 m/sec or above, were also suppressed. MRN stimulation only inhibited the KnJ, CEM and polysynaptic spinal reflexes with transmission velocities between 25 and 60 m/sec, but facilitated spinal reflexes with conduction velocities below 10 m/sec. On the other hand, DMD stimulation resulted in small suppression of KnJ, CEM and inhibition of polysynaptic spinal reflexes with conduction velocities between 25 and 60 m/sec. Even though MRN and DMD partially inhibited polysynaptic spinal reflexes, the magnitude of such inhibition was much smaller than that produced by PRN (-20% and -22% vs. -48%). The above-mentioned PRN effects on SAP and EVRR persisted in chronic animals decerebellated 9-12 days before.(ABSTRACT TRUNCATED AT 250 WORDS)

Frequency coding ability of the somatosensory thalamocortical system and its modulation by anesthesia depth.

The purposes of the present study were to characterize and compare the mid-tail cortical and thalamic somatosensory evoked potentials (SEPs), and to examine how the depth of the barbiturate anesthesia affected them. After the tail representative locations of sacrococcygeal dorsal root (S2 or S3), thalamus (ventroposterior lateral nucleus, VPL) and primary somatosensory cortex (SI) were set up for recording, the rats were infused serially with diluted sodium pentobarbital solution beginning from light (5 to 10 mg/kg/hr) to deep (30 to 40 mg/kg/hr) and then stop infusion (recovery). The effects of anesthetic depth on SEPs were examined of dorsal root, thalamic and cortical field potentials evoked by mid-tail stimulation of various stimulation intensities (100 microA to 2mA, step 100 pA, at 2 Hz) and frequencies (0.5 to 11 Hz, step 0.5 to 1 Hz, at 3T). The depth of anesthesia did not affect the strength-response curves of the SEPs. In contrast, the depth of anesthesia differentially influenced the frequency following capabilities of different recording sites. Under light anesthesia, thalamic SEP was only significantly affected with stimulation frequencies higher than 8 Hz, whereas cortical SEP was significantly affected with 2 Hz or higher. Under deep anesthesia, thalamic SEP evoked by low frequency tail stimulation was not significantly changed. In contrast, cortical SEP was affected much strongly so that under 1 Hz stimulation, a significant difference could be observed. We concluded, therefore, that thalamus was only partially responsible for the limited frequency following capability of the SI, and that the main effect of pentobarbital was on the cortical level. From the data obtained, an exponentially decaying curve could be observed for the cortical SEP under different stimulation frequencies. The decay constant showed a 50% change with a change in anesthesia depth. We propose that the decay constant could be used as a sensitive index for the monitoring of anesthetic depth.

Surface temperature change, cortical evoked potential and pain behavior elicited by CO2 lasers.

The performance of a self-designed CO2 laser stimulator, TL#2, was evaluated against a commercial product, model DE20XL of the Direct Energy Inc. (Irvine). The major items evaluated were the temperature change of the irradiated surface and the electrophysiological and behavior changes in the rat elicited by single laser pulse irradiation. Single shots of TL#2 produced a profile of surface temperature change similar to those of the DE20XL, as quantified by their maximal temperature change, rate of rise (half time to maximum) and rate of temperature drop. TL#2 and DE20XL elicited the same pain behaviors and the same pattern of cortical evoked potential in awake, behaving rats. TL#2 differed from the DE20XL in its laser beam shape and focal depth. The cross sectional energy profile of the TL#2 was a Gaussian shape, i.e., most intense at its center point, whereas that of the DE20XL with the FL20XL attachment had a shape of an inverted Gaussian, i.e., most intense in the periphery. Consequently, the peak energy of the center of the TL#2 laser beam grows rapidly with an increase in the pulse intensity. Caution must be taken not to use this machine at high intensity or for long duration less permanent damage should be produced on tested animal or human subject. In summary, TL#2 when used properly, should be a useful tool in the study of pain mechanism.

Cardiac and pulmonary vagal neurons receive excitatory chemoreceptor input.

