Biomedical subjects

D J Bennett

Publications and source records attributed to D J Bennett.

At least 19 recordsLinked to original sources

PubMed2006

Serotonin facilitates a persistent calcium current in motoneurons of rats with and without chronic spinal cord injury.

In the months after spinal cord transection, motoneurons in the rat spinal cord develop large persistent inward currents (PICs) that are responsible for muscle spasticity. These PICs are mediated by low-threshold TTX-sensitive sodium currents (Na PIC) and L-type calcium currents (Ca PIC). Recently, the Na PIC was shown to become supersensitive to serotonin (5-HT) after chronic injury. In the present paper, a similar change in the sensitivity of the Ca PIC to 5-HT was investigated after injury. The whole sacrocaudal spinal cord from acute spinal rats and spastic chronic spinal rats (S2 level transection 2 mo previously) was studied in vitro. Intracellular recordings were made from motoneurons and slow voltages ramps were applied to measure PICs. TTX was used to block the Na PIC. For motoneurons of chronic spinal rats, a low dose of 5-HT (1 microM) significantly lowered the threshold of the Ca PIC from -56.7 +/- 6.0 to -63.1 +/- 7.1 mV and increased the amplitude of the Ca PIC from 2.4 +/- 1.0 to 3.0 +/- 0.73 nA. Higher doses of 5-HT acted similarly. For motoneurons of acute spinal rats, low doses of 5-HT had no significant effects, whereas a high dose (about 30 microM) significantly lowered the threshold of the L-Ca PIC from -58.5 +/- 14.8 to -62.5 +/- 3.6 mV and increased the amplitude of the Ca PIC from 0.69 +/- 1.05 to 1.27 +/- 1.1 nA. Thus Ca PICs in motoneurons are about 30-fold supersensitive to 5-HT in chronic spinal rats. The 5-HT-induced facilitation of the Ca PIC was blocked by nimodipine, not by the I(h) current blocker Cs(+) (3 mM) or the SK current blocker apamin (0.15 microM), and it lasted for hours after the removal of 5-HT from the nCSF, even increasing initially after removing 5-HT. The effects of 5-HT make motoneurons more excitable and ultimately lead to larger, more easily activated plateaus and self-sustained firing. The supersensitivity to 5-HT suggests the small amounts of endogenous 5-HT below the injury in a chronic spinal rat may act on supersensitive receptors to produce large Ca PICs and ultimately enable muscle spasms.

Anesthetics, Local↗

PubMed2006

Endogenous monoamine receptor activation is essential for enabling persistent sodium currents and repetitive firing in rat spinal motoneurons.

The spinal cord and spinal motoneurons are densely innervated by terminals of serotonin (5-HT) and norepinephrine (NE) neurons arising mostly from the brain stem, but also from intrinsic spinal neurons. Even after long-term spinal transection (chronic spinal), significant amounts (10%) of 5-HT and NE (monoamines) remain caudal to the injury. To determine the role of such endogenous monoamines, we blocked their action with monoamine receptor antagonists and measured changes in the sodium currents and firing in motoneurons. We focused on persistent sodium currents (Na PIC) and sodium spike properties because they are critical for enabling repetitive firing in motoneurons and are facilitated by monoamines. Intracellular recordings were made from motoneurons in the sacrocaudal spinal cord of normal and chronic spinal rats (2 mo postsacral transection) with the whole sacrocaudal cord acutely removed and maintained in vitro (cords from normal rats termed acute spinal). Acute and chronic spinal rats had TTX-sensitive Na PICs that were respectively 0.62 +/- 0.76 and 1.60 +/- 1.04 nA, with mean onset voltages of -63.0 +/- 5.6 and -64.1 +/- 5.4 mV, measured with slow voltage ramps. Application of 5-HT2A, 5-HT2C, and alpha1-NE receptor antagonists (ketanserin, RS 102221, and WB 4101, respectively) significantly reduced the Na PICs, and a combined application of these three monoamine antagonists completely eliminated the Na PIC, in both acute and chronic spinal rats. Likewise, reduction of presynaptic transmitter release (including 5-HT and NE) with long-term application of cadmium also eliminated the Na PIC. Associated with the elimination of the Na PIC in monoamine antagonists, the motoneurons lost their ability to fire during slow current ramps. At this point, the spike evoked by antidromic stimulation was not affected, suggesting that activation of the transient sodium current was not impaired. However, the spike evoked after a slow ramp depolarization was slightly reduced in height and rate-of-rise, suggesting decreased sodium channel availability as a result of increased channel inactivation. These results suggest that endogenous monoamine receptor activation is critical for enabling the Na PIC and decreasing sodium channel inactivation, ultimately enabling steady repetitive firing in both normal and chronic spinal rats.

