Search

This review briefly summarizes recent developments in our understanding of the role of androgens in maintaining normal hippocampal structure. Studies in rats and vervet monkeys have demonstrated that removal of the testes reduces the density of synaptic contacts on dendritic spines of cornu ammonis 1 (CA1) pyramidal neurons. This effect is rapidly reversed by treatment with either testosterone or the non-aromatizable androgen dihydrotestosterone, suggesting that maintenance of normal synaptic density is androgen-dependent, via a mechanism that does not require intermediate estrogen biosynthesis. Similar effects of these androgens are observed in ovariectomized female rats, except that in the female the actions of testosterone include a substantial contribution from estrogen formation. The ability to stimulate hippocampal spine synapse density is not directly related to systemic androgenic potency: thus, weak androgens such as dehydroepiandrosterone exert effects that are comparable to those of dihydrotestosterone; while partial agonist responses are observed after injection of the synthetic antiandrogen, flutamide. These data provide a morphological counterpart to observations that androgens enhance cognitive function and mood state, suggesting that these effects may result at least in part from hippocampal neurotrophic responses. The unusual specificity of these responses raises the possibility that effects of androgens on the brain may be mediated via different mechanisms than the masculinizing actions of these steroids in non-neural androgen target organs.

The important effects of estrogen on the morphology of hippocampal neurons are well established. The mechanisms leading to such changes, nevertheless, have proved confusingly complex, since interactions between glia and neurons, as well as neuronal influences from other brain fields, are involved. This study addresses the possibility that estrogen-sensitive projections from the medial septum/diagonal band of Broca induce astroglial reactions. Estrogen- and cholesterol-filled (controls) cannulae were implanted into the medial septum/diagonal band of Broca of adult ovariectomized rats. Comparative semiquantitative immunohistochemical analysis on the density of the glial fibrillary acidic protein-containing processes and cells were performed on hippocampal slices of locally estrogen-treated and control animals. Rats that received estrogen-filled cannulae showed a lower density of glial processes in the hippocampal CA1 and CA3 subfields than animals of the control group. These effects could not be observed in the dentate gyrus. Cell counts revealed no significant difference in the number of glial fibrillary acidic protein-positive cells in any of the examined areas. Two major conclusions can be drawn from these results. First, the data show that estrogen, in fact, has an indirect influence on hippocampal cells through septo-hippocampal projections. Furthermore, estradiol can have an indirect negative effect on hippocampal astrocytes, causing a reduction in the density of their processes.

This study investigated the question of whether grafted dopamine cells in the striatum of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated monkeys form synapses and, if they do, whether their postsynaptic targets were the same as those in control monkeys or in previous studies in rats. Electron-microscopic single immunostaining was performed for tyrosine hydroxylase on vibratome sections prepared from the head of the caudate nucleus of controls and MPTP-treated African green monkeys (Cercopithecus aethiops sabaeus) that received a graft. Furthermore, correlated light- and electron-microscopic double immunostaining was carried out for tyrosine hydroxylase and calbindin in the same brain area of MPTP-treated plus grafted animals. In control monkeys, the majority (97%) of dopamine boutons terminate on spines that were also synaptic targets of immunonegative boutons forming asymmetric synaptic contacts: synaptic triads. In MPTP-treated, grafted animals, the majority of transplanted dopamine cells terminate on dendritic shafts (67%) and somata (32%), and only a few (1.33%) form axospine synapses. The results of the double immunostaining experiments indicated that these newly formed axosomatic and axodendritic synapses are associated with calbindin-immunoreactive, medium-sized, spiny striatonigral projection neurons. These observations indicate that: (1) dopamine from transplanted embryonic tissue acts via synaptic contacts on host neurons; (2) the primary synaptic targets of transplanted dopamine cells are not spines but dendrites and somata of host neurons; (3) these target neurons are the same as in control animals; and (4) comparing these observations with results of control and grafted rats, there are major species differences between rats and monkeys in the dopamine innervation of both control and transplanted animals.

