D-2-amino-5-phosphonovalerate, and NMDA receptor antagonist, blocks induction of associative long-term potentiation of the flexion reflex in spinal cat.

The effect of the NMDA receptor antagonist D-2-amino-5-phosphonovalerate (APV) on classical conditioning of the flexion reflex in spinal cat was examined. Animals perfused intrathecally with artificial cerebral spinal fluid (CSF) containing APV exhibited flexion reflex potentiation during the conditioning period that was not significantly different from cats receiving artificial CSF alone. However, the APV group exhibited no signs of reflex potentiation during the 2.5 h retention period, in contrast to the CSF alone group. The results suggest that NMDA receptor activation plays a critical role in the induction of associative long-term potentiation of flexion reflexes in spinal cat.

Retrograde labeling of lumbosacral interneurons following injections of red and green fluorescent microspheres into hindlimb motor nuclei of the cat.

In order to map the laminar and segmental positions of lumbosacral interneurons that project to L7 motor nuclei, red and green fluorescent latex microspheres ("beads") were pressure-injected through micropipettes into the deep peroneal or posterior biceps-semitendinosus motoneuron pools of cats. Micropipette tips were positioned by recording the antidromic field potentials evoked by electrical stimulation of the muscle nerves. Projecting interneurons were identified by the presence of retrogradely transported beads. Small bead injections labeled large numbers of neurons. These were observed in all the spinal segments examined, L1-S2, and were most densely concentrated within laminae VI and VII ipsilateral to the injections and lamina VIII contralaterally. In addition, significant numbers of labeled cells were observed in lateral lamina V ipsilaterally and in lamina X. A few cells with bilateral projections were double-labeled following injections of red and green beads on opposite sides of the cord. These were most often observed in midlumbar segments (L3-L5) in medial regions of the gray matter. The results suggest that the intermediate zone (laminae V-VIII and X) of the lumbosacral spinal cord is a major source of interneuronal projections to the L7 ventral horn. This is true for both lateral and medial areas of the intermediate zone, as the fluorescent microspheres labeled neurons in medial regions of the cord largely undetected in previous studies in which other methodologies were employed.

Differential synaptic effects on physiological flexor hindlimb motoneurons from cutaneous nerve inputs in spinal cat.

1. We previously demonstrated in the spinal cat that superficial peroneal cutaneous nerve stimulation produced strong reflex contraction in tibialis anterior (TA) and semitendinosus (St) muscles but unexpectedly produced mixed effects in another physiological flexor muscle, extensor digitorum longus (EDL). The goal of the present study was to further characterize the organization of ipsilateral cutaneous reflexes by examining the postsynaptic potentials (PSPs) produced in St, TA, and EDL motoneurons by superficial peroneal and saphenous nerve stimulation in decerebrate, spinal cats. 2. In TA and St motoneurons, low-intensity cutaneous nerve stimulation that activated only large (A alpha) fibers [i.e., approximately 2-3 times threshold (T)], typically produced biphasic PSPs consisting of an initial excitatory phase and subsequent inhibitory phase (EPSP, IPSP). Increasing the stimulus intensity to activate both large (A alpha) and small (A delta) myelinated cutaneous fibers supramaximally (15-45 T) tended to enhance later excitatory components in TA and St motoneurons. 3. In EDL motoneurons, 2-3 T stimulation of the superficial peroneal nerve evoked initial inhibition (of variable magnitude) in 7/10 EDL motoneurons tested, with either excitation (n = 2) or mixed effects (n = 1) observed in the remaining EDL motoneurons. Saphenous nerve stimuli produced excitation either alone, or preceded by an inhibitory phase in EDL. Increasing the stimulus intensity enhanced later inhibitory influences from superficial peroneal and excitatory influences both from superficial peroneal and saphenous nerve inputs in EDL motoneurons. 4. Short-latency (less than 1.8 ms) EPSPs were observed in a few motoneurons in all reflex pathways examined, except for EPSPs in EDL motoneurons evoked by saphenous stimulation. IPSPs with central latencies less than 1.8 ms were also produced by both saphenous (TA, n = 1; EDL, n = 2) and superficial peroneal (EDL, n = 4) nerve stimulation. 5. The results, in comparison with other reports employing spinal and nonspinal preparations, suggest that removal of influences from higher centers reveals inhibitory circuits from the superficial peroneal and saphenous nerves to EDL motoneurons in the spinal preparation. The inhibitory inputs observed are thought to reflect the activation of "specialized" reflex pathways. Additionally, the demonstration of short-latency EPSPs and IPSPs suggest that the minimal linkage in both the excitatory and inhibitory cutaneous reflex pathways examined is disynaptic. The results are discussed in relation to previous studies on classically conditioned flexion reflex facilitation in spinal cat.