Animals↗

PubMed2006

5-HT2 receptor activation facilitates a persistent sodium current and repetitive firing in spinal motoneurons of rats with and without chronic spinal cord injury.

We examined the modulation of persistent inward currents (PICs) by serotonin (5-HT) in spinal motoneurons of normal and chronic spinal rats. PICs are composed of both a TTX-sensitive persistent sodium current (Na PIC) and a nimodipine-sensitive persistent calcium current (Ca PIC), and we focused on quantifying the Na PIC (and its action on the total PIC), which is known to be critical in enabling repetitive firing. Intracellular recordings were made from motoneurons of the whole sacrocaudal spinal cord of normal adult rats after the cord was acutely transected at the S2 spinal level (acute spinal rat condition), removed from the animal, and then maintained in vitro. In vitro motoneuron recordings were likewise made from rats that had a sacral spinal transection 2 mo previously (chronic spinal rats). In motoneurons from acute spinal rats, moderately high doses of 5-HT (> or = 10 microM), or the 5-HT2 receptor agonist DOI (> or = 30 microM), significantly increased the total PIC, hyperpolarized the PIC onset voltage, and hyperpolarized the spike threshold, whereas lower doses had no effect. Both 5-HT and DOI specifically increased the Na PIC portion of the total PIC (tested with nimodipine blocking the Ca PIC). Additionally, 5-HT, but not DOI, depolarized the resting membrane potential (Vm) and increased the input resistance (Rm) in a dose-dependent manner. Therefore 5-HT2 receptor activation facilitated the Na PIC, whereas other 5-HT receptors modulated Vm and Rm. Motoneurons of chronic spinal rats responded to 5-HT and DOI in the same way, but with larger responses and at much lower doses (0.3-1 microM), thus exhibiting a 30-fold supersensitivity to 5-HT. Specifically the Na PIC was supersensitive to 5-HT2 receptor activation with DOI. Also, Rm and Vm were supersensitive to 5-HT. Consistent with the known critical role of the Na PIC in repetitive firing, enhancement of the Na PIC by DOI or 5-HT facilitated the repetitive firing evoked by steady current injection and enabled repetitive firing in a subpopulation of motoneurons of acute spinal rats that were initially unable to produce sustained repetitive firing. We suggest that after spinal transection, residual endogenous spinal sources of 5-HT help facilitate the Na PIC and repetitive firing. With chronic injury, the developed 5-HT supersensitivity more than compensates for lost brain stem 5-HT, so that the Na PIC is large and motoneurons are very excitable, thus contributing to spasticity.

Amphetamines↗

PubMed2005

Persistent sodium currents and repetitive firing in motoneurons of the sacrocaudal spinal cord of adult rats.

Months after sacral spinal transection in rats (chronic spinal rats), motoneurons below the injury exhibit large, low-threshold persistent inward currents (PICs), composed of persistent sodium currents (Na PICs) and persistent calcium currents (Ca PICs). Here, we studied whether motoneurons of normal adult rats also exhibited Na and Ca PICs when the spinal cord was acutely transected at the sacral level (acute spinal rats) and examined the role of the Na PIC in firing behavior. Intracellular recordings were obtained from motoneurons of acute and chronic spinal rats while the whole sacrocaudal spinal cord was maintained in vitro. Compared with chronic spinal rats, motoneurons of acute spinal rats were more difficult to activate because the input resistance was 22% lower and resting membrane potential was hyperpolarized 4.1 mV further below firing threshold (-50.9 +/- 6.2 mV). In acute spinal rats, during a slow voltage ramp, a PIC was activated subthreshold to the spike (at -57.2 +/- 5.0 mV) and reached a peak current of 1.11 +/- 1.21 nA. This PIC was less than one-half the size of that in chronic spinal rats (2.79 +/- 0.94 nA) and usually was not large enough to produce bistable behavior (plateau potentials and self-sustained firing not present), unlike in chronic spinal rats. The PIC was composed of two components: a TTX-sensitive Na PIC (0.44 +/- 0.36 nA) and a nimodipine-sensitive Ca PIC (0.78 +/- 0.82 nA). Both were smaller than in chronic spinal rats (but with similar Na/Ca ratio). The presence of the Na PIC was critical for normal repetitive firing, because no detectable Na PIC was found in the few motoneurons that could not fire repetitively during a slow ramp current injection and motoneurons that had large Na PICs more readily produced repetitive firing and had lower minimum firing rates compared with neurons with small Na PICs. Furthermore, when the Na PIC was selectively blocked with riluzole, steady repetitive firing was eliminated, even though transient firing could be evoked on a rapid current step and the spike itself was unaffected. In summary, only small Ca and Na PICs occur in acute spinal motoneurons, but the Na PIC is essential for steady repetitive firing. We discuss how availability of monoamines may explain the variability in Na PICs and firing in the normal and spinal animals.