Substance P containing neurons was visualized by immunocytochemistry in the monkey hippocampus, subicular complex, and entorhinal cortex. Immunoreactive neurons were found solely in the hilar region of the dentate gyrus, and in strata oriens and pyramidale of Ammon's horn. In the subicular complex, immunoreactive neurons were located in those layers which were close to the alveus, whereas in the entorhinal cortex most of the substance P-positive neurons appeared in the second and third layers above the lamina dissecans. The majority of substance P-containing neurons were large multipolar cells, but small bipolar and multipolar cells also occurred in Ammon's horn, subiculum and entorhinal cortex. Dendrites of immunoreactive cells were smooth and displayed a few small, faintly stained spines which were hard to identify in the light microscopic preparations, but were visible with electron microscopy. Substance P-positive dendrites were exclusively found in the hilar region and never observed in the upper two-thirds of the molecular layer of the dentate gyrus. Moreover, immunoreactive dendrites rarely penetrated the stratum lacunosum-moleculare of Ammon's horn. In the electron microscopic preparations, somal and dendritic features of substance P-positive neurons were similar to those observed for GABAergic local circuit neurons. Axons of the substance P-immunoreactive local circuit neurons were thin and richly arborized in the upper two-thirds of the molecular layer of the dentate gyrus, in the stratum lacunosum-moleculare of Ammon's horn as well as in the subpial layers of the subicular complex and entorhinal cortex. Their terminals formed exclusively symmetric synapses with dendrites and spines. However, substance P-immunoreactive boutons were not found to make symmetric, axosomatic synapses on the granule cells of the dentate gyrus and very few were present on the pyramidal neurons of Ammon's horn, subicular complex, and entorhinal cortex. Hippocampal neurons, which were immunoreactive for substance P, also contained the neuropeptide somatostatin. However, not all of the somatostatin-containing neurons were substance P-immunoreactive. Thus, substance P-positive neurons are a subpopulation of somatostatin immunoreactive, GABAergic neurons. In conclusion, substance P-immunoreactive neurons are ideally suited for feed-back dendritic inhibition which may control the effectiveness of the main excitatory cortical input to the granule cells of the dentate gyrus and pyramidal neurons of the Ammon's horn.

Our light and electron microscopic studies (Seress L., Nitsch R. and Leranth C. (1993) Neuroscience 55 , 775–796.) indicated that in the hippocampus of the African Green monkey, calretinin is exclusively present in non-pyramidal cells. Calretinin-positive axons formed a prominent band at the border of the dentate molecular and granule cell layers and in the pyramidal layer of CA2, and established asymmetric synapses with different postsynaptic targets. The goal of this study is to determine the cells of origin of this presumably extrinsic innervation, and subsequently, the characterization of their neurochemical features. We were able to demonstrate that calretinin-immunoreactive axon terminals in the inner molecular layer of the dentate gyrus and in the pyramidal layer of CA2 disappear 10 days after fimbria-fornix transection. Retrograde tracing revealed their cells of origin to be in the supramammillary nucleus. Co-localization studies employing the cryostat consecutive, semithin section technique provided evidence that these large projecting neurons contained both calretinin and substance-P but lack GABA as an inhibitory transmitter. In contrast, co-localization studies revealed that almost all of the intrinsic calretinin-positive neurons in different areas of the primate hippocampus contained GAD or GABA. These results suggest that there are two separate calretinin-containing systems in the primate hippocampus, i.e. an intrinsic inhibitory and an extrinsic excitatory one, the latter deriving from the supramammillary nucleus of the hypothalamus.

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 Ammon's 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 Ammon's 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 Ammon's 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.

Light and electron microscopic triple immunostaining was performed on coronal vibratome sections prepared from the hypothalamus of ovariectomized (OVX) and OVX plus estrogen-treated African green monkeys (Cercopithecus aethiops). Immunoreactivity for progesterone receptors (PRs) and neuropeptide-Y (NPY) was visualized by a dark blue to black nickel diaminobenzidine reaction, while the tyrosine hydroxylase-containing perikarya were labeled with a light brown diaminobenzidine reaction. In the OVX plus estrogen-treated material, 30% of the tyrosine hydroxylase-immunoreactive neurons contained PR-immunopositive nuclei. The majority of these cells were found in the central portion of the periventricular area, and a few could be observed in the anterior hypothalamus and the arcuate and dorsomedial hypothalamic nuclei. These tyrosine hydroxylase-immunoreactive PR-containing cells were surrounded with NPY-immunoreactive axon terminals. A correlated electron microscopic analysis of the same sections revealed synaptic contacts between these NPY-immunoreactive boutons and the PR-containing tyrosine hydroxylase-immunoreactive neurons. In contrast, in the OVX animals, no PR-containing tyrosine hydroxylase-immunoreactive neurons could be detected. In these monkeys, the frequency of synaptic contacts between the NPY-immunoreactive axon terminals and tyrosine hydroxylase-immunopositive cells was similar to that in the OVX plus estrogen-treated monkeys. These observations indicate that in a population of hypothalamic dopamine cells, the presence of nuclear PRs is estrogen dependent, show that these cells are innervated by NPY axons, and suggest that these estrogen-induced PR-containing dopamine neurons are involved in mediation of the effect of NPY on hypophyseal hormone secretion, including ovarian steroid hormone-dependent LH and PRL release.

