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Notes: Summary We have devised a protocol for the cloning of our cell lines, and have demonstrated that a cloned line may contain cells of widely differing morphology — epithelial, fibroblast-like, and lamellocyte-like. These different morphologies must therefore represent diversity in the microenvironment of the culture rather than diversity in the cellular origin of the line.

Notes: Summary We have examined the metamorphosis of the wing imaginal disc of Drosophila during culture in vitro in the continuous presence of 20-hydroxy ecdysone (0.1 μg/ ml). We find that the sequence of cellular changes in the wing blade during culture closely match those occurring in situ, involving two periods at which the dorsal and ventral surfaces are joined only by cell processes containing trans-alar microtubule arrays. Good pupal and imaginal cuticle secretion is found in this system.

Notes: Summary We have investigated the action of the moulting hormone 20-hydroxy ecdysone (20-HOE) on our leg and wing imaginal disc cell lines. At the morphological level, cells stop dividing and there is some cell death. The remaining cells elongate and aggregate, often producing long processes which form connections between different aggregates. 20-HOE acts within the first one or two days of a passage, at an optimum concentration of 10 ng/ml, this being about 1/100 of the optimum for ecdysone. One cloned wing cell line, C9, has been found to be relatively insensitive to the action of 20-HOE. We have been able to select for resistance to 20-HOE by growing cells in gradually increasing concentrations of hormone followed by passages in hormone-free medium. This has enabled us to isolate a wing cell line C1.8R from its parent cloned line C1.8+. This shows no response to 20-HOE, and cell growth continues even at hormone concentrations as high as 150 ng/ml. We have measured chitin synthesis by the incorporation of radioactive glucosamine into a cell fraction resistant to extensive alkali hydrolysis. The residue was incubated with chitinase, which resulted in a 50% reduction in labelled product. Treatment with 10 ng/ml of 20-HOE dramatically increased chitin synthesis in line C1.8+, but had no effect in the line C1.8R, selected for resistance to hormone.

Notes: Summary Pairs of eye-antennal discs, attached to the cephalic ganglia, were cultured in vitro with a concentration of β-ecdysone optimal for imaginal differentiation. The eye-antennal discs fused to form a vesicle inside which the antennae were partially everted, and on the inner surface of which imaginal structures differentiated. The epithelium of the discs was continuous, and an integrated pattern of bristles and hairs differentiated in vitro. In particular, the median ocellus, a unified structure derived partially from each disc, differentiated normally.

Notes: Summary The thin region of the peripodial membrane is confined to the area overlying the distal anlagen in thoracic discs. During the early stages of evagination the peripodial membrane is greatly stretched, but does not rupture. The appendage then evaginates through the stalk, probably by means of a contraction of the peripodial membrane. The cells of the peripodial membrane of leg and wing discs persist and differentiate sheets of trichomes characteristic of the ventral and lateral thorax. This is discussed in relation to imaginal disc fate maps.

Notes: Summary The fusion of the eye-antennal discs during culturein vitro has been investigated, and the complex morphogenetic movements which occur during the formation of the head capsule of the insect are described. The initial contact between the eye anlagen is by means of cell processes spanning the gap between the two discs. Subsequently the two epithelia become firmly apposed, and then the integrity of the epithelium in the region of fusion breaks down, cells appearing to move to new positions in order to form an epithelium which unites the two discs. The epithelium eventually secretes a pattern of cuticular structures which is continuous between the derivatives of the two discs. Bristles on either side of the line of fusion are perfectly aligned, and structures such as the median ocellus, which are formed jointly by the cells of the two discs, differentiate normally. This is also found when left and right eye-antennal discs of different genotypes are placed side-by-side, indicating that processes of pattern regulation can occur in culture.