Animals↗

PubMed2004

Effects of baclofen on spinal reflexes and persistent inward currents in motoneurons of chronic spinal rats with spasticity.

In the months after spinal cord injury, motoneurons develop large voltage-dependent persistent inward currents (PICs) that cause sustained reflexes and associated muscle spasms. These muscle spasms are triggered by any excitatory postsynaptic potential (EPSP) that is long enough to activate the PICs, which take > 100 ms to activate. The PICs are composed of a persistent sodium current (Na PIC) and a persistent calcium current (Ca PIC). Considering that Ca PICs have been shown in other neurons to be inhibited by baclofen, we tested whether part of the antispastic action of baclofen was to reduce the motoneuron PICs as opposed to EPSPs. The whole sacrocaudal spinal cord from acute spinal rats and spastic chronic spinal rats (with sacral spinal transection 2 mo previously) was studied in vitro. Ventral root reflexes were recorded in response to dorsal root stimulation. Intracellular recordings were made from motoneurons, and slow voltage ramps were used to measure PICs. Chronic spinal rats exhibited large monosynaptic and long-lasting polysynaptic ventral root reflexes, and motoneurons had associated large EPSPs and PICs. Baclofen inhibited these reflexes at very low doses with a 50% inhibition (EC50) of the mono- and polysynaptic reflexes at 0.26 +/- 0.07 and 0.25 +/- 0.09 (SD) microM, respectively. Baclofen inhibited the monosynaptic reflex in acute spinal rats at even lower doses (EC50 = 0.18 +/- 0.02 microM). In chronic (and acute) spinal rats, all reflexes and EPSPs were eliminated with 1 microM baclofen with little change in motoneuron properties (PICs, input resistance, etc), suggesting that baclofen's antispastic action is presynaptic to the motoneuron. Unexpectedly, in chronic spinal rats higher doses of baclofen (20-30 microM) significantly increased the total motoneuron PIC by 31.6 +/- 12.4%. However, the Ca PIC component (measured in TTX to block the Na PIC) was significantly reduced by baclofen. Thus baclofen increased the Na PIC and decreased the Ca PIC with a net increase in total PIC. By contrast, when a PIC was induced by 5-HT (10-30 microM) in motoneurons of acute spinal rats, baclofen (20-30 microM) significantly decreased the PIC by 38.8 +/- 25.8%, primarily due to a reduction in the Ca PIC (measured in TTX), which dominated the total PIC in these acute spinal neurons. In summary, baclofen does not exert its antispastic action postsynaptically at clinically achievable doses (< 1 microM), and at higher doses (10-30 microM), baclofen unexpectedly increases motoneuron excitability (Na PIC) in chronic spinal rats.

Acute Disease↗

PubMed2004

Spastic long-lasting reflexes in the awake rat after sacral spinal cord injury.