Both fresh and cryopreserved-thawed human fetal ventral mesencephalon have been used for preclinical research and implantation into the brains of patients with Parkinson's disease. Further characterization and an evaluation of the effects of cryopreservation on immunocytochemical and neurochemical markers of monoamine neurons in human fetal ventral mesencephalic tissue are reported here. Fresh and cryopreserved-thawed human fetal mesencephalic tissue of 7-10 weeks fetal age was analyzed for the presence of tyrosine hydroxylase-like immunoreactivity and levels of dopamine (DA), norepinephrine, serotonin, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindole acetic acid. After fixation, cryopreserved-thawed mesencephalic tissue exhibited cellular tyrosine hydroxylase-like immunoreactivity identical to that seen in fresh tissue. The levels of DA, norepinephrine, serotonin, and 5-hydroxyindole acetic acid in the cryopreserved thawed tissue were the same as the levels in fresh tissue. The levels of DOPAC were higher, and those of HVA were lower, in the cryopreserved-thawed tissue compared to the levels in fresh tissue. The changes in the levels of the DA metabolities, DOPAC and HVA, without corresponding change in the levels of the parent monoamine in cryopreserved-thawed tissue, indicate ongoing metabolic activity in DA-containing neurons. These results further suggest that cryopreservation and subsequent thawing does not have a measurable adverse effect on DA biosynthesis in the human fetal mesencephalon. The presence of the monoamines and their metabolites in the ventral mesencephalon at 7-10 weeks of fetal age coincides well with the early presence of immunocytochemical markers of monoamine neurons and reflects the early function of the nuclear groups containing specific monoamine neurotransmitters.

Pharmacological data suggest that opiates, acting indirectly via the catecholaminergic system, are involved in the inhibition of LH release and the stimulation of PRL secretion. The aim of this study was to demonstrate on the ultrastructural level whether beta-endorphin-immunoreactive fibers form synaptic contacts with hypothalamic dopaminergic neurons. Light and electron microscopic double immunostaining experiments were performed on vibratome sections prepared from the hypothalamus of acrolein-fixed female rat brains. Immunoreactivity for beta-endorphin was visualized by a dark blue to black nickel ammonium sulfate-intensified diaminobenzidine reaction, and in a consecutive immunostaining procedure, the tyrosine hydroxylase-immunoreactive dopamine cells were labeled with the brown diaminobenzidine reaction product. Under the light microscope, beta-endorphin axon terminals were found to contact dopamine cell bodies and dendrites throughout the hypothalamus. The majority of opiate target dopamine neurons were found in the periventricular area, retrochiasmatic area, and lateral part of the zona incerta. A much smaller number was observed in the dorsomedial hypothalamic nucleus and the anterior hypothalamus, and only a very few dopamine cells could be detected in contact with beta-endorphin axons in the arcuate nucleus (particularly in the posterior part where the beta-endorphin cells are located) and the medial part of the zona incerta. After light microscopic examination and color photography, the double immunostained sections were embedded for correlated electron microscopy to verify and characterize the putative synaptic connections. Electron microscopy revealed symmetric synaptic connections between beta-endorphin-immunoreactive boutons and tyrosine hydroxylase-immunopositive cell bodies and dendrites. These results together with the observation of dopamine innervation of LHRH-producing neurons and progesterone receptor-containing cells indicate that neurons of the hypothalamic dopaminergic system probably mediate opiate effects on hypophyseal hormone secretion.

Progestin receptor-containing cells in the hypothalamus of the adult female green monkey (Cercopithecus aethiops) were examined by double-label immunocytochemical methods to determine their anatomical location, neurotransmitter content and afferent connections. Animals were ovariectomized and administered either estradiol valerate or the oil injection vehicle, and were sacrificed after 10 days of treatment. Using a monoclonal antibody raised against rabbit uterine progestin receptor (PR), the distribution of PR-immunoreactive cells in the mediobasal hypothalamus and the effect of estrogen treatment on this distribution was determined. PR-immunoreactive cells were found throughout the ventromedial nucleus (VMN), in the area between the VMN and fornix, and in the medial portion of the infundibular nucleus. Estrogen treatment dramatically increased both the number of labeled cells and the intensity of immunoreaction product in these regions. In double-immunostained sections, boutons immunoreactive for antigens indicative of serotonin, pro-opiomelanocortin derived peptides, GABA, catecholamine, neuropeptide Y, substance P, cholecystokinin, and somatostatin were demonstrated to establish synaptic contact with the soma of PR-immunoreactive hypothalamic neurons. In colchicine-pretreated animals, all PR-containing neurons in the mediobasal hypothalamus were found to contain immunoreactivity for glutamic acid decarboxylase, the enzyme required for synthesis of GABA. No evidence of colocalization with other antigens, including LHRH, was observed. Because LHRH neurons are known to receive a rich GABAergic innervation PR-containing GABAergic cells may represent steroid-sensitive sites of integration for inputs from other neural systems involved in the control of gonadotropin secretion.

