Motor function of the cerebellorubrospinal system.
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Biomedical subjects
J Keifer
Publications and source records attributed to J Keifer.
Intrinsic and synaptic properties of turtle red nucleus neurons in vitro.
Burst discharges in the red nucleus are correlated with discrete limb movements. Intracellular recordings from red nucleus neurons in the in vitro turtle brainstem-cerebellum was performed to elucidate mechanisms underlying these bursts. Depolarizing intracellular current injection failed to demonstrate endogenous membrane currents that might produce burst discharges, and neurons did not exhibit significant spike frequency adaptation, which is a characteristic of synaptically driven bursts. Responses of red nucleus neurons to synaptic input demonstrated a late, slow depolarizing synaptic potential (slow EPSP) having a latency of 9-12 ms, and a maximal duration of 600 ms. it is concluded that neither intrinsic membrane responses, nor the duration of the slow EPSP, can fully account for the behavior of red nucleus neurons during burst discharge. We hypothesize that activity in the red nucleus is driven by a gradual recruitment of NMDA receptors, and lpr by polysynaptic excitatory pathways.
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Anatomy of the turtle cerebellorubral circuit studied in vitro using neurobiotin and biocytin.
We have combined the rapid anterograde and retrograde transport of neurobiotin and biocytin with the extended viability of the isolated turtle brainstem-cerebellum to conduct in vitro studies of the chelonian cerebellorubral circuit. Tracers were pressure injected in 15-25 nl quantities and the optimal transport time was 16 h. Tissue sections were incubated with avidin-biotin-HRP complex and reacted with DAB. Retrogradely labeled soma, dendrites and axons, and anterogradely labeled axons and to a lesser extent terminals were visible with both tracers. Red nucleus injections resulted in dense retrograde label in the contralateral lateral cerebellar nucleus and a heavily labeled contralateral rubrospinal tract. Cerebellar nucleus injections revealed light retrograde and dense terminal label in the contralateral red nucleus, together with retrograde label in a cell cluster in the ipsilateral ventrolateral medullary reticular formation, an area we identify as the lateral reticular nucleus. Injections into this medullary region resulted in heavy mossy fiber input to the ipsilateral cerebellum and moderate retrograde label in the contralateral red nucleus. These results identify prominent recurrent projections between the lateral cerebellar nucleus, red nucleus and lateral reticular nucleus, in addition to revealing other features of the cerebellorubral circuit.
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In vitro eye-blink reflex model: role of excitatory amino acids and labeling of network activity with sulforhodamine.
Evidence is presented suggesting that the neural correlate of the eye-blink reflex can be evoked in an in vitro brainstem-cerebellum preparation from the turtle by using electrical rather than natural stimulation of cranial nerve inputs. Abducens nerve discharge is triggered by brief electrical stimulation of the ipsilateral trigeminal nerve. This discharge corresponds behaviorally to EMG recordings of extraocular muscles and eye retraction recorded in situ, in a reduced preparation. The abducens nerve discharge has two components: a short-duration response having a latency of 3-6 ms and a duration of 50-100 ms, followed by a long-duration component having a latency of 12-20 ms and a duration of several seconds. The long-duration component of the reflex is blocked by the NMDA receptor antagonist APV, while both reflex responses are blocked by the non-NMDA glutamate receptor antagonist CNQX. To visualize the spatial distribution of activity during the abducens nerve reflex, bath application of the activity-dependent dye sulforhodamine was used. During reflex activity, neurons in the ipsilateral trigeminal nucleus, principal abducens nucleus, and presumed interneurons ventrolateral to the principal abducens nucleus, labeled with the dye, in addition to areas in the raphe nucleus and reticular formation. In conditions where the long-duration component of the reflex was suppressed, sulforhodamine label was absent in the principal abducens nucleus and in the caudal brainstem. From these data it is hypothesized that the region of interneurons and the accessory abducens nucleus participate in the short-duration component of the reflex. This response is mediated by non-NMDA receptors. The principal abducens nucleus is postulated to contribute also to the short-duration portion of the reflex, but is primarily involved in the generation of the long-duration component. This component of the reflex is mediated principally by NMDA receptors. Sustained reflex activity is further postulated to originate from recurrent excitation in pathways within the caudal brainstem, particularly the reticular formation. This interpretation is consistent with the observed patterns of sulforhodamine label, the effects of local microinjections of APV, and the elimination of sustained activity when the caudal brainstem is transected. These data have implications for pathways that may underlie conditioning of the eye-blink response.
