Development of Cultured Entorhino-Hippocampal Slices Innervated by Diffuse Ascending Transmitter Systems
This work was done at the University of California at Irvine
 

Goal: Many psychoactive agents act by modulating one or more of the diffuse ascending systems; i.e., serotonin, acetylcholine, dopamine, and norepinephrine. These compounds facilitate or retard transmission but do not act as agonists or antagonists and often have only subtle effects on transmitter binding. Because of this, detection of modulatory agents is often best accomplished with an assay that incorporates synaptic transmission. This presents a severe problem in the case of the diffuse systems because the pertinent axons are both very sparse and disconnected from their cell bodies. It is thus difficult to establish that a stimulating electrode is in contact with the targeted fibers or that an evoked response reflects monosynaptic transmission involving the intended projections. One solution to these problems is to co-culture the cell bodies that give rise to the diffuse system of interest with an appropriate anatomical target of the system. However, co-cultures have not been used with the cultured slice technique recently introduced by Stoppini et al. The new method uses slices prepared from rat brains in the second post-natal week, a time point at which the major anatomical systems have been laid down. Moreover, the slices gradually take on a much more adult-like state than is the case with traditional organotypic cultures. These are important considerations with regard to investigating how a given agent or manipulation affects the adult, as opposed to developing, brain. Accordingly, a primary objective of the first year work in this project has been to determine if serotoninergic, cholinergic, and dopaminergic cells will innervate cultured entorhino-hippocampal slices.

  Progress: Figure 1 consists of micrographs of sections through two cultured slices, one (left side) co-cultured with the median raphe nucleus of the brainstem and the other co-cultured with the medial septal nucleus. The raphe-hippocampal sections have been processed for immunocytochemistry using antibodies against serotonin while the septal-hippocampal sections were processed for acetylcholinesterase histochemistry. Serotoninergic and cholinergic cell bodies can be seen in the boxes marked 'B' in the survey micrographs in the top panels of the figure. Labelled fibers arising from these neurons are evident in the higher power images in the middle panels. The bottom panels show the serotoninergic and cholinergic fibers forming dense plexuses above and below the cell body layers within hippocampus, the pattern of innervation found in situ. The dense puncta in these figures correspond in size, appearance, and distribution to axon terminals. In all, cultured slices prepared relatively late in development provide excellent targets for the diffuse projections.

 

Success in the co-culture experiment opened the way to tests of how the diffuse, ascending projections affect network level operations in hippocampus and retrohippocampal cortex. Figure 2A shows a septal/hippocampal culture sitting atop a 64-electrode array while figure 2B provides typical recordings of spontaneous physiological activity. Note the triplets of large biphasic waves that are particularly prominent at electrodes #36 and #44. Based on their shape and distribution, these potentials appear to be synchronous post-synaptic responses; i.e., the result of a sizeable number of CA3 pyramidal neurons firing at about the same time. Figure 2C shows first results obtained with physostigmine, a psychoactive drug that modulates cholinergic transmission by blocking the catalytic enzyme acetylcholinesterase. As is evident, the drug triggered rhythmic EEG activity at sites throughout the hippocampus, an effect similar to that it produces in vivo. Analyses now in progress indicate that multiple frequencies are promoted by physostigmine and that the response to the drug is regionally differentiated. But as they stand, the above results demonstrate the feasibility of using cultured slices to detect compounds that modulate ascending diffuse projections. Rhythmic activity of a type not seen in conventional slices has also been recorded in raphe/hippocampal preparations (data not shown) and experiments with modulatory drugs will begin shortly.

 

Figure 1: Raphe-hippocampal slice (left) and septo-hippocampal slice (right) cocultures. Immunohistochemistry with anti-serotonin antibodies (left) and histochemistry with cholinesterase (right).

 

 

 

 

Figure 2: Example of a septo-hippocampal slice co-culture (top) and spontaneous electrical activity recorded under control conditions (middle) and in the presence of phsysostigmine (lower).