Electron microscopic immunocytochemistry was combined with acute anterograde axon degeneration, following transection of the fimbria-fornix, to describe the innervation of somatospiny neurons by vasopressin-immunoreactive and degenerated hippocamposeptal axon terminals in the rat lateral septal area. Vasopressin-immunopositive boutons characterized by symmetric synaptic membrane specializations, and the degenerated hippocamposeptal axon terminals which form asymmetric synaptic contacts, frequently terminate on the same dendritic and somatic profiles, and particularly on the somata of somatospiny neurons. Although hippocamposeptal fibers predominantly form axospinous synapses in the lateral septal area, they terminate mainly on the dendritic shafts and soma of the vasopressin-receptive neurons. Of 720 vasopressin-immunoreactive terminals in the mediolateral part of the lateral septal area, 80% form synaptic contacts with dendritic shafts: 50% on small (distal) dendritic profiles and 30% on large (proximal) dendrites. Synaptic contacts between vasopressin-immunoreactive terminals and dendritic spines were not observed. The remaining 20% of immunoreactive boutons formed axosomatic synaptic contacts with a total of 58 neurons; 31% of these neurons exhibited somatic spines in the plane of the section analysed. Previous studies have demonstrated that in the lateral septal area vasopressin modulates the action of the excitatory amino acid-containing hippocamposeptal fibers, and also plays a role in the maintenance of long term potentiation evoked by fimbria-fornix stimulation. The convergent vasopressinergic and hippocampal input onto the same somatospiny neurons of the lateral septal area suggests that these neurons are targets of these physiological actions.

Neural transplantation offers the potential of treating Parkinson’s disease by grafting fetal dopamine neurons to depleted regions of the brain. However, clinical studies of neural grafting in Parkinson’s disease have produced only modest improvements. One of the main reasons for this is the low survival rate of transplanted neurons. The inadequate supply of critical neurotrophic factors in the adult brain is likely to be a major cause of early cell death and restricted outgrowth of fetal grafts placed into the mature striatum. Glial derived neurotrophic factor (GDNF) is a potent neurotrophic factor that is crucial to the survival, outgrowth and maintenance of dopamine neurons, and so is a candidate for protecting grafted fetal dopamine neurons in the adult brain. We found that implantation of adeno-associated virus type 2 encoding GDNF (AAV2-GDNF) in the normal monkey caudate nucleus induced over-expression of GDNF that persisted for at least 6 months after injection. In a 6-month within-animal controlled study, AAV2-GDNF enhanced the survival of fetal dopamine neurons by 4-fold, and increased the outgrowth of grafted fetal dopamine neurons by almost 3-fold in the caudate nucleus of MPTP-treated monkeys, compared with control grafts in the other caudate nucleus. Thus, the addition of GDNF gene therapy to neural transplantation may be a useful strategy to improve treatment for Parkinson’s disease.

The concept of estrogen-induced "sexual differentiation of the brain" has been a useful focus for investigation. However, it has become clear that the action of estrogen on the rat brain is lifelong, including effects on neurogenesis in the fetus, synaptogenesis in the newborn, and synaptic remodeling in the adult. Estrogen imparts sex differences in the rat's brain by shaping synaptology, postsynaptic membranes, and glia within the arcuate nucleus. These effects of estrogen on the arcuate nucleus also could underlie sexual maturation in both sexes and the development of senescent constant estrus in females.

This study investigated the contribution of sub-cortical afferent input to the effects of testosterone (T) on spine synapse density in the CA1 subfield of the hippocampus, in adult male rats. Gonadectomized (GDX) male rats exhibited a considerably lower density of spine synapses in the CA1 region than control, intact males. The effects of GDX were reversed by treatment with testosterone propionate (TP; 500 microg/day, for 2 days). Transection of the fimbria/fornix (FF) had no significant effect on the synaptic density in non-GDX males. However, FF transection partially inhibited the responses to TP in GDX animals. These data suggest that the effects of T on spine synapse density in the CA1 region of the male rat hippocampus are partially, but not completely, dependent on afferent sub-cortical input.

