Joyce Keifer

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.

Somatosensory and movement-related properties of red nucleus: a single unit study in the turtle

Extracellular recordings were performed from turtle red nucleus neurons to examine their responsiveness to peripheral somatic stimulation and to study differences between rubral sensory and movement-related responses. In pentobarbital sodium-anesthetized or decerebrate turtles, red nucleus neurons could be divided into two categories based on their response characteristics. The first group, which included 87% of neurons studied, had low spontaneous rates of activity and responded with excitation to electrical stimulation of the spinal cord or the cerebellum, or during active movement of the contralateral limbs. Neurons in this category were likely to be rubrospinal cells. The remaining 13% of cells studied had higher rates of spontaneous discharge and were inhibited by electrical stimulation or during active movement. These cells might be rubral GABAergic interneurons. Single red nucleus neurons responded with excitation and/or inhibition to somatosensory stimulation. Unlike the motor fields, which were restricted to a single contralateral limb, red nucleus sensory receptive fields were wide and often bilaterally distributed. Rubral responsiveness to sensory stimulation was found to be significantly diminished during active limb movements, thereby suggesting that sensory inputs to the red nucleus are not used for the on-line modification of motor commands. Inactivation of the cerebellar cortex enhanced the sensory responsiveness of rubral neurons and expanded the size of red nucleus receptive fields. These results suggest that the red nucleus receives substantial sensory input, and that the cerebellar cortex can modify the flow of sensory information to the red nucleus.

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 turtless 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.

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.

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.

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.

Positive Feedback in the Cerebro-Cerebellar Recurrent Network May Explain Rotation of Population Vectors

It has been postulated that descending motor commands from the motor cortex and cerebellum are generated by recurrent networks driven by positive feedback. Computer simulation of a reaching task was used to examine this theory. Twelve two-cellt reciprocally connected elements were connected in a loop, and each element was assigned a preferred direction. Cerebellar inhibition levels were set for each element to produce a movement in a desired direction. When a stimulus was given in the desired direction, the population vector grew in the desired direction. When a stimulus was given 90° away from the desired direction, the population vector rotated to the desired direction as seen in data from neuronal recordings in the motor cortex of monkeys during movement. These results demonstrate that networks driven by positive feedback can account for the rotation of the direction vector observed in the motor cortex during reaching

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.