Following chronic sacral spinal cord transection in rats the affected tail muscles exhibit marked spasticity, with characteristic long-lasting tail spasms evoked by mild stimulation. The purpose of the present paper was to characterize the long-lasting reflex seen in tail muscles in response to electrical stimulation of the tail nerves in the awake spastic rat, including its development with time and relation to spasticity. Before and after sacral spinal transection, surface electrodes were placed on the tail for electrical stimulation of the caudal nerve trunk (mixed nerve) and for recording EMG from segmental tail muscles. In normal and acute spinal rats caudal nerve trunk stimulation evoked little or no EMG reflex. By 2 wk after injury, the same stimulation evoked long-lasting reflexes that were 1) very low threshold, 2) evoked from rest without prior EMG activity, 3) of polysynaptic latency with >6 ms central delay, 4) about 2 s long, and 5) enhanced by repeated stimulation (windup). These reflexes produced powerful whole tail contractions (spasms) and developed gradually over the weeks after the injury (< or =52 wk tested), in close parallel to the development of spasticity. Pure low-threshold cutaneous stimulation, from electrical stimulation of the tip of the tail, also evoked long-lasting spastic reflexes, not seen in acute spinal or normal rats. In acute spinal rats a strong C-fiber stimulation of the tip of the tail (20 x T) could evoke a weak EMG response lasting about 1 s. Interestingly, when this C-fiber stimulation was used as a conditioning stimulation to depolarize the motoneuron pool in acute spinal rats, a subsequent low-threshold stimulation of the caudal nerve trunk evoked a 300-500 ms long reflex, similar to the onset of the long-lasting reflex in chronic spinal rats. A similar conditioned reflex was not seen in normal rats. Thus there is an unusually long low-threshold polysynaptic input to the motoneurons (pEPSP) that is normally inhibited by descending control. This pEPSP is released from inhibition immediately after injury but does not produce a long-lasting reflex because of a lack of motoneuron excitability. With chronic injury the motoneuron excitability is increased markedly, and the pEPSP then triggers sustained motoneuron discharges associated with long-lasting reflexes and muscle spasms.

Anesthesia↗

PubMed2004

Spastic long-lasting reflexes of the chronic spinal rat studied in vitro.

Over the months following sacral spinal cord transection in adult rats, a pronounced spasticity syndrome emerges in the affected tail musculature, where long-lasting muscle spasms can be evoked by low-threshold afferent stimulation (termed long-lasting reflex). To develop an in vitro preparation to examine the neuronal mechanisms underlying spasticity, we removed the whole sacrocaudal spinal cord of these spastic chronic spinal rats (>1 mo after S(2) sacral spinal transection) and maintained it in artificial cerebral spinal fluid in a recording chamber. The ventral roots were mounted on monopolar recording electrodes in grease, and the reflex responses to dorsal root stimulation were recorded and compared with the reflexes seen in the awake chronic spinal rat. When the dorsal roots were stimulated with a single pulse, a long-lasting reflex occurred in the ventral roots, with identical characteristics to the long-lasting reflex in the awake spastic rat tail. The reflex response was low threshold (T), short latency, long duration ( approximately 2 s), and enhanced by repeated stimulation. Brief high-frequency stimulation trains (0.5 s, 100 Hz, 1.5 x T) evoked even longer duration responses (5-10 s), with repeated bursts of activity that were similar to the repeated muscle spasms evoked in awake rats with stimulation trains or manual skin stimulation. Stimulation of a given dorsal root evoked long-lasting reflexes in both the ipsilateral and contralateral ventral roots. Long-lasting reflexes did not occur in the sacrocaudal spinal cord of acute spinal rats (S(2) transection), which is similar to the areflexia seen in awake acute spinal rats. However, long-lasting reflexes could be made to occur in the acute spinal rat by altering K(+) (7 mM) or Mg(2+) (0 mM) concentrations, or by application of high doses of the neuromodulators norepinephrine (NE, >20 microM) or serotonin (5-HT, >20 microM). In chronic spinal rats, much lower doses of these neuromodulators (0.1 microM) enhanced the long-lasting reflexes, suggesting a denervation supersensitivity to 5-HT and NE following injury. Higher doses of NE or 5-HT produced a paradoxical inhibition of the long-lasting reflexes. The high dose inhibition by NE was mimicked by the alpha(2)-adrenergic receptor agonist clonidine but not the alpha(1)-adrenergic receptor agonist methoxamine. In summary, the sacral spinal in vitro preparation offers a new approach to the study of spinal cord injury and analysis of antispastic drugs.

Adrenergic alpha-Agonists↗

PubMed2002

Differential distribution of interneurons in the neural networks that control walking in the mudpuppy (Necturus maculatus) spinal cord.