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.

Galanin (GAL) and neuropeptide Y (NPY) have been shown to play important roles in the regulation of pituitary hormone secretion, as well as ingestive and sexual behaviors, by acting within the hypothalamus. While the mechanism of action of these regulatory peptides is under intensive investigation, less attention has been paid to the possible interaction between them in influencing these central regulatory processes. Because NPY and GAL augment pituitary gonadotropin release, the present study was undertaken to evaluate the nature of morphological and functional relationships between these excitatory hypothalamic peptidergic systems. Double immunolabeling for NPY and GAL was carried out on vibratome sections taken from the hypothalamus of colchicine-pretreated female rats. Avidinbiotin peroxidase technique and a dark blue diaminobenzidine reaction was used to visualize NPY profiles, while the GAL neurons were labeled with a light brown diaminobenzidine reaction using either the avidin-biotin peroxidase or the peroxidase antiperoxidase technique. Light microscopic examination of the immunostained material showed that in the arcuate nucleus, paraventricular nucleus, supraoptic nucleus, anterior hypothalamus, and medial preoptic area, an abundant network of NPY-immunoreactive axons surrounded GAL-immunostained cells. Numerous dark blue NPY-containing putative boutons were observed in close proximity to GAL-immunolabeled cell bodies and dendrites. Correlated light and electron microscopic examination revealed that most of the immunoreactive NPY axon terminals established synaptic connections with GAL-expressing cells. Synaptic connections were most frequently found in the medial preoptic area and in the magnocellular region of the paraventricular nucleus and arcuate nucleus. Fewer connections were observed in the supraoptic nucleus. These morphological observations demonstrate the existence of a strong NPY input to hypothalamic GAL neurons, thereby suggesting a modulatory role for NPY in monitoring GAL release. To evaluate the functional relevance of this anatomical relationship, the effects of intraventricular injection of a GAL receptor antagonist, galantide, were examined on NPY-induced LH release in ovarian steroid-primed ovariectomized rats. As expected, intraventricular injection of NPY readily stimulated LH release. Although, while on its own, galantide was ineffective in altering basal LH release, it markedly attenuated the NPY-induced LH response, thereby suggesting that GAL released in response to NPY administration may, in part, mediate the excitatory effects of NPY. These experimental results, taken together with the morphological observations, document the involvement of an NPY --GAL signaling modality in the release of gonadotropins and, likewise, raise the possibility of a similar signaling process in the release of other pituitary hormones and elicitation of behavioral effects attributed to NPY and GAL.

1. Studies conducted on the rat arcuate nucleus, an area involved in the development and control of LH and FSH secretion, have shown the existence of hormonally regulated developmental sex differences in synaptic patterns and estrogen-induced synaptic plasticity during adult life. Several questions raised by these findings are examined in this review: 2. The mechanisms of estrogen-regulated developmental synaptogenesis. These include the role of glycocalyx glycoproteins in neuronal membranes, neural cell adhesion molecules, and insulin-like growth factor I. 3. The relationship among circulating estrogen, gonadotropin levels, and hypothalamic synaptic plasticity. Recent evidence for the role of GABAergic and dopaminergic synaptic inputs and POMC projections from the arcuate nucleus to the GnRH cells is discussed. 4. The synaptologic basis of age-related failure of positive feedback. The role of the cumulative effect of repeated preovulatory synaptic retraction and reapplication cycles on sensescent constant estrus is analyzed. © 1996 Plenum Publishing Corporation.