Notes: Abstract This study examined changes in the performance of a single-joint, elbow task produced by reversible inactivation of local regions within the proximal forelimb representation in area 4γ of motor cortex (MCx) and the red nucleus (RN) of the cat. Inactivation was carried out by microinjecting lidocaine, γ-aminobutyric acid, or muscimol into sites where microstimulation evoked contraction of elbow muscles. Reaction time, amplitude, and speed (velocity or dF/dt) of position and force responses elicited during inactivation were compared to control values obtained immediately prior to inactivation. In addition, we assessed qualitatively the effects of inactivation on reaching, placing reactions, and proprioceptive responses to imposed limb displacement. In the single-joint task, injections in MCx did not increase reaction time (simple or choice) and produced modest and inconsistent reductions in response amplitude (mean-8%) and speed (mean -19%). In contrast, injections of the same amounts of inactivating agents in the forelimb representation of RN consistently increased reaction time (34.4%), and increased the reaction time coefficient of variability (32%). There were small reductions in response amplitude (-4%) and speed (-10%) which were less than those produced by MCx inactivation. During reaching, however, these same injections in MCx and RN produced a substantial loss of accuracy. For MCx, this was due, in part, to systematic hypometria: for RN, inaccuracy resulted from increased variability in paw paths. Placing reactions and corrective responses to imposed limb displacements were also depressed by the cortical and rubral injections. Our results suggest that the forelimb representation in RN plays a role in the initiation of the single-joint, elbow tracking response examined here. The RN may mediate cerebellar regulation of response timing, a function that is likely to be important for interjoint coordination. Although neurons in the forelimb representations of MCx may contribute to force generation in single-joint movements, their contribution to multijoint control appears to be more important and is examined in the subsequent report (Martin and Ghez 1993).

Notes: Summary (1) Participation of the motor cortex in initiating muscle contraction in an isometric tracking task was assessed in cats trained to make accurate force adjustments using forelimb muscles, in response to a vibrissal/visual display stimulus. Behavior in the task was characterized by short reaction times. While the task was performed, recordings of single cortical units were made in zones within area 4γ defined by the effects of microstimulation in forelimb muscles and by receptive fields on the forelimb. (2) Two types of receptive fields with different regional distributions were observed. Cells with simple receptive fields (superficial or deep) were seen throughout the area sampled, consisting of the lateral half of the anterior and posterior sigmoid gyri. Cells whose receptive fields had complex features (directional specificity, temporal lability, multiple foci, etc.) were preferentially located in the cortex rostral to the cruciate sulcus. (3) The area of motor cortex rostral to the cruciate sulcus also differed from the area caudal to the cruciate sulcus in the timing of task-related activity. Neurons that were active before response onset (lead cells), and could therefore contribute to response initiation, were preferentially located in the rostral cortex, and, in general, had complex receptive fields. (4) Lead cells were active at a constant latency from the stimulus, rather than being timed to response onset. However, the modulation of their activity was related to both the direction and magnitude of the force response. (5) These results suggest that the pericruciate motor cortex of the cat contains two functional subdivisions: a caudal one concerned with ongoing movement, perhaps under the control of specific sensory inputs from the responding limb, and a rostral one involved in initiating movement. Because behaviorally relevant stimuli can rapidly activate a specialized population of cells in the rostral cortex, this area is able to participate in responses with short reaction times.

