Quantitative analyses of neurons projecting to primary motor cortex zones controlling limb movements in the rat.

The objective was to determine if projections of single neurons to primary motor cortex preferentially terminate in several efferent zones that could form synergies for the execution of limb movements. Intracortical microstimulation was used to identify zones evoking hip flexion (HF), elbow flexion (EF), and both plantarflexion (PF) and dorsiflexion (DF) about the ankle. Histological examination showed that the zones from which some movements were evoked extended beyond the agranular cortex into granular cortex. Fluorogold, Fast blue, and propridium iodide or rhodamine-labeled dextran were injected into three of these four efferent zones in each rat. There was a virtual absence of multiple-labeled cells despite having an intermingling of different-colored cells of which 15% in frontal cortex were less than 1.2 mm away from a neighboring neuron that projected to a different efferent zone. This suggests that single neurons projecting to the motor cortex do not hard-wire specific synergies but rather project to single efferent zones in order to offer the greatest degree of freedom for the generation of movements. The distribution of ventral posterolateral and ventrolateral thalamic nucleus labeling depended on whether the injections were in granular or agranular cortex. Conversely, frontal cortex projections to motor efferent zones were made irrespective of their location in either granular or agranular cortex and thereby supporting their presumed role in the control of movements. Hindlimb motor cortex injections yielded retrograde labeling that extended into the more localised distribution of frontal cortex neurons retrogradely labeled from forelimb injections. This may allow hindlimb movements to be synchronized by forelimb movements during walking on challenging terrain.

Response patterns and postspike effects of premotor neurons in cervical spinal cord of behaving monkeys.

Most of our information about spinal neurons has been derived from experiments with anesthetized or surgically. reduced preparations. To investigate these neurons under normal behavioral conditions, we recorded the activity of single afferent units in cervical dorsal root ganglia and of single interneurons in the cervical spinal cord of macaque monkeys, as they generated alternating flexion and extension torques about the wrist. Spike-triggered averages of rectified electromyographic activity were used to identify "premotor" (PreM) units associated with correlated postspike effects in active muscles. In addition to postspike effects, some spike-triggered averages showed early increases in average muscle activity, which were attributed to synchronous discharges in other PreM units. In recordings of peripheral afferents, 49% of the task-related dorsal root ganglia units produced postspike facilitation (PSF) of at least one forearm muscle, with a mean PSF latency of 5.8 +/- 0.3 ms (SE). The PSF amplitude was measured as the mean percent increase (MPI): the average increase of the PSF as a percentage of the prespike baseline mean. PreM afferent units produced PSF with an average MPI of 4.6 +/- 0.3%. In a study of cervical interneurons, about 13% (72/562) of the task-related cells showed postspike effects. These PreM interneurons had a mean PSF latency of 7.2 +/- 0.3 ms and a mean MPI of 4.6 +/- 0.2%. The MPI values for spinal neurons were similar to the MPIs reported for rubromotoneuronal and corticomotoneuronal cells. PreM neurons usually facilitated a subset of the coactivated muscles called the unit's "muscle field." The PreM afferents facilitated an average of 46% of the synergistically coactivated muscles, while PreM interneurons facilitated an average of 37%. These are comparable with the percentage of muscles facilitated by corticomotoneuronal (40%) and rubromotoneuronal (50%) cells. During the step-tracking task the monkeys generated ramp-and-hold torques about the wrist. The PreM afferents typically became active during either flexion or extension of the wrist, although a few were bidirectionally active. The most common response pattern in PreM afferents was a tonic discharge, followed by phasic and phasic-tonic discharge. The most common patterns exhibited by PreM interneurons were tonic and phasic-tonic responses. PreM afferent units began to discharge on average 51 +/- 13 ms before activation of their target muscle. This early onset supports our hypothesis that these PreM afferents arose from muscle spindles, which is also consistent with their short-latency PSF and their responses to perturbations that stretched their target muscles. The results reveal some salient differences between the discharge properties of dorsal root ganglia neurons, spinal interneurons, and supraspinal PreM cells in the motor cortex and red nucleus. All four PreM populations include tonic, phasic-tonic, and phasic cells, but in significantly different proportions. Most PreM afferents resembled corticomotoneuronal cells in being active only with their target muscles, unlike rubromotoneuronal cells and spinal PreM interneurons, which tended to exhibit more bidirectional discharges.