The electrophysiological observations that substance P administration to the lateral septal area elicits both excitatory and inhibitory responses, together with earlier reports on the multiple sources of substance P innervation of the septum, implies that these axons with distinct origins have different functions. This prompted us to examine the origin and neurochemical character of substance P afferents to the lateral septal area. Chronic surgical isolation of the septum from its ventral afferents and retrograde tracer experiments using wheat germ agglutinin-conjugated horseradish peroxidase, both followed by an immunostaining for substance P, were employed to elucidate the origin of these axon terminals. In order to assess the possible co-existence of substance P with other neurotransmitter substances in the parent cells of the septopetal projections, co-localization studies for substance P and choline acetyltransferase, as well as substance P and GABA, were performed. The comparative distribution of substance P fibers and septal calbindin-containing neurons was also investigated using correlated light and electron microscopic double immunostaining. The results are summarized as follows: (i) the substance P innervation of the lateral septal area derives from several hypothalamic nuclei (including the lateral and lateroanterior hypothalamic area, tuber cinereum and ventromedial hypothalamic nucleus) and tegmental nuclei (the majority of fibers from the laterodorsal and a few from the pedunculopontine tegmental nucleus), as well as intrinsic septal cells; (ii) the septopetal substance P fibers of tegmental origin are cholinergic; intraseptal substance P neurons located in the dorsolateral part of the lateral septum also contain GABA, while substance P neurons seen on the border between the medial and lateral septal area and septopetal hypothalamic substance P cells do not contain GABA or acetylcholine; (iii) substance P fibers from pericellular baskets around calbindin-containing lateral septal neurons with a high degree of selectivity; (iv) approximately 90% of the entire calbindin cell population are postsynaptic targets of substance P axons; (v) their terminals contact the soma and the dendrites of these cells, among them the somatospiny neurons; and (vi) the extrinsic substance P boutons establish asymmetric, while the intrinsic substance P axon terminals form symmetric membrane specializations. Because neurons in the lateral septal area receive hippocampal input and project massively to hypothalamic areas, the different types of substance P input on these neurons can modify the information flow arriving from the hippocampus to diencephalic brain structures at the level of the lateral septal area.

Surprisingly little is known about the synaptic architecture of the cholinergic innervation in the primate cerebral cortex in spite of its acknowledged relevance to cognitive processing and Alzheimer's disease. To address this knowledge gap, we examined serially sectioned cholinergic axons in supra- and infragranular layers of the macaque prefrontal cortex by using an antibody against the acetylcholine synthesizing enzyme, choline acetyltransferase (ChAT). The tissue bound antibody was visualized with both immunoperoxidase and silver-enhanced diaminobenzidine sulfide (SEDS) techniques. Both methods revealed that cholinergic axons make synapses in all cortical layers and that these synapses are exclusively symmetric. Cholinergic axons formed synapses primarily on dendritic shafts (70.5%), dendritic spines (25%), and, to a lesser extent, cell bodies (4.5%). Both pyramidal neurons and cells exhibiting the morphological features of GABAergic cells were targets of the cholinergic innervation. Some spiny dendritic shafts received multiple, closely spaced synapses, suggesting that a subset of pyramidal neurons may be subject to a particularly strong cholinergic influence. Analysis of synaptic incidence of cholinergic profiles in the supragranular layers of the prefrontal cortex by the SEDS technique revealed that definitive synaptic junctions were formed by 44% of the cholinergic boutons. An unexpected finding was that cholinergic boutons were frequently apposed to spines and small dendrites without making any visible synaptic specializations. These same spines and dendrites often received asymmetric synapses, presumably of thalamocortical or corticocortical origin. Present ultrastructural findings suggest that acetylcholine may have a dual modulatory effect in the neocortex: one through classical synaptic junctions on dendritic shafts and spines, and the other through nonsynaptic appositions in close vicinity to asymmetric synapses. Further physiological studies are necessary to test the hypothesis of the nonsynaptic release of acetylcholine in the cortex.

Reciprocal connections between lateral septal neurons and neurons in the lateral hypothalamus/lateral preoptic area were studied in the rat. The anterograde tracer Phaseolus vulgaris leucoagglutinin (PHA-L) and the retrograde tracer Fluoro-Gold (FG) were simultaneously injected into the lateral septum. After double-immunocytochemistry, PHA-L-labeled terminals were found in synaptic contact with dendrites of retrogradely FG-labeled neurons in the lateral hypothalamic/lateral preoptic area.

Central administration of neuropeptide-Y (NPY) inhibits pituitary LH release in ovariectomized rats and stimulates LH release in intact and ovariectomized rats pretreated with ovarian steroids. Although the precise neural mechanism of this dual effect of NPY is not known, experimental evidence suggests an underlying interaction between hypothalamic NPY and the inhibitory beta-endorphin (beta END) systems in the neuroendocrine regulation of pituitary LH release in the rat. The present study was undertaken to examine the morphological basis of the interaction between these two peptidergic systems in the hypothalamus. Sections of the mediobasal hypothalamus of colchicine-pretreated female rats were double immunostained for NPY and beta END and examined by light and electron microscopy. The light brown diaminobenzidine reaction was used to visualize beta END cells, while NPY neurons were labeled with a dark blue nickel ammonium sulfate-intensified diaminobenzidine reaction. Under the light microscope, a dense network of NPY-immunoreactive axons and axon terminals was observed in close apposition with beta END-immunoreactive neurons throughout the medial basal hypothalamus. Electron microscopic examination revealed that NPY-immunoreactive boutons formed axosomatic and axo-dendritic synaptic connections with beta END cells. A majority of these synaptic membrane specializations appeared asymmetrical [corrected]. In light of the previous evidence of excitatory and inhibitory effects on LH release and the existence of direct synaptic connections between NPY and LHRH neurons in the hypothalamus, the current results imply that the dual effects of NPY on LH secretion may involve modulation of LHRH secretion, both by the direct route and indirectly through the hypothalamic beta END system.