Locomotor behavior is believed to be produced by interneuronal networks that are intrinsically organized to generate the underlying complex spatiotemporal patterns. In order to study the temporal correlation between the firing of individual interneurons and the pattern of locomotion, we utilized the spinal cord-forelimb preparation from the mudpuppy, in which electrophysiological recordings of neuronal activity were achieved during walking-like movement of the forelimb induced by bath application of N-methyl- D-aspartate (NMDA). Intra- and extracellular recordings were made in the C2 and C3 segments of the spinal cord. These segments contain independent flexor and extensor centers for the forelimb movement about the elbow joint during walking. Among the 289 cells recorded in the intermediate gray matter (an area between the ventral and dorsal horns) of the C2 and C3 segments, approximately 40% of the cells fired rhythmically during "walking." The firing rates were 6.4+/-0.4 impulses/s (mean +/- SE). These rhythmically active cells were classified into four types based on their phase of activity during a normalized step cycle. About half the rhythmic cells fired in phase with either the flexor (F) or extensor (E) motoneurons. The rest fired in the transitions between the two phases (F-->E and E-->F). Longitudinal distributions of the four types of interneurons along the spinal cord were in agreement with observations that revealed distinct but overlapping flexor and extensor centers for walking. Some cells triggered short-latency responses in the elbow flexor or extensor muscles and may be last-order interneurons. These observations suggest that there is a differential distribution of phase-specific interneurons in the central pattern generator of the mudpuppy spinal cord for walking.

Action Potentials↗

PubMed2001

Alpha-amino acid phenolic ester derivatives: novel water-soluble general anesthetic agents which allosterically modulate GABA(A) receptors.

In the search for a novel water-soluble general anesthetic agent the activity of an alpha-amino acid phenolic ester lead, identified from patent literature, was markedly improved. In addition to improving in vivo activity in mice, good in vitro activity at GABA(A) receptors was also conferred. Within the series of compounds good enantioselectivity for both in vitro and in vivo activity was found, supporting a protein-mediated mechanism of action for anesthesia involving allosteric modulation of GABA(A) receptors. alpha-Amino acid phenolic ester 19, as the hydrobromide salt Org 25435, was selected for clinical evaluation since it retained the best overall anesthetic profile coupled with improved stability and water solubility. In the clinic it proved to be an effective intravenous anesthetic in man with rapid onset of and recovery from anesthesia at doses of 3 and 4 mg/kg.

Allosteric Regulation↗

PubMed2001

Plateau potentials in sacrocaudal motoneurons of chronic spinal rats, recorded in vitro.

Intracellular recordings were made from sacrocaudal tail motoneurons of acute and chronic spinal rats to examine whether plateau potentials contribute to spasticity associated with chronic injury. The spinal cord was transected at the S2 level, causing, over time, exaggerated long-lasting reflexes (hyperreflexia) associated with a general spasticity syndrome in the tail muscles of chronic spinal rats (1-5 mo postinjury). The whole sacrocaudal spinal cord of chronic or acute spinal rats was removed and maintained in vitro in normal artificial cerebral spinal fluid (ACSF). Hyperreflexia in chronic spinal rats was verified by recording the long-lasting ventral root responses to dorsal root stimulation in vitro. The intrinsic properties of sacrocaudal motoneurons were studied using intracellular injections of slow triangular current ramps or graded current pulses. In chronic spinal rats, the current injection triggered sustained firing and an associated sustained depolarization (plateau potential; 34/35 cells; mean, 5.5 mV; duration >5 s; normal ACSF). The threshold for plateau initiation was low and usually corresponded to an acceleration in the membrane potential just before recruitment. After recruitment and plateau activation, the firing rate changed linearly with current during the slow ramps [63% of cells had a linear frequency-current (F-I) relation] despite the presence of the plateau. The persistent inward current (I(PIC)) producing the plateau and sustained firing was estimated to be on average 0.8 nA as determined by the reduction in injected current needed to stop the sustained firing [DeltaI = -0.8 +/- 0.6 (SD) nA], compared with the current needed to start firing (I = 1.7 +/- 1.5 nA; 47% reduction). In motoneurons of acute spinal rats, plateaus were rarely seen (3/22), although they could be made to occur with bath application of serotonin. In motoneurons of chronic spinal rats there were no significant changes in the mean passive input resistance, rheobase or amplitude of the spike afterhyperpolarization (AHP) as compared with acute spinal rats. However, there were significant increases in AHP duration and initial firing rate at recruitment and decreases in minimum firing rate and F-I slope. We suggest that the higher initial firing rate resulted from the plateau activation at recruitment and the lower F-I slope resulted from an increase in active conductance during firing, due to I(PIC). Brief dorsal root stimulation also triggered a plateau and sustained discharge (long-lasting reflexes; 2-5 s) in motoneurons of chronic (but not acute) spinal rats. When the plateau was eliminated by a hyperpolarizing current bias, the reflex response was significantly shortened (to 1 s). Thus plateaus contributed substantially to the long-lasting reflexes in vitro and therefore should contribute significantly to the corresponding exaggerated reflexes and spasticity in awake chronic spinal rats.

