Possible glutamatergic/aspartatergic projections to the supramammillary nucleus and their origins in the rat studied by selective [(3)H]D-aspartate labelling and immunocytochemistry.

The supramammillary neurons projecting directly to the hippocampus or indirectly via the septum participate in the regulation of hippocampal theta activity. Inputs to the supramammillary nucleus are only partly specified neurochemically. Glutamate appears to be an excitatory transmitter in this cell group, however, the origin of the glutamatergic afferents is unknown. The present investigations were devoted to study this question. The transmitter-selective [(3)H]D-aspartate retrograde transport method was used injecting the tracer into the lateral subregion of the nucleus. The radioactive tracer was visualized by autoradiography. Non-selective retrograde tracing experiments were also performed for reference injecting wheat germ agglutinin-conjugated colloidal gold into the same supramammillary region. Retrogradely radiolabelled neurons in various numbers were detected in several brain regions including medial septum-diagonal band complex, lateral septum, rostral part of medial and lateral preoptic areas, lateral habenula, ventral premammillary nucleus, apical subregion of interpeduncular nucleus, laterodorsal tegmental nucleus, and dorsal and median raphe nuclei. Radiolabelled neurons in the mentioned raphe nuclei were serotonin-immunonegative. In the non-selective retrograde tracing experiments combined with immunocytochemistry, about 50% of the retrogradely labelled neurons in the raphe nuclei was serotonin-immunonegative, showing that not only serotonergic raphe neurons project to the supramammillary nucleus. The findings indicate that a significant part of the afferents from telencephalic, diencephalic and brainstem regions to the supramammillary nucleus may contain glutamate/aspartate as neurotransmitter. The most important functional implications of these observations concern the role of the supramammillary nucleus in controlling the electrical activity of the hippocampus, and in particular the generation and maintenance of the theta rhythm.

The supramammillo-hippocampal and supramammillo-septal glutamatergic/aspartatergic projections in the rat: a combined [3H]D-aspartate autoradiographic and immunohistochemical study.

It is well established that the supramammillary nucleus plays a critical role in hippocampal theta rhythm generation/regulation by its direct and indirect (via the septal complex) connections to the hippocampus. Previous morphological and electrophysiological studies indicate that both the supramammillo-hippocampal and supramammillo-septal efferents contain excitatory transmitter. To test the validity of this assumption, transmitter specific retrograde tracer experiments were performed. [3H]D-aspartate was injected into different locations of the hippocampus (granular and supragranular layers of the dentate gyrus and CA2 and CA3a areas of the Ammon's horn) and septal complex (medial septum and the area between the medial and lateral septum) that are known targets of the supramammillary projection. Consecutive vibratome sections prepared from the entire length of the posterior hypothalamus, including the supramammillary area, were immunostained for calretinin, tyrosine hydroxylase, or calbindin, and further processed for autoradiography. Radiolabeled, radiolabeled plus calretinin-containing, and calretinin-immunoreactive neurons were plotted at six different oro-caudal levels of the supramammillary area. The results demonstrated that following both hippocampal and septal injection of the tracer, the majority of the retrogradely radiolabeled (glutamatergic/aspartatergic) cells are immunoreactive for calretinin. However, non-radiolabeled calretinin-containing neurons and radiolabeled calretinin-immunonegative cells were also seen, albeit at a much lower density. These observations clearly indicate the presence of glutamatergic/aspartatergic projections to both the hippocampus and septal complex. It may be assumed that this transmitter could play a role in hippocampal theta rhythm generation/regulation.

Topographic localization of calretinin, calbindin, VIP, substance P, CCK and metabotropic glutamate receptor immunoreactive neurons in the supramammillary and related areas of the rat.

A detailed description of the localization of neurons containing various neuropeptides in the supramammillary complex (SUM) is provided. Further, the neurochemical character of supramammillohippocampal and supramammilloseptal projecting neurons was investigated. The following experiments were performed: (a) immunocytochemistry for each of the eight different neuropeptides investigated, in animals pretreated or not with colchicine, and perfused in fixative containing or lacking acrolein; (b) a thorough mapping study of the localization of immunolabelled neurons at three rostrocaudal levels; (c) double-tracing retrograde labelling for two-directional neuronal projections combined with immunocytochemistry, to study neurochemical character of the projecting neurons. The observations are: (1) each type of immunolabelled elements, such as calretinin, calbindin, VIP, substance P, CCK and metabotropic glutamate receptor 1a immunopositive neurons has a characteristic localization; (2) no parvalbumin- and enkephalin-containing neurons are present in the SUM; and (3) a small population of calretinin-containing and a small number of calretinin-negative supramammillohippocampal neurons located in the lateral area also project to the medial septum-diagonal band region of the septal complex.