Electron microscopy and immunocytochemistry with a monoclonal antibody against parvalbumin (PV) were combined to analyze the distribution and morphology of PV-immunoreactive (PV-IR) neurons and the synaptology of PV-IR processes in the principal sulcus of the macaque prefrontal cortex. Parvalbumin-IR neurons are present in layers II-VI of the macaque principal sulcus (Walker's area 46) and are concentrated in a band centered around layer IV. PV-IR cells are exclusively non-pyramidal in shape and are morphologically heterogeneous with soma sizes ranging from less than 10 microns to greater than 20 microns. Well-labeled neurons that could be classified on the basis of soma size and dendritic configuration resembled large basket and chandelier cells. A novel finding is that supragranular PV-IR neurons exhibit dendritic patterns with predominantly vertical orientations, whereas infragranular cells exhibit mostly horizontal or oblique dendritic orientations. PV-IR cells within layer IV exhibit a mixture of dendritic arrangements. Vertical rows of PV-IR puncta, 15-30 microns in length, resembling the "cartridges" of chandelier cell axons were most dense in layers II, superficial III, and the granular layer IV but were not observed in the infragranular layers. Cartridges were often present beneath unlabeled, presumed pyramidal cells. PV-IR puncta also formed pericellular nests around pyramidal cell somata and proximal dendrites, suggestive of basket cell innervation. PV-IR axons were occasionally observed in the white matter underlying the principal sulcus. Electron microscopic analysis revealed that PV-IR somata and dendrites are densely innervated by nonimmunoreactive terminals forming asymmetric (Gray type I) synapses as well as by fewer terminals forming symmetric (Gray type II) synapses. The majority of terminals forming symmetric synapses with PV-IR post-synaptic structures were not immunolabeled; however, some of these boutons did contain PV-immunoreactivity. PV-IR boutons exclusively form symmetric synapses and heavily innervate layer II/III pyramidal cells. PV-IR axon cartridges formed numerous axo-axonic synapses with the axon initial segments of pyramidal cells 15-20 microns beneath the axon hillock and also terminated on large axonal spines of the initial segment. Furthermore, we failed to observe a mixture of PV-immunoreactive and non-immunoreactive boutons composing a single axon cartridge. Pyramidal cell somata and proximal dendrites were also heavily innervated by PV-IR boutons forming symmetric synapses, again, consistent with basket cell innervation. In addition, PV-IR axon terminals frequently formed symmetric synapses with dendritic shafts and spines of unidentified neurons.(ABSTRACT TRUNCATED AT 400 WORDS)

This study deals with the synaptology, morphologically identified postsynaptic targets, and origin of somatostatin (SOM) fibers in the rat lateral septal area (LSA) with special reference to those forming pericellular baskets. Septal vibratome sections were immunostained for SOM-14 in 3 experimental groups: control animals, rats subjected to a chronic transection of the ascending afferents to the septum, and animals with acute fimbria-fornix lesion. Light microscopy revealed that the SOM-immunoreactive fibers form pericellular baskets predominantly in the intermediate and ventral parts of the caudal half of the LSA. Electron microscopic analysis showed that the somatospiny neurons are postsynaptic targets of these pericellular baskets. Eight days after a unilateral cut placed at the ventral border of the septum, virtually all SOM-immunoreactive axon terminals disappeared from the ipsilateral intermediate and ventral LSA, and they were substantially reduced in the dorsal LSA. However, in these rats SOM-positive neurons could be observed in the LSA on the lesioned, but not on the contralateral side. Furthermore, on the lesion side of the anterior periventricular hypothalamus an increase was detected both in the number and the intensity of immunostaining of SOM-positive neurons. Thirty-six h following a unilateral transection of the fimbria-fornix, the SOM-immunoreactive axon terminals in the LSA remained intact; only immunonegative degenerated hippocamposeptal boutons were detected forming synaptic contacts with somatospiny neurons. Axosomatic synapses of SOM-positive boutons regularly appeared at the neck of somatic spines which were postsynaptic to degenerated hippocamposeptal fibers. The results indicate that the septal SOM fibers are of multiple origin. Those forming pericellular baskets in the LSA originate in ventral extraseptal, probably periventricular hypothalamic areas. SOM fibers scattered in the dorsal LSA are most likely processes of local SOM neurons. The accumulation of immunoreactive SOM in some cells of the undercut septum is a sign of axonal lesion, indicating that these neurons project outside the septum. The SOM innervation of somatospiny neurons which also receive hippocampal input and have been reported to contain γ-aminobutyric acid (GABA) may be a morphological substrate of the SOM-related disinhibition in the LSA.

