C. Leranth

Immunohistochemical evidence for synaptic connections between pro-opiomelanocortin-immunoreactive axons and LH-RH neurons in the preoptic area of the rat

Connections between adrenocorticotropic hormone (ACTH)-immunoreactive neurons in the arcuate nucleus and the preoptic area were studied in the female rat. ACTH-immunopositive terminals were observed in the medial preoptic area in contact with dendritic shafts, while in the ventrolateral preoptic area the majority of ACTH-immunoreactive synapses were found on dendritic spines. Double-label electron microscopic immunocytochemistry using peroxidase and avidin-ferritin as contrasting electron-dense markers revealed numerous synaptic contacts between ACTH-immunopositive boutons and luteinizing hormone-releasing hormone (LH-RH)-immunoreactive dendritic shafts in the medial preoptic area. Following injection of horseradish peroxidase (HRP) into the medial preoptic area, retrogradely HRP-labeled perikarya were observed throughout the arcuate nucleus. Double-staining experiments revealed that a proportion of these retrogradely labeled cells, in the ventromedial arcuate nucleus, are also immunoreactive for ACTH. These results suggest that pro-opiomelanocortin peptide-producing neurons in the ventromedial arcuate nucleus project to the medial preoptic area. Some of these neurons establish direct synaptic contacts with LH-RH-immunoreactive cells.

Immunocytochemical evidence for direct synaptic connections between corticotrophin-releasing factor (CRF) and gonadotrophin-releasing hormone (GnRH)- containing neurons in the preoptic area of the rat

Electron microscopic double-label immunostaining with peroxidase and avidin-ferritin was used to study connections between corticotrophin-releasing factor (CRF) and gonadotrophin-releasing hormone (GnRH) immunoreactive elements in the medial preoptic area of the rat. Synaptic contacts were observed between CRF-immunoreactive axon terminals and the dendrites of GnRH-immunopositive neurons. These results suggest that the inhibitory effects of stress-induced CRF release on reproductive function may involve a direct CRF input to the GnRH-producing cells.

AMPA receptors in the rat and primate hippocampus : a possible absence of GLUR2/3 subunits in most interneurons

Amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors are assembled from the four subunits GluR1, 2, 3, 4 (or GluRA, B, C, D). AMPA channels that do not contain the GluR2 subunit are permeable to calcium. Recent studies indicate that excitotoxic as well as epileptic and ischemic cell damage may be mediated not only by N-methyl-Daspartate receptors, but also by AMPA receptors. The majority of interneurons in the hippocampus are resistant, but subsets of interneurons are consistently damaged in different disease states. Single immunolabeling using antibodies against AMPA receptor subunits, together with double immunolabeling for calcium-binding proteins (parvalbumin, calbindin and calretinin) and the neuropeptide somatostatin, were performed to study GluR1-4 immunoreactivity in interneuronal populations and principal cells. The ultrastructure of GluR1-4 labeled neurons was also examined using electron microscopy. With the exception of calbindin-positive interneurons, GluR2/3 was absent from hippocampal interneurons in both rat and monkey. In the rat, interneurons were more strongly immunoreactive against GluR1 than principal cells. In the monkey, immunoreactivity for GluR4 in interneurons was stronger than for GluR1. All GluR subunits were confined to spines, dendritic membrane and cytoplasm surrounding the nucleus but absent from axons and presynaptic terminals. Our findings suggest that hippocampal principal cells and interneurons express different complements of AMPA receptor subunits. Furthermore, the absence of GluR2 and/or GluR3 in both vulnerable and resistant interneurons subtypes indicates that knowledge of receptor subunit composition is not sufficient to predict neuronal vulnerability.

The LH-RH-containing neuronal network in the preoptic area of the rat: demonstration of LH-RH-containing nerve terminals in synaptic contact with LH-RH neurons.

The existence of a luteinizing hormone-releasing hormone (LH-RH)-containing local neuronal network in the preoptic area of the rat was demonstrated by electron microscopic immunocytochemistry. LH-RH-immunostained presynaptic boutons were observed in synaptic contact with LH-RH-immunoreactive dendrites and perikarya.

Hormonal regulation of hippocampal spine synapse density involves subcortical mediation

It is well established that estrogen has positive effects on the density of pyramidal cell spines in the hippocampal CA1 subfield. This study explored whether afferent connections of the hippocampus that come from estrogen-sensitive subcortical structures, including the septal complex, median raphe and supramammillary area, play a role in this estrogen-induced hippocampal synaptic plasticity. These particular subcortical structures have major influences on hippocampal activity, including theta rhythm and long-term potentiation. The latter also promotes the formation of new synapses. All of the rats were ovariectomized; the fimbria/fornix, which contains the majority of subcortical efferents to the hippocampus, was transected unilaterally in each, and half of the animals received estrogen replacement. Using unbiased electron microscopic stereological methods, the CA1 pyramidal cell spine synapse density was calculated. In the estrogen-treated rats, contralateral to the fimbria/fornix transection, the spine density of CA1 pyramidal cells increased dramatically, compared to the spine density values of both the ipsilateral and contralateral hippocampi of non-estrogen-treated animals and to that of the ipsilateral hippocampus of the estrogen replaced rats. These observations indicate that fimbria/fornix transection itself does not considerably influence CA1 area pyramidal cell spine density and, most importantly, that the estrogenic effect on hippocampal morphology, in addition to directly affecting the hippocampus, involves subcortical mediation.