Action Potentials↗

PubMed2001

Evidence for plateau potentials in tail motoneurons of awake chronic spinal rats with spasticity.

Motor units of segmental tail muscles were recorded in awake rats following acute (1-2 days) and chronic (>30 days) sacral spinal cord transection to determine whether plateau potentials contributed to sustained motor-unit discharges after injury. This study was motivated by a companion in vitro study that indicated that after chronic spinal cord injury, the tail motoneurons of the sacrocaudal spinal cord exhibit persistent inward currents (I(PIC)) that cause intrinsically sustained depolarizations (plateau potentials) and firing (self-sustained firing). Importantly, in this companion study, the plateaus were fully activated at recruitment and subsequently helped sustain the firing without causing abrupt nonlinearities in firing. That is, after recruitment and plateau activation, the firing rate was modulated relatively linearly with injected current and therefore provided a good approximation of the input to the motoneuron despite the plateau. Thus in the present study, pairs of motor units were recorded simultaneously from the same muscle, and the firing rate (F) of the lowest-threshold unit (control unit) was used as an estimate of the synaptic input to both units. We then examined whether firing of the higher-threshold unit (test unit) was intrinsically maintained by a plateau, by determining whether more synaptic input was required to recruit the test unit than to maintain its firing. The difference in the estimated synaptic input at recruitment and de-recruitment of the test unit (i.e., change in control unit rate, DeltaF) was taken as an estimate of the plateau current (I(PIC)) that intrinsically sustained the firing. Slowly graded manual skin stimulation was used to recruit and then de-recruit the units. The test unit was recruited when the control unit rate was on average 17.8 and 18.9 Hz in acute and chronic spinal rats, respectively. In chronic spinal rats, the test unit was de-recruited when the control unit rate (re: estimated synaptic input) was significantly reduced, compared with at recruitment (DeltaF = -5.5 Hz), and thus a plateau participated in maintaining the firing. In the lowest-threshold motor units, even a brief stimulation triggered very long-lasting firing (seconds to hours; self-sustained firing). Higher-threshold units required continuous stimulation (or a spontaneous spasm) to cause firing, but again more synaptic input was needed to recruit the unit than to maintain its firing (i.e., plateau present). In contrast, in acute spinal rats, the stimulation did not usually trigger sustained motor-unit firing that could be attributed to plateaus because DeltaF was not significantly different from zero. These results indicate that plateaus play an important role in sustaining motor-unit firing in awake chronic spinal rats and thus contribute to the hyperreflexia and hypertonus associated with chronic injury.

Action Potentials↗

PubMed2000

Gi/Go couple met-enkephalin to inhibition of cholinergic and beta-adrenergic stimulation of lacrimal secretion.

PURPOSE: To determine whether G-protein-mediated inhibition of secretion by met-enkephalin involves cyclic adenosine monophosphate (cAMP)-dependent events and to identify the G proteins that couple met-enkephalin to inhibition of lacrimal secretion. METHODS: Secretion of protein was measured in 3-day primary cultures of rabbit lacrimal acini exposed to vehicle, the cholinergic agonist carbachol (Cch), the beta-adrenergic agonist isoproterenol (Isop), vasoactive intestinal peptide (VIP), or forskolin (FSK) with or without the enkephalin analog D-ala2-met-enkephalinamide (DALA). In separate experiments, cells were pretreated with pertussis toxin or polyclonal antibodies against the alpha subunits of Gi/Go to determine the physiologic role of G proteins in met-enkephalin inhibition of the release of lacrimal protein. Adenylyl cyclase (AC) activity was measured by a cAMP-dependent protein kinase binding assay in lacrimal membranes in response to the same agonists used in the secretion studies. RESULTS: Cch resulted in a significant increase in protein release from cultured lacrimal acini. Increased secretion also occurred with Isop, VIP, and FSK. Cch- and Isop-stimulated secretion was inhibited by DALA to near-basal values. However, DALA did not inhibit VIP- or FSK-stimulated secretion. The inhibitory effect of DALA on Cch and Isop stimulation of secretion was reversed by pertussis toxin. Inhibition of Cch-stimulated secretion was blocked by antibody specific to a common peptide sequence of Gialpha1 and Gialpha2 but was not blocked by anti-Gialpha1 antibody. The inhibitory effect on Cch-stimulated secretion was also blocked by anti-Gialpha3 and anti-Goalpha. Similar experiments resulted in a reversal of DALA inhibition of beta-adrenergic stimulation of secretion by immunoneutralization of Gialpha1/2 and Goalpha but not by immunoneutralization of Gialpha1 or Gialpha3. VIP, Isop, and FSK significantly stimulated AC. However, Cch had no effect on the activity of the enzyme. In addition, DALA had no effect on AC activity under any conditions. CONCLUSIONS: These results show that enkephalin inhibition of cholinergic and beta-adrenergic stimulation of secretion is mediated by Gi2, Gi3, and Go. The effector coupled by the G proteins is not AC. However, we suggest a role for met-enkephalin in G-protein-coupled modulation of ion channels important for cholinergic and beta-adrenergic stimulation of lacrimal secretion.