2-Amino-5-phosphonovalerate↗
Distributed motor commands in the limb premotor network.
Neuroanatomical studies have demonstrated extensive interconnections between the motor cortex, red nucleus and cerebellum, forming a premotor network for controlling limb movement. Single-unit studies indicate that command signals for limb movements are distributed broadly throughout this network. Cellular studies have demonstrated multiple recurrent loops in this network, and the presence of excitatory and inhibitory amino acid neurotransmitters. A recent model suggests that movement commands are initiated by sensory inputs to these loops, and that positive feedback, regulated by inhibition from cerebellar Purkinje cells, distributes commands throughout the limb premotor network. This model offers a new framework for exploring relationships between basic neural mechanisms and concepts of motor performance that derive from experimental psychology.
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Evidence for GABAergic interneurons in the red nucleus of the painted turtle.
Immunocytochemical and electrophysiological evidence supporting the presence of GABAergic interneurons in the turtle red nucleus is presented. Injections of HRP into the spinal cord produced labeling of large neurons in the contralateral red nucleus. The peroxidase-antiperoxidase (PAP) method revealed smaller cells immunoreactive to an antibody against glutamate decarboxylase (GAD), the synthetic enzyme for the inhibitory neurotransmitter GABA, that were interspersed among larger immunonegative neurons. Similar small neurons were densely immunostained by antibodies to GABA-glutaraldehyde conjugates obtained from different sources and applied according to pre-embedding and postembedding protocols. Rubrospinal neurons retrogradely labeled with HRP measured 16 and 27 microns in mean minor and major cell body diameters, while GABA-like immunopositive neurons situated within the red nucleus measured 7 and 13 microns. There was very little overlap in soma size between the two cell populations. Therefore, we suggest that the GAD- and GABA-positive neurons may be local inhibitory interneurons. This notion is further supported by observations of pre-embedding immunostaining for GAD and postembedding immunostaining for GABA showing that the turtle red nucleus is amply innervated by immunoreactive axon terminals. These puncta are closely apposed to cell bodies and dendrites of both immunonegative large neurons and immunopositive small neurons. Moreover, immunogold staining at the electron microscopic level demonstrated that GABA-like immunoreactive axon terminals with pleomorphic synaptic vesicles formed symmetric synapses with cell bodies and dendrites of the two types of red nucleus cells. These ultrastructural features are commonly assumed to indicate inhibitory synapses. A moderately labeled bouton with round vesicles and asymmetric synapses was also observed. In addition, the two types of red nucleus neurons received asymmetric axosomatic and axodendritic synapses with GABA-negative boutons provided with round vesicles, features usually associated with excitatory functions. To obtain electrophysiological evidence for inhibition, intracellular recordings from red nucleus neurons were conducted using an in vitro brainstem-cerebellum preparation from the turtle. Small, spontaneous IPSPs were recorded from 7 out of 14 red nucleus cells studied. These morphological and physiological results provide strong support for concluding that the turtle red nucleus, like its mammalian counterpart, contains GABAergic inhibitory interneurons. While we have not identified the main source of input to these interneurons, in view of the scarce development of the reptilian cerebral cortex, this input is unlikely to come from the motor cortex as it does in mammals.(ABSTRACT TRUNCATED AT 400 WORDS)
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Sulforhodamine labeling of neural circuits engaged in motor pattern generation in the in vitro turtle brainstem-cerebellum.
A fluorescent molecular probe was used in combination with a novel in vitro preparation to study spatial patterns of neural activity associated with motor pattern generation. The in vitro brainstem-cerebellum preparation takes advantage of the turtle's unusual resistance to anoxia to preserve the entire neural network that connects the cerebellum, red nucleus, and reticular formation. This preparation was bathed in a 0.01% solution of sulforhodamine while it was activated unilaterally by electrical stimulation of the dorsal quadrant of the spinal cord for 1 hr. Sulforhodamine is a small, sulfonated, highly charged fluorescent molecule that is taken up by endocytosis. To examine its distribution in the cerebellum and brainstem, coronal sections were prepared and viewed under epifluorescence illumination. Distinctive spatial patterns of labeling were associated with unilateral electrical stimulation of the in vitro network, suggesting that dye uptake was activity dependent. Blockade of uptake with altered magnesium and calcium concentrations indicated that single spike discharge evoked ortho- or antidromically was insufficient to induce dye uptake. Instead, sulforhodamine staining correlated with the presence of burst discharge that was recorded extracellularly from the red nucleus. Blockade of burst discharge with excitatory amino acid receptor antagonists prevented dye uptake in the red nucleus, the lateral cerebellar nucleus, and other structures that are known to be interconnected by recurrent anatomical pathways. These results suggest that sulforhodamine is internalized by intensely active neurons. The spatial distributions of label support the hypothesis that burst discharges in the turtle red nucleus are mediated by excitatory amino acid neurotransmitters and sustained by recurrent excitation in cerebellorubral synaptic pathways. Positive feedback in these recurrent pathways may provide an important driving force for the generation of motor programs that control limb movements.