The effects of hypercapnia and hypocapnia on the activities of the cardiac and pulmonary vagal single fibers were examined in the decerebrated, unanesthetized, paralyzed, and vagotomized cats. The animals breathed 100% O2. Fractional end tidal CO2 concentration was raised to 9% by adding CO2 into the O2 inlet. Average discharge rate of efferent cardiac vagal units (n=10) increased from 1.0+/-0.3 to 2.2+/-0.3 Hz. Hypocapnia apnea was produced by hyperventilation. Activities of cardiac vagal units tested (n = 4) showed dramatic decrease (0.1+/-0.0 Hz). Mean arterial blood pressure did not change significantly under these conditions. In contrast, only instantaneous firing rate during inspiration was significantly increased for efferent pulmonary vagal units (n = 11) during hypercapnia. The activities of the 3 pulmonary vagal units tested with hypocapnia decreased significantly. We concluded that cardiac and pulmonary vagal neurons were excited by chemoreceptor input.

Mechanisms underlying the cardioinhibitory and pressor responses elicited from the medullary neurons in the gigantocellular tegmental field of cats.

A stimulation of the gigantocellular tegmental field (FTG) in the medulla oblongata often increases systemic arterial blood pressure (SAP) and decreases heart rate (HR). We investigated if the cardioinhibitory/depressor areas, including the nucleus ambiguus (NA), the dorsal motor nucleus of vagus (DMV) and the caudal ventrolateral medulla (CVLM), underlied the functional expression of FTG neurons in regulating cardiovascular responses. In 73 chloralose-urethane anesthetized cats, the HR, SAP and vertebral nerve activity (VNA) were recorded. Neurons in the FTG, NA, DMV and CVLM were stimulated by microinjection of sodium glutamate (25 mM Glu, 70 nl). To study if the NA, DMV, and CVLM relayed the cardioinhibitory messages from the FTG, 24 mM kainic acid (KA, 100 nl) was used as an excitotoxic agent to lesion neurons in the NA, DMV or CVLM. We found that the cardioinhibition induced by FTG stimulation was significantly reduced by KA lesioning of the ipsilateral NA or DMV. Subsequently, a bilateral KA lesion of NA or DMV abolished the cardioinhibitory responses of FTG. Compared to the consequence of KA lesion of the DMV, only a smaller bradycardia was induced by FTG stimulation after KA lesion of the NA. The pressor response induced by Glu stimulation of the FTG was reduced by the KA lesion of the CVLM. Such an effect was dominant ipsilaterally. Our findings suggested that both NA and DMV mediated the cardioinhibitory responses of FTG. The pressor message from the FTG neurons might be partly working via a disinhibitory mechanism through the depressor neurons located in the CVLM.

Evoked potential elicited by periaqueductal grey stimulation in the pressor sites of dorsal and ventrolateral medulla in cats.

Evoked potential methods were used to compare the patterns of responses to periaqueductal grey (PAG) stimulation in two major medullary sympathoexcitatory areas, namely, ventrolateral medulla (VLM) and dorsal medulla (DM). Adult cats were anesthestized with chloralose and urethane, paralyzed, ventilated and bilaterally vagotomized. Pressor points in the PAG were identified with rectangular pulses in 80 Hz, 200 microA, 0.1 ms and used as stimulation points. Pressor points in VLM and DM were identified by injection of 100 nl of 0.25 M glutamate solution. Adjacent points 1 mm away from these pressor points where injected the same amount of glutamate solution elicited very little blood pressure changes were used as control recording points. Stimulation of the PAG pressor points with rectangular pulse in 1 Hz, 0.1 ms, 200 microA elicited complex evoked field potentials in both VLM and DM. In 9 of the 13 pressor-control pairs tested, distinctive evoked potential of long latency were seen in the pressor point only. These evoked potentials, averaged 3.5 ms in the DM, and 5.6 ms in the VLM. Therefore, it was postulated that pressor points of PAG had separate and independent connections with DM and VLM.