Notes: Summary In a previous study in the cat, we have reported that motor cortex neurons discharging before the initiation of an aimed forearm response (lead cells) are better timed to movement of a display (stimulus) than to the response. The present study was done to distinguish the coding of stimulus and response features in the discharge patterns of such early activity in motor cortex. Single neurons were recorded in the arm area of motor cortex in three cats performing the same pair of responses (forearm flexion and extension) but to display movements in either of the two directions by changing display polarity. The modulation of lead cell activity was contingent on the occurrence of the learned motor response and timed to the stimulus in all conditions. The majority of lead cells (88%, n = 50) fell into one of two distinct classes. In one class of neurons, force-direction (56%, n = 32), activity was contingent on a single direction of forelimb response (flexion or extension) and was thus independent of the direction of the display stimulus. The only muscles whose patterns matched the activity of this class of response-related neurons were forelimb flexors and extensors. In these neurons, the onset of modulation was timed to one or the other of the two stimuli according to the stimulus direction which elicited the appropriate response. Thus, the display-related input to these neurons varied according to the response required. In the second class of neurons, stimulus-direction (32%, n = 18), modulation was associated with a specific stimulus direction rather than the response direction. The pattern of activity of these neurons was similar to the pattern of EMG signals of shoulder and neck muscles during the different task conditions. The contraction of proximal and axial muscles corresponded to a second response elicited by the stimulus, namely attempts at head rotation towards the moving display and was independent of the conditioned forelimb response in both time of onset and direction. To test the possibility that stimulus-direction neurons participated in the control of head rotation we trained two of the animals to also produce isometric changes in neck torque in the direction of the moving display without making the forelimb response. The activity of stimulus-direction neurons was similarly modulated during performance of the neck task. By contrast, force-direction neurons examined during the neck task were either unmodulated or discharged after the neck response. These data suggest that force-direction neurons participate in response initiation and that their activity is triggered by stimuli specific for the task. The reorganization of the inputs to motor cortex is likely to result from gating mechanisms associated with behavioral set. Such neural gates could provide for the efficient transfer of any member of an array of behaviorally relevant stimuli to restricted sectors of the somatotopically organized motor areas.

Notes: Summary In the present study we recorded the activity of single neurons in the forelimb area of red nucleus (RN) during performance of three step-tracking tasks designed to dissociate the coding of stimulus and response variables in the discharge of recorded neurons. In two of these tasks, the standard and stimulus-reversal arm tasks, elbow flexion and extension were elicited by different stimuli enabling us to distinguish activity correlated with the forelimb response from the stimulus eliciting it. The third task (neck task) allowed us to determine whether neuronal modulation was related to an unconditioned orienting response that occurred concurrently with the forelimb response. We have previously reported that these three tasks separate neurons in MCx whose modulation precedes the response (lead cells) into three distinct classes in which task-related activity either is correlated with the direction of the forelimb response, correlated with the stimulus, or not correlated with either (Martin and Ghez 1985). All lead cells, however, remained timed to the stimulus rather than to the response. The present results show that RN lead cells can be subdivided into the same three classes as those in MCx and their discharge was also contingent on the subsequent production of a behavioral response. (1) Force-direction neurons (35%; n = 16) showed changes in activity correlated with the production of forearm force in a particular direction suggesting that they could participate in selecting the appropriate forelimb response. The onset of task-related modulation of activity was better timed to the response, in contrast to force-direction neurons in MCx, which were better timed to the stimulus. (2) Stimulus-direction neurons (18%; n = 8) modulated their activity in relation to a particular stimulus evoking either flexor or extensor responses and during neck task performance. These neurons could be involved in processing stimulus information or in the production of neck torque. The task-related discharge of these lead cells was better timed to the stimulus than to either the forelimb or the neck response. (3) Nondirectional neurons (47%; n = 21) modulated their activity during all tasks examined. Their discharge did not correlate with any specific feature of the stimulus or response, and as a group, was better timed to the stimulus than to the response. Nondirectional neurons may participate in some aspect of motor preparation. To determine the relative contributions of RN and MCx lead cells to response initiation, we compared the amount of response latency variance that could be explained by variation in the latency of the unit modulation to the stimulus for the present data and the data in the earlier MCx study (Martin and Ghez 1985). Between 38% and 53% of response latency variance (for trials examined during performance of the standard arm and stimulus reversal tasks) was accounted for by the latency variations of RN force direction neurons; in contrast, 8% and 11% for MCx force-direction neurons. Variations in timing of stimulus-direction neurons in both RN and MCx account for less than 10% of response latency variance. Our findings suggest that, in the tasks examined, RN force-direction neurons play a more direct role than MCx force-direction neurons in initiating and selecting responses to stimuli. We hypothesized that this subcortical control reflects the high degree of stereotypy of the motor response examined.