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Little is known about the neurochemical features of the nucleus reuniens thalami (RE). In the present study, immunocytochemical experiments were performed to characterize the expression pattern of certain neurochemical markers, e.g. the calcium-binding proteins calbindin and calretinin and several neuropeptides. Colocalization studies revealed that half of the calbindin-positive cells express calretinin, and numerous calretinin-immunoreactive neurons contain calbindin. In contrast, immunolabelling for neuropeptides did not reveal cell bodies in the RE. The RE establishes widespread connections with several limbic structures. To correlate these projection patterns with the neurochemical characteristics of RE neurons, the retrograde tracer [3H]D-aspartate, which is selectively taken up by high affinity uptake sites that use glutamate as neurotransmitter, and the nonselective retrograde tracer wheatgerm agglutinin-conjugated colloidal gold was injected into the stratum lacunosum moleculare of the hippocampal CA1 subfield and into the medial septum. The results provide direct anatomical demonstration of aspartatergic/glutamatergic projection from the RE to the hippocampus and to the medial septum. Nearly all of the projecting neurons proved to be calbindin-immunopositive and many of them expressed calretinin. Both retrograde labelling techniques revealed that neurons projecting to the hippocampus were located in clusters in the dorsolateral part of the RE, whereas neurons projecting to the medial septum were mainly distributed in the ventromedial portion of the nucleus, indicating that different cell populations project to these limbic areas. These results suggest that neurons in the RE are heterogeneous and contribute to the excitatory innervation of the septo-hippocampal system.

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