Morphological relationships among extensor digitorum longus, tibialis anterior, and semitendinosus motor nuclei of the cat: an investigation employing the retrograde transport of multiple fluorescent tracers.

To determine the morphological relationships among extensor digitorum longus (EDL), tibialis anterior (TA), and semitendinosus (St) motor nuclei in the spinal cord of the cat, these nuclei were retrogradely labeled with three different fluorescent tracers. The fluorochromes--bisbenzimide, nuclear yellow, and propidium iodide--were applied by intramuscular injection or soaking the muscle nerve. The positions of the labeled motor nuclei were bilaterally symmetrical. The EDL and TA motoneurons were located in close proximity to one another, in the lateral regions of lamina IX in spinal segments L6 and L7. Although the boundaries of each nucleus were tightly opposed, the EDL and TA motor nuclei overlapped minimally, with the somata of EDL motoneurons positioned dorsal to those of TA. The St motor nucleus was located ventromedial to that of EDL and extended from the caudal portion of L6 through S1. Supplemental studies of the reflex effects evoked in EDL, TA, and St muscles by cutaneous nerve stimulation provided physiological observations that may be related to these anatomical results.

Retention of a backward classically conditioned reflex response in spinal cat.

Retention of a backward classically conditioned reflex response was investigated in the spinal cat preparation. Facilitation of the flexion reflex was induced by the pairing of superficial peroneal nerve stimulation (30 Hz, 0.5 s), the US (unconditioned stimulus), with saphenous nerve stimulation (10 Hz, 1.5 s), the CS (conditioned stimulus). Both the US and CS were supramaximal for activation of A delta cutaneous afferent fibers. Experimental animals received 30 paired trials (US preceded CS by 0.25 s) with an intertrial interval (ITI) of three min. Control animals received the same stimuli but in an explicitly unpaired manner. Following acquisition, all animals received 30 additional CS-alone trials at five min intervals. This paradigm, which incorporated ITIs longer than those which had been used previously in backward conditioning studies, induced a long-lasting potentiation of the flexion reflex which appeared to be specific to spinal reflex pathways activated by A alpha cutaneous fibers. The relevancy of these results to a more specific understanding of backward and forward classical conditioning in the spinal cat is discussed.

Evidence that the neural pathways involved in backward conditioning are different from those involved in forward conditioning.

Effects of forward and backward conditioned-unconditioned stimulus (CS-US) intervals on classical conditioning of the flexion reflex were examined in a spinal cat preparation. A less intense conditioned stimulus (CS) was employed (activation of A-alpha cutaneous fibers) compared to that of a previous study (activation of both A-alpha and A-delta cutaneous fibers). Interstimulus intervals (ISIs) ranging from +3.0 to -3.0 sec were examined in 9 experimental groups, and results contrasted to those of an explicitly unpaired control group. The ISI of -0.25 sec produced optimal backward excitatory conditioning, paralleling the previous results. However, in contrast to the previous study, no conditioning was observed in any of the forward ISI groups. These observations support the hypothesis that backward and forward conditioning may be fundamentally different phenomena, induced by CS activation of different spinal reflex pathways: backward conditioning involves spinal reflex pathways activated by A-alpha cutaneous fibers of the CS, while forward conditioning involves spinal reflex pathways activated by A-delta cutaneous fibers of the CS.

Forward and backward classical conditioning of the flexion reflex in the spinal cat.

Effects of forward and backward conditioned-unconditioned stimulus (CS-US) intervals on classical conditioning of the flexion reflex were examined in a cat spinal preparation. Interstimulus intervals (ISIs) ranging from +3.0 to -3.0 sec were employed in 9 experimental groups and the results compared with those of an explicitly unpaired control group. Forward conditioning produced an asymmetrical, inverted U-shaped gradient relating magnitude of conditioning to ISI for both acquisition and extinction. The optimum ISI was 1.0 sec. Backward ISIs also produced excitatory conditioning, with optimal conditioning at -0.25 sec. Unlike forward conditioning, backward conditioning produced little sign of retention during extinction trials. The results, which parallel in several ways those of ISI effects in studies of intact animals, support the hypothesis that backward and forward conditioning may be fundamentally different phenomena, under the control of different neural processes.