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Effects of infant versus adult pyramidal tract lesions on locomotor behavior in hamsters.
The role of the pyramidal tract in locomotion was studied in hamsters by analyzing their locomotor behavior after lesions of the medullary pyramidal tract. Animals with lesions either as adults or as infants were compared to determine whether early pyramidotomy results in greater functional recovery. Normal and pyramidotomized animals were filmed during locomotion on a runway consisting of either smooth or rough terrain to assess whether the uneven surface would accentuate locomotor deficits. Frame-by-frame analysis of the filmed behavior during all phases of the step cycle was carried out to determine positions of the joints of the forelimb and hindlimb during locomotion. Accuracy of limb placement on the rough terrain was determined by observations of consecutive step cycles. The results show that lesions of the pyramidal tract in both infant and adult hamsters affect locomotion first by causing a reduction in the yielding phase of the step cycle and second by producing inaccuracies of forelimb placement. Rough terrain accentuates deficits in forelimb placement during locomotion. Animals with lesions as infants and those with lesions as adults show surprisingly similar deficits in locomotion, with the exception that animals with lesions as infants show some behavioral compensation in hindlimb movement by developing a normal degree of yielding at the knee. In contrast, hamsters with lesions as either adults or infants never recover normal forelimb behavior in either yielding at the elbow or accuracy of forelimb placement. These results emphasize the sensorimotor role of the pyramidal tract, even in a relatively stereotyped behavior such as locomotion.(ABSTRACT TRUNCATED AT 250 WORDS)
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Role of excitatory amino acids in mediating burst discharge of red nucleus neurons in the in vitro turtle brain stem-cerebellum.
1. Bursts of discharge have been recorded in the red nucleus in several species and are thought to represent the expression of motor commands. A cerebellorubral circuit comprised of recurrent connections among the cerebellum, red nucleus, and reticular formation was postulated to function as a positive feedback loop that generates these motor commands and transmits them to the spinal cord via the rubrospinal pathway. We have used an in vitro preparation from the turtle that leaves the circuitry connecting the cerebellum, brain stem, and spinal cord intact to study the role of excitatory amino acid neurotransmitters and recurrent excitation in mediating the generation of burst discharges in the red nucleus. 2. Burst discharges were recorded extracellularly from single cells in the red nucleus in response to single pulse or brief train stimulation of the contralateral spinal cord or brief train stimuli applied to the ipsilateral cerebellar cortex. The firing characteristics and pharmacologic sensitivities of the bursts were independent of the type of stimulus used. The bursts had long durations ranging from 2 to 17 s and showed spike frequency adaptation. 3. Transection of the cerebellar peduncle, which eliminates inhibition impinging onto the cerebellorubral circuit, greatly enhanced the spontaneous activity and burst discharges recorded in the contralateral red nucleus. Furthermore, bath application of a solution containing elevated levels of calcium and magnesium blocked the expression of burst discharges even though synaptic activation of the neurons was not blocked. 4. The possibility that excitatory amino acid receptors mediate burst responses in the red nucleus was investigated in light of the antagonistic effects of elevated magnesium ions on bursting. Bath application of 100 microns DL-2-amino-5-phosphonovaleric acid (APV), a specific N-methyl-D-aspartate (NMDA) receptor antagonist; [10 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)], a specific non-NMDA receptor antagonist; or 100 microM, DL-2-amino-4-phosphonobutyric acid (AP4), an agonist of a fourth class of excitatory amino acid receptor, blocked burst activity in the red nucleus. With a multibarreled pipette for simultaneous ejection of drug and recording, iontophoresis of APV or CNQX into the red nucleus blocked bursting whereas AP4 failed to show a significant effect. These data suggest that red nucleus neurons have both NMDA and non-NMDA receptors. The site of action of the AP4-sensitive receptor appears to be elsewhere in the cerebellorubral circuit. 5. Iontophoretic application of excitatory amino acid receptor agonists NMDA and quisqualate (Q) induced excitation of red nucleus neurons.(ABSTRACT TRUNCATED AT 400 WORDS)
2-Amino-5-phosphonovalerate↗
An in vitro preparation for studying motor pattern generation in the cerebellorubrospinal circuit of the turtle.
