Demonstration of connections between cortex and superior colliculus in vitro by the use of biocytin and HRP.

To study the morphology of neurons in the superior colliculus (SC) and visual cortex (VC) in vitro, the retro- and anterograde tracers biocytin and horseradish-peroxidase (HRP) were applied in cortical and tectal explant co-cultures. Injection of biocytin alone into the SC explant after up to 12 days in vitro revealed the detailed morphology of retrogradely labelled SC cells, comparable to cell types previously described in situ. However, only in 2 co-cultures out of 110 did injections of biocytin alone into the SC result in retrograde labelling of cortical cells; the label was very weak and did not allow detailed analysis of the morphological features. Only with a combination of biocytin and HRP, were the pyramidal cells of layer V adequately stained. Our result show that: 1. It is possible to use actively transported neuronal tracers in cultures, 2. anterograde as well as retrograde labelling reveals intrinsic connections in an SC explant, 3. connections between the two explants (both cortico-tectal as well as tecto-cortical) could be labelled, and 4. a combination of biocytin and HRP resulted in a much better retrograde labelling and thereby demonstration of cellular morphology of layer V pyramidal cells in VC than biocytin alone.

The corticotectal projection of the rat in vitro: development, anatomy and physiological characteristics.

In this study, the formation of the corticotectal projection of the rat in organotypic slice culture was investigated, using both anatomical and physiological approaches. The establishment of fibre connections from visual cortex to superior colliculus explants was monitored after 3, 6, 14, 20 and 30 days in vitro by cortical injections of Dil. As in cortical cultures without cocultured colliculus, fibres anterogradely labelled by this procedure spread radially from the injection site into the surroundings of the explant, without displaying any directional preference. Especially, layer V pyramidal cells could be seen to extend processes not only to the collicular target, but also in the opposite direction, suggesting that no axonal guidance was exerted by the projection target. The total number of fibres projecting in the direction of the colliculus was not higher than of those projecting in the opposite direction. However, fibres projecting into the colliculus were significantly longer. This was also the case when the colliculus was placed next to the pial side of the cortical explant, indicating that outgrowth direction was not related to this observation. We therefore assume a chemotrophic rather than a chemotactic influence of the projection target on cortical axons, which is based on direct contact of axons to the target tissue. It cannot be excluded, however, that the failure to detect chemotactic guidance was caused by the lack of diffusion gradients in our culture system. Innervation of the collicular slice exclusively originated from layer V pyramidal cells, irrespective of the position of the collicular target. Fibre courses suggested that discrimination of the projection target was achieved upon encounter with the collicular surface by direct membrane contact. Inside the collicular tissue, fibre arborizations occurred preferredly in up to three layers perpendicular to the surface. Even after the smallest tracer injections, termination fields were diffusely distributed over the collicular slice. Also, the spatial distribution of retrogradely stained projection neurons did not differ statistically from an equal distribution. Thus, a high degree of convergence and divergence was observed anatomically in the corticotectal projection formed in vitro, corresponding to the immature state in vivo. The functionality of the corticotectal projection was assessed by intracellular recordings from collicular neurons. Electrophysiological properties, such as membrane potential (-68 +/- 11 mV), membrane resistance (35.4 +/- 27.7 M omega) and the time constant (3.0 +/- 2.1 ms) were comparable to reference values, confirming the viability of our culture preparation. The functionality of corticotectal transmission was revealed by intracellularly recorded responses of collicular cells to extracellular cortical stimulation.(ABSTRACT TRUNCATED AT 400 WORDS)

Neural interactions between motor cortical hemispheres during bimanual and unimanual arm movements.

Cortico-cortical connections through the corpus callosum are a major candidate for mediating bimanual coordination. However, aside from the deficits observed after lesioning this connection, little positive evidence indicates its function in bimanual tasks. In order to address this issue, we simultaneously recorded neuronal activity at multiple sites within the arm area of motor cortex in both hemispheres of awake primates performing different bimanual and unimanual movements. By employing an adapted form of the joint peri-stimulus time histogram technique, we discovered rapid movement-related correlation changes between the local field potentials (LFPs) of the two hemispheres that escaped detection by time-averaged cross-correlation methods. The frequency and amplitude of dynamic modifications in correlations between the hemispheres were similar to those within the same hemisphere. As in previous EEG studies, we found that, on average, correlation decreased during movements. However, a subset of recording site pairs did show transiently increased correlations around movement onset (57% of all pairs and conditions in monkey G, 39% in monkey P). In interhemispheric pairs, these increases were consistently related to the mode of coupling between the two arms. Both the correlations between the movements themselves and the interhemispheric LFP correlation increases were strongest during bimanual symmetric movements, and weaker during bimanual asymmetric and unimanual movements. Increased correlations occurred mainly around movement onset, whilst decreases in correlation dominated during movement execution. The task-specific way in which interhemispheric correlations are modulated is compatible with the notion that interactions between the hemispheres contribute to behavioural coupling between the arms.