Early enhancement but no late changes of motor responses induced by intracortical microstimulation in the ketamine-anesthetized rat.

The objectives of this study were to determine whether changes in electromyographic (EMG) responses observed during prolonged intracortical microstimulation (ICMS) were due to local plasticity of the motor system or to global changes in the preparation. Local effects would be expressed as changes only along the activated motor pathway, whereas global effects would be expressed as changes also appearing at distant cortical efferent microzones. The results of ICMS in the ketamine-anesthetized rat showed that the size of consecutive EMG responses increased gradually to a relatively stable magnitude over a period of four to six trains of stimuli. This early enhancement of EMG responses was maintained while continuously providing trains of stimuli at 1 Hz. However, it disappeared after a 5-min period of muscle inactivity. This response enhancement in the presence of ketamine (an NMDA, N-methyl-D-aspartate, receptor blocker) suggests that a neuronal mechanism involving non-NMDA-mediated homosynaptic short-term potentiation (STP) was responsible for the early enhancement of EMG responses. To compare ICMS effects at several time intervals it was necessary to average several evoked EMG responses because there was normal biological variability between single EMG responses. To determine the optimal number of EMG responses that would provide a reliable average EMG response, averages of 5, 10, 15, 20, and 25 EMG responses evoked from a single cortical site were collected at 5-min intervals. The results revealed that averages of 10 responses would provide reliable average EMG responses for all subsequent analyses. There were wide fluctuations in the average EMG responses when periodic injections of ketamine were used to maintain a low reflexive state in the animal. Switching to continuous infusion of ketamine abolished these fluctuations but there remained a small drift in the magnitudes of consecutive EMG responses. To test whether this drift reflected local plastic changes in the motor system induced by stimulation or some global changes, EMG responses evoked from another ICMS site were used as control. The rationale was that global effects would affect all motor output sites equally. The sizes of control EMG responses followed a similar time course to those evoked from the test site. Furthermore, standardizing the test EMG responses with respect to the control responses eliminated the drift in response magnitudes. Thus the drift was due to slow global changes in neuronal excitability possibly produced by the anesthesia. In conclusion, late changes occurring after hours of ICMS were not due to plasticity of the motor system but rather to global changes in the preparation, possibly resulting from the inability to set an ideal anesthetic infusion rate that could maintain a constant level of neuronal excitability over long periods of time. However, there was early enhancement of the EMG responses evoked by ICMS due to neuronal plasticity possibly mediated by a non-NMDA mechanism of homosynaptic STP such as post-tetanic potentiation (PTP). This early enhancement would favor recruitment of the previously activated motor pathway and lead to greater consistency in movement execution.

Producing columnar depositions of neuroanatomical tracer in rat cortex.

A new technique was developed to fill columnar regions of cortex with retrograde neuroanatomical tracer. This technique involved (1) standard iontophoresis to eject tracer from a micropipette in concert with (2) displacement of this micropipette radially through the cortex using a computer controlled microdrive. The displacement of the micropipette left an evenly dense deposit of tracer throughout the cortical thickness forming a column of neuroanatomical tracer. The diameter of the columnar injection site was reliably dependent on the iontophoretic current strength, the micropipette displacement parameters, and the nature of the tracer used. This simple technique provided very reproducible results. Several issues related to the interconnections of functionally identified columns of sensory and motor cortex could be addressed with this injection technique.

Field potential mapping of neurons in the lumbar spinal cord activated following stimulation of the mesencephalic locomotor region.