In vivo studies in mammals have suggested that the cerebellorubrospinal circuit functions as a recurrent excitatory loop that generates motor commands and transmits them to the spinal cord via the rubrospinal pathway. Here we describe an in vitro preparation from the turtle exhibiting functional synaptic connections between the cerebellum, brainstem and upper spinal cord that is suitable for detailed analysis of this circuit. Electrical stimulation of the spinal cord was used to activate the cerebellorubrospinal circuit while activity was sampled with extracellular recordings from single cells in the red nucleus. Single units responded to stimulation with short and long latency synaptic responses, in addition to antidromic activation. Some cells showed bursts of activity lasting several hundred milliseconds suggesting the presence of recurrent excitation. Interruption of Purkinje cell inhibitory input impinging on the cerebellorubrospinal loop prolonged bursting and enhanced spontaneous activity. This preparation should facilitate the examination of the role of the cerebellorubrospinal circuit in motor pattern generation.
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Modulation of spinal reflexes by pyramidal tract stimulation in an in vitro brainstem-spinal cord preparation from the hamster.
Electrophysiological evidence is presented showing that the pyramidal tract (PT) of the hamster modulates spinal reflexes in an in vitro brainstem-spinal cord preparation. Three spinal reflexes were studied. Stimulation of a dorsal root (DR) while recording from a ventral root (VR) of the same spinal segment evoked two reflexes: the monosynaptic reflex, and a long latency polysynaptic reflex. Stimulation of a DR while recording from a DR immediately rostral to it elicited a volley of antidromic discharges characteristic of the dorsal root reflex (DRR). The effect of PT stimulation on reflex transmission was tested by stimulating the PT at varying intervals prior to evoking a reflex. The results show that the amplitude of the monosynaptic reflex is progressively inhibited when preceded at shorter delays by a train of PT stimuli. Similarly, PT stimulation also suppresses the long latency reflex. In contrast, the PT facilities the DRR and repeated stimulation of the PT may evoke antidromic discharges recorded from the DRs. These data from the in vitro brainstem-spinal cord preparation indicate that the PT of the hamster exerts both inhibitory and facilitatory effects on reflex transmission in the spinal cord. The present study shows that it is possible to examine the descending control of spinal circuitry using an in vitro brainstem-spinal cord preparation.
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Chloroacetate esterase positivity in acute lymphoblastic leukemia.
Chloroacetate esterase (CAE) reactivity is associated with acute nonlymphocytic leukemia (ANLL) and, to the author's knowledge, has not been reported in acute lymphoblastic leukemia (ALL). The authors have identified a single patient whose blasts displayed CAE reactivity from a review of 400 newly diagnosed patients with ALL. The diagnosis of ALL in this case was based upon morphologic evaluation (FAB:L1), presence of terminal deoxynucleotidyl transferase (TdT), the absence of reactivity with myeloperoxidase, and the failure of leukemic blasts to mark with a panel of anti-myeloid monoclonal antibodies. Remission was induced promptly with standard ALL therapy. This case demonstrates that punctate CAE positivity rarely can occur in ALL, and, therefore, CAE is not entirely specific for ANLL. The authors conclude that CAE positivity should not be used as a sole criterion to diagnose ANLL in the absence of supporting morphologic, cytochemical, immunocytologic, or clinical information.
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Three forms of the scratch reflex in the spinal turtle: movement analyses.