Adenylyl Cyclases↗

PubMed2000

Activity of hindlimb motor units during locomotion in the conscious rat.

This paper compares the activity of hindlimb motor units from muscles mainly composed of fast-twitch muscle fibers (medial and lateral gastrocnemius: MG/LG, tibialis anterior: TA) to motor units from a muscle mainly composed of slow-twitch muscle fibers (soleus: SOL) during unrestrained walking in the conscious rat. Several differences in the activation profiles of motor units from these two groups of muscles were observed. For example, motor units from fast muscles (e.g., MG/LG and TA) fired at very high mean frequencies of discharge, ranging from 60 to 100 Hz, and almost always were recruited with initial doublets or triplets, i.e., initial frequencies >/=100 Hz. In contrast, the majority of SOL units fired at much lower mean rates of discharge, approximately 30 Hz, and had initial frequencies of only 30-60 Hz (i.e., there were no initial doublets/triplets >/=100 Hz). Thus the presence of initial doublet or triplets was dependent on the intrinsic properties of the motor unit, i.e., faster units were recruited with a doublet/triplet more often than slower units. Moreover, in contrast to units from the slow SOL muscle, the activity of single motor units from the fast MG/LG muscle, especially units recruited midway or near the end of a locomotor burst, was unrelated to the activity of the remainder of the motoneuron pool, as measured by the corresponding gross-electromyographic (EMG) signal. This dissociation of activity was suggested to arise from a compartmentalized recruitment of the MG/LG motoneuron pool by the rhythm-generating networks of the spinal cord. In contrast, when comparing the rate modulation of simultaneously recorded motor units within a single LG muscle compartment, the frequency profiles of unit pairs were modulated in a parallel fashion. This suggested that the parent motoneurons were responsive to changes in synaptic inputs during unrestrained walking, unlike the poor rate modulation that occurs during locomotion induced from brain stem stimulation. In summary, data from this study provide evidence that the firing behavior of motor units during unrestrained walking is influenced by both the intrinsic properties of the parent motoneuron and by synaptic inputs from the locomotor networks of the spinal cord. In addition, it also provides the first extensive description of motor-unit activity from different muscles during unrestrained walking in the conscious rat.

Action Potentials↗

PubMed1999

Alpha(2) adrenoceptor agonists as potential analgesic agents. 1. (Imidazolylmethyl)oxazoles and -thiazoles.

A series of (imidazolylmethyl)oxazoles and -thiazoles were prepared and evaluated as alpha(2) adrenoceptor agonists. These compounds were also tested in in vivo paradigms that are predictive of analgesic activity. Variations in both the imidazole and thiazole portions of the molecule were investigated. Some of the more potent compounds such as 22, 26, 45, and 53 displayed alpha(2) receptor binding in the 10-20 nM range and also had significant antinociceptive activity in the mouse abdominal irritant test (MAIT).

Adrenergic alpha-2 Receptor Antagonists↗

PubMed1999

Spasticity in rats with sacral spinal cord injury.