The spinal neurons involved in the control of locomotion in mammals have not been identified, and a major step that is necessary for this purpose is to determine where these cells are likely to be located. The principal objective of this study was to localize lumbar spinal interneurons activated by stimulation of the mesencephalic locomotor region (MLR) of the cat. For this purpose, extracellular recordings of MLR-evoked cord dorsum and intraspinal field potentials were obtained from the lumbosacral enlargement during fictive locomotion in the precollicular-postmammillary decerebrate cat preparation. Potentials recorded from the dorsal surface of the cord between the third lumbar (L3) and first sacral (S1) segments typically showed four short-latency positive waves (P1-P4). These P-waves were largest between the L4-L6 segments. The amplitude of the P2-4 waves increased with the appearance of locomotion and displayed rhythmic modulation during the locomotor step cycle. Microelectrode recordings from the L4-L7 spinal segments during fictive locomotion revealed the presence of both positive and negative short-latency MLR-evoked intraspinal field potentials, and were used to construct isopotential maps of the evoked potentials. Positive field potentials were observed throughout the dorsal horn of the L4-L7 spinal segments with the largest amplitude potentials occurring in laminae III-VI. Negative field potentials were found in laminae VI-X of the lumbar cord. The shortest latency negative field potentials were observed in lamina VII and at the border between laminae VI and VII and were considered to be evoked monosynaptically from the arrival of the descending volley. Short-latency mono- and disynaptic negative field potentials were also observed in lamina VIII. Longer latency, tri- and polysynaptic field potentials were observed in laminae VII and VIII. Many of the longer latency negative waves observed in laminae VII and VIII followed shorter latency negative potentials recorded from the same location. Laminae VII and VIII negative field potentials were largest in the L5-6 and L4-5 spinal segments, respectively. Negative field potentials were also evoked in the motor nuclei of the L4-7 spinal segments. The segmental latencies for these potentials indicate that they were evoked di- and trisynaptically.(ABSTRACT TRUNCATED AT 400 WORDS)

Use of spike triggered averaging of muscle activity to quantify inputs to motoneuron pools.

1. The goal of this study was to determine the extent to which postspike facilitation (PSpF) of electromyograms (EMGs) could be used to estimate the inputs to separate motoneuron pools, under conditions where there was wide variability in the parameters of muscle activity. These parameters included cancellation of motor unit action potentials (MUAPs), variations in EMG noise, and changes in MUAP amplitude and duration. A systematic series of computer simulations with increasing complexity were used to achieve this goal. The initial simulations (model I) included a premotoneuronal (PreM) cell connected to a single postsynaptic motoneuron (Mn), which in turn projected to a muscle. The next simulations (model II) included other target motoneurons with their efferents each projecting to separate muscles. The last simulations (model III) included more than one postsynaptic motoneuron per Mn-pool, as is the case in mammalian neuromuscular systems. 2. A sample simulation (model I) was performed to determine if the PreM-evoked effects were within physiologically observed values. A cross-correlogram (XC) calculated from a PreM cell and its target Mn, receiving a PreM-evoked excitatory postsynaptic potential (EPSP) of 0.5 mV, produced a XC peak area of 0.04 Mn-spikes/PreM-trigger. The PSpF of EMG activity evoked by this PreM cell had a mean percent increase of 4.6% (MPI = mean bin amplitude of PSpF above baseline/mean baseline level x 100). These XC and PSpF values were within the range of values previously obtained from animal experiments. 3. The magnitude of MUAP cancellation in the EMG was tested by calculating two spike-triggered averages (SpTAs) of EMGs from Mn-triggers (not PreM-triggers as in the other SpTAs): one using typical bipolar MUAPs and another using their rectified counterpart of only positive polarity to eliminate the possibility of MUAP cancellation. The PSpF calculated from bipolar spikes was 24.8% smaller than the one calculated using unipolar spikes. This cancellation could be greater or smaller depending on the state of parameters, such as the shape and number of MUAPs, that determine the probability of overlap between MUAP components of opposite polarity. All subsequent computer simulations used typical bipolar MUAPs. 4. A series of increasing motoneuron EPSP amplitudes were used to determine the relationship between PreM-Mn connection strength and PSpF area. A nearly perfect linear relationship between EPSP amplitude and PSpF area was obtained for SpTAs of rectified EMGs (r = 0.99). An equally linear relationship was obtained when averaging nonrectified EMGs (r = 0.99), but the smaller EPSPs or weaker synaptic connections were not detected.(ABSTRACT TRUNCATED AT 400 WORDS)