The scratch reflex is a motor task exhibited by the hindlimb of a turtle after complete transection of the spinal cord just posterior to the forelimb enlargement. The scratch is elicited by applying gentle tactile stimulation to a site innervated by spinal segments caudal to the level of the transection. During the scratch response, the ipsilateral hindlimb reaches toward and rubs against the stimulated site. Each scratch response consists of either a single rub or a rhythmic series of rubs. There are three types or "forms" of the scratch reflex in the spinal turtle: rostral scratch (previously termed the scratch reflex in Refs. 5, 43, 45), pocket scratch, and caudal scratch. We have characterized each scratch form according to which portion of the hindlimb is used to rub against the stimulated site. During a rostral scratch, the dorsum of the foot and/or toes is used to rub against the stimulated site. During a pocket scratch, the side of the thigh, knee, and/or calf is used to rub. During a caudal scratch, the heel or side of the foot is used to rub. Each scratch form has a distinct receptive field on the turtle shell and skin. A rhythmic scratch response to maintained tactile stimulation consists of as many as 10-25 cycles. We have divided each cycle of movement into a prerub, rub, and postrub phase. Extension of the knee joint occurs during the rub phase of each scratch cycle for all scratch forms. The timing of the extension phase of the knee joint movement within the protraction-retraction (flexion-extension) cycle of the hip joint is different for each form of the scratch. During a rostral scratch, extension of the knee joint occurs when the hip is protracted. During a pocket scratch, knee extension occurs when the hip is retracting. During a caudal scratch, knee extension occurs when the hip is retracted. The spinal turtle therefore shifts the timing of movements of a distal joint (knee) with respect to the timing of movements of a proximal joint (hip) to obtain appropriate scratching movements to different regions of the body. A narrow transition zone exists between the receptive field for one form of the scratch and the nearby receptive field for another form of the scratch. There is a rostral-pocket transition zone and caudal-pocket transition zone in the spinal turtle.(ABSTRACT TRUNCATED AT 400 WORDS)
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Three forms of the scratch reflex in the spinal turtle: central generation of motor patterns.
A turtle with a complete transection of the spinal cord, termed a spinal turtle, exhibits three types or "forms" of the scratch reflex: the rostral scratch, pocket scratch, and caudal scratch (21). Each scratch form is elicited by tactile stimulation of a site on the body surface innervated by afferents entering the spinal cord caudal to the transection. We recorded electromyographic (EMG) potentials from the hindlimb during each of the three forms of the scratch in the spinal turtle (see Fig. 1). Common to all scratch forms is the rhythmic alternation of the activity of the hip protractor muscle (VP-HP) and hip retractor muscle (HR-KF). Each form of the scratch displays a characteristic timing of the activity of the knee extensor muscle (FT-KE) with respect to the cycle of activity of the hip muscles VP-HP and HR-KF. In a rostral scratch, activation of FT-KE occurs during the latter portion of VP-HP activation. In a pocket scratch, activation of FT-KE occurs during HR-KF activation. In a caudal scratch, activation of FT-KE occurs after the cessation of HR-KF activation. The timing characteristics of these muscle activity patterns correspond to the timing characteristics of changes in the angles of the knee joint and the hip joint obtained with movement analyses (21). We recorded electroneurographic (ENG) potentials from peripheral nerves of the hindlimb during each of the three forms of the "fictive" scratch in the spinal turtle immobilized with neuromuscular blockade (see Fig. 4). Common to all forms of the fictive scratch is the rhythmic alternation of the activity of hip protractor motor neurons (VP-HP) and hip retractor motor neurons (HR-KF). Each form displays a characteristic timing of the activity of knee extensor motor neurons (FT-KE) with respect to the cycle of VP-HP and HR-KF motor neuron activity. The timing characteristics of these motor neuron activity patterns are similar to the timing characteristics of the muscle activity patterns obtained in the preparation with movement (cf. Figs. 1 and 4). The motor pattern for each scratch form is generated centrally within the spinal cord. In the spinal immobilized preparation, neuromuscular blockade prevents both limb movement and phasic sensory input, and complete spinal transection isolates the cord from supraspinal input.(ABSTRACT TRUNCATED AT 400 WORDS)
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Erythroleukemic infiltration of a lymph node: use of hemoglobin immunohistochemical techniques in diagnosis.
Isolated submandibular adenopathy developed in a patient who had subacute erythroleukemia. Lymph node biopsy revealed a proliferation of immature cells and scattered foci of dyserythropoietic normoblasts. Since a review of the literature revealed no detailed description of an erythroleukemic lymph node infiltrate, a histopathologic study of this node was undertaken. The immature cells were identified as erythroblasts and pronormoblasts by histochemical staining (periodic acid-Schiff reagent and chloroacetate esterase), immunohistochemical reaction (directed against hemoglobin), and electron microscopy. The diagnosis of erythroleukemic lymph node infiltrate may be missed in the absence of a high index of suspicion. It is concluded that the detection of hemoglobin within malignant cells is most helpful in establishing the diagnosis.
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In vitro motor program for the rostral scratch reflex generated by the turtle spinal cord.
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