Retention of a classically conditioned reflex response in spinal cat.

Retention of classically conditioned flexion reflex facilitation was examined in unanesthetized, decerebrate, acute spinal cats. Flexion reflex facilitation, recorded from the tibialis anterior muscle, was obtained by pairing saphenous nerve stimulation (the conditioned stimulus) with superficial peroneal nerve stimulation (the unconditioned stimulus). The flexion reflex declined in control animals receiving the same number of nerve stimuli over the same time span, but in an explicitly unpaired sequence. To investigate retention, conditioned stimuli were presented at 5-min intervals following acquisition for a 2 1/2-h period. During this time a significant difference between conditioning and control groups was maintained even to the last trial, with no indication that the difference was subsiding over time. The results support the possibility that a classical conditioning paradigm applied to the spinal cord can induce alterations in spinal reflexes of long duration. Furthermore, the results appear to rule out post-tetanic potentiation as a mechanism producing the observed long-term effects.

Conditioned stimulus intensity: role of cutaneous fiber size in classical conditioning of the flexion reflex in the spinal cat.

Three intensities of the conditioned stimulus (CS) were used to investigate the role of cutaneous fiber size on classical conditioning of the flexion reflex in the spinal cat. Conditioning occurred when the CS activated A alpha and A delta cutaneous fibers; CS activation of only the largest myelinated (A alpha) fibers or CS recruitment of unmyelinated (C) fibers in addition to A alpha and A delta fibers did not lead to significant conditioning. Comparison with two unpaired CS-US control treatment groups showed that the facilitation of reflex output during conditioning was specific to pairing of the CS and US and was not a consequence of sensitization or differential habituation rates between paired (conditioning) and unpaired (control) groups. These results were comparable to those from intact animal conditioning studies and thus lend further support to the spinal conditioning preparation as an appropriate simplified system for investigations of neural mechanisms of learning.

Features of laminar and somatotopic organization of lumbar spinal cord units receiving cutaneous inputs from hindlimb receptive fields.

Single-unit recordings from 312 units of lamina I-VII of the lumbar spinal cord of unanesthetized, decerebrate, T8 spinal cats were used to determine the somatotopic and laminar organization of spinal neurons responding to cutaneous stimulation of the hindlimb. Properties of cells confined to different Rexed laminae (I-VII) were shown to differ in several respects, including responses to variations in stimulus intensity, receptive-field areas, spontaneous frequencies, and central delays. Spinal cord neurons with similarly localized cutaneous receptive fields were found to be organized in sagittally oriented rectangular columns. These columns were 7 to at least 20 mm long (rostral-caudal axis), 0.5-1.0 mm wide, and could encompass laminae I-VII in depth. Touch, pressure, and pinch were effective excitatory inputs into each column subserving a given receptive-field location. A map of the somatotopic organization of units in the horizontal plane is presented, which in general confirms previous reports and in particular deals with the organization of units with receptive fields on the plantar cushion and individual toes.

Classical conditioning of the flexion reflex in spinal cat: features of the reflex circuitry.

Classically conditioned facilitation of the flexor withdrawal reflex of spinal cat occurs in knee and ankle flexor muscles but not in a flexor muscle of the toes. Furthermore, the spinal circuitry activated by a component of the conditioned stimulus (A alpha cutaneous fibers) is not by itself involved in the reflex conditioning. The results suggest that increases in both cutaneous afferent output and motoneuron excitability may be eliminated as mechanisms contributing to conditioning and point to certain interneuronal pools as the locus of learning in this preparation.

Classically conditioned alterations in single motor unit activity in the spinal cat.

Muscle tension and single motor unit EMG recordings from a flexor muscle of acute spinal cats were obtained during presentation of classical conditioning and control paradigms. Conditioned increases in muscle tension were similar to previously obtained results. Motor unit recordings suggested that this conditioned reflex facilitation is brought about by an increased probability firing of motoneurons initially responsive to the conditioned stimulus, as well as orderly, size-dependent recruitment of initially non-responsive motoneurons. These results indicate that the same physiological mechanisms for grading motor output may apply for conditioned responses as have been demonstrated for reflexive and voluntary movements.

Dimensions of medical students' perceptions of instruction.

Factor analysis of medical student ratings of basic science instruction yielded three dimensions of student perception of instruction: faculty-student rapport, outside work, and aspects of student comprehension. While these factors are similar to those identified in other studies, they differ in respects probably reflecting differences between medical students and other students and differences in questionnaire design.