Comparison of cerebellar and motor cortex activity during reaching: directional tuning and response variability.

1. The responses of 262 motor cortex cells and 223 cerebellar neurons were recorded during whole-arm reaching movements toward targets lights in eight evenly distributed directions radiating from a common central starting position. The reaching movements were followed by a 2-s target hold period where a fixed arm posture was actively maintained to stabilize the hand over the target light. 2. Cerebellar neurons had a higher mean tonic discharge rate while holding over the starting position (22.9 imp/s) than did motor cortex cells (12.5 imp/s). The mean population response curve describing the changes in activities with movement direction was likewise shifted toward higher frequencies in the cerebellum compared with the motor cortex, but the amplitude of the two curves was about equal. Therefore, the baseline discharges of cerebellar neurons were higher, but their changes in activity during movement were similar to those of motor cortical cells. 3. Motor cortex neurons were more strongly related to active maintenance of different arm postures than were cerebellar units. This was shown by a larger posture-related population response curve in the motor cortex (half-wave amplitude of cosine function was 11.2 imp/s, compared with 7.0 imp/s for cerebellar neurons), which represented the average response curve calculated from all the cells of the population. Furthermore, the motor cortex population had a higher percentage of single cells with tonic responses while the hand was held over different targets (tonic and phasic-tonic cells composed 57% of motor cortex population, compared with 38% of cerebellar population). Proportionately more cerebellar cells were phasically related to the movements. 4. The majority of motor cortex cells (58%) showed reciprocal changes relative to the center-hold time activity where the activity increased for movements in the preferred direction and decreased for movements in the opposite direction. Most of the remaining cells (40%) showed graded changes where the activity increased gradually as reaching was directed closer to the preferred direction. In contrast, the most common cerebellar response pattern was graded (38%). Only 26% were reciprocal and 18% were non-directional. The remaining 2% of motor cortical cells and 18% of cerebellar neurons could not be readily assigned to any of these three response classes. 5. Sector widths were calculated to measure the dispersion of individual cerebellar and motor cortical cell activities about the eight movement directions. Sector widths calculated from the absolute activities were always broader for cerebellar neurons (i.e., the cells were more broadly tuned).(ABSTRACT TRUNCATED AT 400 WORDS)

Response patterns and postspike effects of peripheral afferents in dorsal root ganglia of behaving monkeys.