We have investigated sacral spinal cord lesions in rats with the goal of developing a rat model of muscular spasticity that is minimally disruptive, not interfering with bladder, bowel, or hindlimb locomotor function. Spinal transections were made at the S2 sacral level and, thus, only affected the tail musculature. After spinal transection, the muscles of the tail were inactive for 2 weeks. Following this initial period, hypertonia, hyperreflexia, and clonus developed in the tail, and grew more pronounced with time. These changes were assessed in the awake rat, since the tail is readily accessible and easy to manipulate. Muscle stretch or cutaneous stimulation of the tail produced muscle spasms and marked increases in muscle tone, as measured with force and electromyographic recordings. When the tail was unconstrained, spontaneous or reflex induced flexor and extensor spasms coiled the tail. Movement during the spasms often triggered clonus in the end of the tail. The tail hair and skin were extremely hyperreflexive to light touch, withdrawing quickly at contact, and at times clonus could be entrained by repeated contact of the tail on a surface. Segmental tail muscle reflexes, e.g., Hoffman reflexes (H-reflexes), were measured before and after spinalization, and increased significantly 2 weeks after transection. These results suggest that sacral spinal rats develop symptoms of spasticity in tail muscles with similar characteristics to those seen in limb muscles of humans with spinal cord injury, and thus provide a convenient preparation for studying this condition.

Animals↗

PubMed1999

Activation patterns of hindlimb motor units in the awake rat and their relation to motoneuron intrinsic properties.

The activity of hindlimb motor units from the lateral gastrocnemius and tibialis anterior muscles in the awake rat was compared during locomotion and during slow, sinusoidal muscle stretch. The majority of units were activated with high initial frequencies and often commenced firing with an initial doublet or triplet, even when activated by slow muscle stretch. The high firing rates at recruitment occurred without jumps in the firing rates of other concurrently activated units, the firing rate profiles of which were used as a measure of the net synaptic drive onto the motoneuronal pool. This suggested that the sharp recruitment jumps were not due to an abrupt increase in synaptic drive but rather due to intrinsic properties of the motoneuron. In addition, motor units that were activated phasically by the muscle stretch fired more action potentials during muscle shortening than during muscle lengthening, resulting in rightwardly skewed, asymmetrical firing profiles. In contrast, when the same units fired tonically during the imposed muscle stretch, the frequency profiles were modulated symmetrically and no nonlinearities were observed. Tonically firing units were modulated symmetrically throughout a wide range of firing frequencies, and discrete jumps in rate (i.e., bistable firing) were not observed. The sharp recruitment jumps during locomotion and muscle stretch are proposed to have resulted from the additional depolarization produced by the activation of plateau potentials at recruitment. Likewise, the sustained activation of plateaus subsequent to recruitment may have produced the prolonged firing of the motor units during sinusoidal muscle stretch.

Animals↗

PubMed1998

Decerebration by global ischemic stroke in rats.

In this study we present a fast and simple technique to decerebrate rats. By injecting polyvinylsiloxane (PVS) into both common carotid arteries we occluded the circle of Willis and all cerebral arteries, thereby completely interrupting the blood supply to the cerebrum. High viscosity PVS was used so that it only entered the large arteries, and did not pass into the capillary beds. This procedure reliably resulted in an anemic decerebration, without interfering with the blood supply to the brainstem. Long-term survival was achieved by reducing intracranial pressure by the application of steroids and/or opening the skull.

Animals↗

PubMed1998

Identification, localization, and modulation of neural networks for walking in the mudpuppy (Necturus maculatus) spinal cord.

We tested the hypothesis that the neural networks for walking in the mudpuppy can be divided into a flexor and an extensor center, each of which contains collections of interneurons localized in the vicinity of their motoneuron pools. Combining a battery of techniques, we identified and localized the elbow flexor center and its motoneuron pool in the C2 segment and the elbow extensor center and its motoneuron pool in the C3 segment. Rhythmic flexion or extension of the limb in isolation could be induced by continuous trains of current pulses of the C2 or C3 segments, respectively. Independent activation could also occur after application of glutamate receptor agonist NMDA. Part of segment C2 in isolation generated rhythmic elbow flexor bursts, whereas part of segment C3 in isolation generated rhythmic elbow extensor bursts. An isolated region spanning the C3 roots generated both flexor and extensor bursts. The step cycle was modulated in a phase-dependent manner by stimulation of the dorsal roots, the ventral roots, or either of the two centers. The effects of ventral root stimulation were removed by deafferentation to block reafferent input attributable to muscle contraction induced by the stimulation. We conclude that the neural networks for walking contain at least a flexor and an extensor generator that are localized in close apposition to the motoneuron pools, that the two centers can work independently despite the fact that there are reciprocal inhibitory interconnections between them, and that sensory input interacts with the spinal neural networks to reset the ongoing walking rhythm in a phase-dependent manner.

Animals↗