Oligodendrocytes differentiate in organotypic cultures of rat visual cortex and myelinate efferent axons.

We have investigated the presence and function of glia cells, especially of oligodendrocytes (OL) in organotypic cultures of rat visual cortex grown for 1-6 weeks in vitro. OL identified by strong Galactocerebroside-immunoreactivity (GalC-ir) displayed rather small somata and elaborately ramified processes. They were most concentrated in layers VIa and VIb and the remnant of the white matter. Silver staining revealed long descending or oblique processes in layers V and VI, which were often arranged in patches, and horizontal processes in the white matter. Proximal processes of OL cell bodies were connected to these long processes. DiI-labeling revealed very similar patches of processes, termed OL domains. They were identified as membraneous sheaths formed by processes of single OL around axons passing the OL domain. Confocal microscopy revealed single axons running through the membrane sheaths. We compared the molecular differentiation of glial cells in cultures to the in vivo situation with protein blots and immunohistochemistry for glial cell marker molecules. In homogenates of visual cortex in vivo, protein blots revealed the increase in expression by OL of myelin basic protein (MBP) during the fourth postnatal week. The astrocytic marker glial fibrillary acidic protein (GFAP), blotted as a control, increased over time in vivo, beginning at P14, indicating the differentiation of astrocytes. In homogenates of organotypic cortex cultures, the times course of expression of GFAP was very similar: it increased dramatically during the first 10 DIV, and remained fairly constant in older cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Local field potentials related to bimanual movements in the primary and supplementary motor cortices.

We recorded local field potentials (LFP) in primary (MI) and supplementary (SMA) motor areas of rhesus monkey cortex in order to compare movement-evoked potentials (mEP) in bimanual and unimanual movements with single-unit activity recorded concurrently. The mEP was often different during bimanual and unimanual movements (a "bimanual-related" effect), but, unlike the single units, the size of the mEP in both MI and SMA was always greater during bimanual movements than during unimanual movements. This increase primarily reflected an increase in the late positive peak of the mEP, a result that may reflect greater overall cortical activation during bimanual movements. In addition, analysis of the mEP revealed differences between MI and SMA not seen in the single-unit activity. mEP in MI had greater contralateral preference than in SMA. Also, SMA mEP was more correlated to the single-unit activity than in MI. This greater correlation was also more apparent in the late peaks of the mEP than in the early peaks and may reflect a greater influence of recurrent activation in SMA than in MI. Our results further reinforce the idea that unimanual and bimanual movements are represented differently both in MI and in SMA and also show that a complex relationship between spikes of individual neurons and LFP may reflect the different input-output relations of different cortical areas.

Timing of bimanual movements in human and non-human primates in relation to neuronal activity in primary motor cortex and supplementary motor area.

This study investigates the timing of bimanual movements in a combined behavioral and physiological approach. Human subjects and rhesus monkeys performed the same bimanual task. In monkeys, we simultaneously recorded neuronal activity in the two hemispheres of primary motor cortex (MI) or supplementary motor area (SMA), and related it to bimanual coordination in the temporal domain. Both for monkeys and humans, the reaction times of bimanual movements never significantly exceeded the reaction times of the slower arm in unimanual movements. Consistent with this, the longest delay between neural activity onset in SMA and MI and movement initiation was observed in unimanual movements of the slower arm and not in bimanual movements. Both results suggest that the programming of bimanual movements does not require more processing time than unimanual movements. They are also consistent with the view that bimanual movements are programmed in a single process, rather than by combining two separate unimanual movement plans. In both humans and monkeys, movement initiation was highly correlated between the arms. However, once movements began, the temporal correlation between the arms progressively declined. Movement decorrelation was accompanied by a net decorrelation of neuronal population activity in MI and SMA, suggesting a functional connection between neuronal interactions and the level of bimanual coupling and decoupling. The similarity of neuronal activities in MI and SMA in relationship to behavioral timing lends support to the idea that both areas are involved in the temporal coordination of the arms.