1. The activity of single afferent units was recorded in cervical dorsal root ganglia (DRG) in two macaque monkeys as they generated alternating flexion and extension torques about the wrist during a step-tracking task. During these isometric and auxotonic muscle contractions, electromyographic (EMG) activity was recorded with electrode pairs in up to 12 independent forearm muscles. Spike-triggered averages (STAs) of rectified EMG activity were used to identify afferents that were associated with correlated facilitation of active muscles. 2. Our aim was to find peripheral afferents producing postspike effects in muscles and to compare their properties with those of corticomotoneuronal (CM) and rubromotoneuronal (RM) cells previously obtained under identical behavioral conditions. We documented the timing, magnitude and distribution of their postspike facilitation (PSF) of forearm muscles and investigated the response properties of task-related units. 3. Of 125 afferent units tested with STAs, 68 showed PSF of EMG activity in at least one muscle. Fifty-nine DRG units provided sufficiently long recordings to generate averages with greater than or equal to 2,000 triggers, the minimum number considered to demonstrate reliable effects. Of these 59 units, 29 (49%) were associated with facilitation of forearm muscle activity. 4. Many STAs showed a gradual increase in EMG activity starting before or near the afferent trigger spike; often superimposed on this broad facilitation was a sharply rising PSF starting at a longer latency. The earliest poststimulus facilitation evoked by single microstimuli delivered in DRG occurred in stimulus-triggered averages at a latency of 3.5 ms. In STAs the broad facilitation beginning at latencies shorter than the responses to electrical stimulation was attributed to synchronous discharges in other afferent units. The sharper postspike EMG increases occurring with latencies of greater than or equal to 3.5 ms were identified as PSF produced by the afferent. The PSF parameters documented in this study were measured after subtracting the effects of synchrony facilitation. 5. PSF of EMG activity began at a mean latency of 5.8 +/- 0.3 (SE) ms and peaked at a mean latency of 7.5 +/- 0.3 (SE) ms. In previous studies, the PSFs from CM and RM cells had mean onset latencies of 6.3 and 5.6 ms, respectively, and mean peak latencies of 10.2 and 9.1 ms. 6. A measure of the PSF amplitude is the mean percent increase (MPI), defined as the increase of the PSF above its base measured as a percentage of the prespike baseline mean.(ABSTRACT TRUNCATED AT 400 WORDS)

Cerebellar neuronal activity related to whole-arm reaching movements in the monkey.

1. Three monkeys were trained to make whole-arm reaching movements from a common central starting position toward eight radially arranged targets disposed at 45 degrees intervals. A sample of 312 cerebellar neurons with proximal-arm receptive fields or discharge related to shoulder or elbow movements was studied in the task. The sample included 69 Purkinje cells, 115 unidentified cortical cells, 65 interpositus neurons, and 63 dentate units. 2. The reaching task was divided into three movement-related epochs: a reaction time, a movement time, and holding over the target. All neurons demonstrated significant changes in discharge during one or more of these three epochs. Almost all of the cells (95%) showed a significant change in activity during the movement, whereas 68-69% of the cells showed significant changes from premovement activity during the reaction time and holding periods. 3. During the combined reaction time-movement period, 231/312 cells were strongly active in the task. Of these, 151 cells (65.4%) demonstrated unimodal directional responses. Sixty-three had a reciprocal relation to movement direction, whereas 88 showed only graded increases or decreases in activity. A further 37 cells (16.0%) were nondirectional, with statistically uniform changes in discharge in all eight directions. The remaining 43 cells (18.6%) showed significant differences in activity for different directions of movement, but their response patterns were not readily classifiable. 4. The proportion of directional versus nondirectional cells was consistent across the four cell populations. However, graded response patterns were more common and reciprocal responses less common among Purkinje and dentate neurons than among unidentified cortical cells and interpositus neurons. 5. The distribution of preferred directions of the population of cerebellar neurons covered all possible movement directions away from the common central starting position in the horizontal plane. When the preferred direction of each cell in the sample population was aligned, the mean direction-related activity of the cerebellar population formed a bell-shaped tuning curve for the activity recorded during both the reaction time and the movement, as well as during the time the arm maintained a fixed posture over the targets. A vector representation also showed that the overall activity of the cerebellar population during normal reaching arm movements generated a signal that varied with movement direction. 6. These results demonstrate that the cerebellum generates a signal that varies with the direction of movement of the proximal arm during normal aimed reaching movements and is consistent with a role in the control of the activity of muscles or muscle groups generating these movements.

Locomotor deficits in the mutant mouse, Lurcher.