Ultrastructural evidence of amygdalofugal axons terminating on cholinergic cells of the rostral forebrain.

In the present study a double-label ultrastructural procedure was used to study amygdalofugal fibers contacting cholinergic cells of the rostral forebrain. Following horseradish peroxidase (HRP) injections into the basolateral amygdala, anterogradely transported HRP was detected in axon terminals contacting the dendrites of choline acetyltransferase-containing cells in the ventral pallidum.

Calretinin immunoreactivity in the monkey hippocampal formation—I. Light and electron microscopic characteristics and co-localization with other calcium-binding proteins

Calretinin-containing neurons were visualized by immunocytochemistry in the monkey hippocampal formation, subicular complex, and entorhinal cortex. Calretinin-immunoreactivity was present exclusively in non-granule cells of the dentate gyrus and in non-pyramidal cells of Ammons horn, subiculum and entorhinal cortex. Most frequently, calretinin-positive neurons were found at the hilar border of the dentate granule cell layer and in the stratum radiatum of CA1-3 areas. In the subicular complex, immunoreactive neurons were evenly distributed in all layers, whereas in the entorhinal cortex, they were accumulated in external layers above the lamina dissecans. Distinct bands of calretinin-positive fibers occupied the supragranular zone of the molecular layer in dentate gyrus, the pyramidal cell layer of the CA2 area in Ammons horn and the upper two layers of presubiculum. The majority of calretinin-immunoreactive neurons were small, bipolar or fusiform neurons with a dendritic tree oriented parallel to the dendrites of principal cells (granule cells in dentate gyrus and pyramidal neurons elsewhere). Dendrites were smooth or sparsely spiny, displaying small spines of conventional type. Co-existence studies showed that these neurons were completely devoid of other calcium-binding proteins, parvalbumin and calbindin. Electron microscopic analysis revealed somata of immunoreactive neurons which contained a large nucleus and a small cytoplasmic rim, which contained only few organelles. The nucleus displayed deep infoldings and intranuclear rods. Input synapses of immunoreactive neurons were rare both on somata and dendrites and large surface areas were frequently apposed by glial processes. This was very prominent in the dentate gyrus and Ammons horn. Axons of calretinin-positive neurons were thin, arborized in all layers and had small varicosities. Their terminals formed symmetric synaptic contacts mainly with dendrites and less frequently with somata of principal cells. Axon terminals of calretinin-immunoreactive fiber bundles in the supragranular layer, as well as in the pyramidal layer of the CA2 area, formed asymmetric synaptic contacts with dendritic shafts. In addition, they established asymmetric axospinous and axosomatic synaptic contacts with granule cells of the dentate gyrus. In the presubiculum, the calretinin-positive axon bundle included a large number of immunoreactive myelinated axons, as well as axon terminals. The characteristic location and features of synapses suggests that these fibers derive from extra-hippocampal afferents (Nitsch, R. and Leranth C. (1993) Neuroscience 55, 797-812) and not from the calretinin-immunoreactive neurons of the hippocampal formation.

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 Ammons 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.

The cellular effects of estrogens on neuroendocrine tissues.

Estrogen action on sensitive neurons in the rat diencephalon has been studied by morphologic techniques; evidence of estrogen action at every level is presented, including tracts, cells, circuitry and subcellular organelles. The demonstration in the arcuate nucleus of estrogen-induced synaptic remodelling, estrogen-induced postsynaptic membrane phenotypes, changes in intracellular membranes and rapid estrogen actions on neuronal endo-exocytosis indicates that cellular estrogen actions may underlie the neuronal control of reproduction.

The entorhino-septo-supramammillary nucleus connection in the rat : Morphological basis of a feedback mechanism regulating hippocampal theta rhythm

Recent electrophysiological observations suggest that, in addition to the medial septal area pacemaker system, several alternative or additional mechanisms are involved in the generation/regulation of hippocampal theta activity. Discharging neurons phase-locked to hippocampal theta waves have been observed in the dorsal raphe, nucleus reticularis pontis oralis and especially in the supramammillary region of rats. Since these areas are reciprocally interconnected with the hippocampal formation, including the entorhinal cortex, it would aid our understanding of limbic function to elucidate the location and neurochemical content of the entorhino-septal and septo-supramammillary projection neurons, as well as that of their postsynaptic targets. Light and electron microscopic immunostaining for calretinin, in combination with antero- and retrograde tracer techniques, postembedding immunostaining for GABA and the transmitter specific [3H]D-aspartate retrograde radiolabeling, as well as a co-localization experiment for calretinin and glutamate decarboxylase in rat supramammillary and septal neurons, demonstrated that: (i) a large population of entorhinal cells that forms asymmetric synaptic contacts on calretinin-containing neurons located at the border between the medial and lateral septal areas contains calretinin and are aspartate/glutamatergic; (ii) the overwhelming majority of calretinin-immunoreactive cells located at the border between the lateral and medial septal area are GABAergic; (iii) these neurons can be retrogradely labeled from the supramammillary area; (iv) anterogradely labeled axons originating in the border between the medial and lateral septum are GABAergic and (v) terminate on supramammillary area non-GABAergic, calretinin-containing neurons, which are known to project to the septal complex and hippocampus. These observations indicate that a large population of cells participating in the hippocampal feedback regulation of theta regulation/generation contain the same calcium-binding protein. Furthermore, entorhinal excitatory transmitter-containing neurons can depress the activity of supramammillary theta generating/regulating cells via septal inhibitory neurons.