The effect of total Purkinje cell degeneration on treadmill locomotion was studied in the cerebellar mutant mouse Lurcher. Other movements such as swimming and scratching were also studied in order to evaluate the cerebellar control of rhythmic actions. Cinematographic and electromyographic recordings were taken from normal and Lurcher mice that were subsequently perfused to obtain a Purkinje cell count. Walking deteriorated progressively and was clearly abnormal in 30 day old Lurchers with 90% Purkinje cell degeneration. In adult Lurcher mice in which Purkinje cells were totally absent, walking was characterized by short steps with exaggerated hindlimb flexion in the swing phase. Also, both the interlimb step ratio, defined as the step length of the reference limb divided by the step length of the opposite limb, and the interlimb coupling, defined as the temporal relation of one footfall with respect to the footfall of another limb, varied more than in normal mice. Furthermore, the locomotion of Lurcher mice displayed increased vertical displacement of the hip and an inability to produce continuous step cycles without stumbling. Both the EMG onset relative to foot contact and the EMG burst duration were highly variable, and a greater overlap in the activities of antagonist muscles at the transition from ankle extension to flexion was evident. Although both walking and swimming involve cyclical limb movements, the disorganization of the cycle and the irregular EMG pattern seen in the Lurcher during walking were not observed during swimming. Furthermore, scratching was well executed in the Lurcher mice. However, a consistently higher tonic extensor activity at the ankle appeared during walking, swimming and scratching. These results suggest that, in contrast to swimming and scratching, the requirements of walking depend to a greater degree on a functional cerebellar cortex for successful performance.

Influence of low extracellular Ca2+ ions on the cardiac function changes induced by verapamil in the perfused rabbit heart.

We tested the hypothesis that the myocardial effects of verapamil (VER) could be enhanced by decreasing the extracellular Ca2+ concentration ([Ca2+]o) in the isolated rabbit heart at 37 degrees C. After perfusion with standard Krebs - bicarbonate solution containing 1.27 mM Ca2+, for a 30-min period of stabilization and 15 min of control, groups of hearts were perfused for an additional 60 min with solutions containing one of the following: 1.27 mM Ca2+ (control group), 0.23 mM Ca2+ (low [Ca2+]o group), 1.27 mM Ca2+ plus 10(-7) M VER (VER group), or 0.23 mM Ca2+ plus 10(-7) M VER (combination, CBN group). These concentrations of [Ca2+]o and VER produce submaximal responses in our preparation. We found that the heart rate - LV pressure product (RPP) in the CBN group fell rapidly to 0 in the first 2-3 min of perfusion, this response being significantly lower than in the other two groups for the first 15 min. Electromechanical dissociation (EMD) appeared in one of six hearts at 60 min and in four of six hearts at 30 min in the low [Ca2+]o and VER groups, respectively, whereas it occurred in the CBN group in all hearts at 3 min. Depolarization rate (DR) fell by 10% in the low [Ca2+]o and VER groups versus a reduction of 45% in the CBN group (P less than 0.05) during the last 45 min of perfusion. The PR interval increased by 300% in the CBN group, a much greater and significant change (P less than 0.05) than in the hearts exposed to VER or low [Ca2+]o.(ABSTRACT TRUNCATED AT 250 WORDS)

Low extracellular Ca2+ and enzyme release in the perfused rabbit heart.

Previous studies have shown that the well-oxygenated perfused rabbit heart releases creatine kinase when treated with the calcium antagonist drug verapamil (VER) in a dose-related manner. It is possible that this effect is related to Ca2+ ion deprivation of the sarcolemma. This possibility was explored by perfusing hearts with low Ca2+ (0.5, 0.23, 0.15, and 0 mM) versus a control group (1.27 mM Ca2+) for 60 min. Low Ca2+ perfusion was associated with reduction in the heart rate--left ventricular systolic pressure product and O2 consumption, tendency for the coronary sinus flow to increase, electromechanical dissociation, prolongation of atrioventricular conduction and QT interval, and decrease in myocardial glycogen. Lower total adenosine nucleotides were found only in the 0 mM Ca2+ group. As the Ca2+ concentration was reduced, the hearts lost increasing amounts of creatine kinase, aspartate aminotransferase, and lactate dehydrogenase. These results confirm the importance of Ca2+ ions in contractile and electrical cardiac functions and show that decreased availability of this cation leads to increasing enzyme leakage resembling that seen in VER-treated hearts.