In order to study the synaptic connections of neurons identified by their projection target and neurotransmitter content, we have adapted a method of combining retrograde tracing of horseradish peroxidase (HRP) and immunocytochemistry at the electron microscopic level. HRP was injected into the rat amygdala. Sections from the rostral forebrain were processed according to the 3,3'-diaminobenzidine/glucose oxidase reaction followed by choline acetyltransferase (ChAT) localization. Neurons in the ventral pallidum which contained both the diffuse immunoperoxidase reaction product (ChAT) and large electron dense bodies characteristic of retrogradely transported HRP were defined as double labeled, i.e. cholinergic neurons that project to the amygdaloid body.

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.

Dopamine (DA)-containing projections to the cerebral cortex are considered to play an important role in cognitive processes. Using a recently developed monoclonal antiserum directed against DA and an antibody directed against tyrosine hydroxylase in combination with Golgi impregnation and electron microscopy, we have observed that DA and tyrosine hydroxylase afferents establish symmetric membrane specializations with the soma, dendritic shafts, and spines of identified pyramidal cells in the prefrontal, cingulate, and motor cortex of primates. The axospinous contacts invariably formed part of a synaptic complex in which the dendritic spine of a pyramidal neuron was the target of both a DA-positive symmetric and an unlabeled asymmetric bouton. This arrangement allows direct DA modulation of the overall excitability of cortical projection neurons by altering local spine responses to excitatory inputs.

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.

Double immunostaining was performed for electron microscopy to analyze the synaptic connections between glutamate decarboxylase (GAD)-immunoreactive axons and cholecystokinin (CCK)-immunoreactive neurons in the hilar region of the rat hippocampal formation. Following immunostaining for CCK, the diaminobenzidine (DAB) reaction product was silver-intensified and gold-substituted. In a subsequent second immunostaining for GAD, the immunoreactive elements were labeled using a single DAB reaction. Electron microscopic analysis of the double-stained Vibratome sections demonstrated that the single DAB-labeled GAD-immunoreactive boutons form symmetrical synaptic connections on the soma and primary dendrites of the DAB-gold-labeled CCK-immunoreactive neurons.

Insulin (INS) specific binding sites were detected in the myometrium of pregnant rats. The apparent equilibrium dissociation constants for the higher and lower affinity INS binding components were 0.01 X 10(-9) M and 2.5 X 10(-9) M, respectively. During the latter half of pregnancy INS binding increased gradually and peaked on the day of parturition (159.9 +/- 19.8, 196.3 +/- 24.6, 254.7 +/- 36.8, and 272.6 +/- 29.1 and femtomolars per mg protein, mean +/- SEM on days 15, 20, 21, and 22 of gestation, respectively). The pattern of increased INS binding paralleled the prepartum increase in myometrial cell nuclear estrogen receptors as well as myometrial glycogen levels. In contrast, low INS binding was associated with elevated cell nuclear progestin receptor levels, on day 15 and immediately postpartum. These results suggest that, during the latter half of pregnancy, myometrial INS binding capacity and glycogen concentration gradually increase. Since INS receptor levels begin to increase in parallel with increasing nuclear estrogen receptors, INS receptor synthesis may be under estrogen control.

Electronmicroscopic immunocytochemical analysis of the types and patterns of synaptic contacts formed by cholecystokinin (CCK)-containing terminals in the CA1 and CA3 region of the rat and monkey hippocampus reveals numerous symmetric synaptic contacts on cell bodies and dendritic shafts of pyramidal cells in both species. In the monkey, however, CCK-immunoreactive terminals also form asymmetric synaptic contacts with dendritic spines, such contacts are absent or very rare in the rat hippocampus. The present finding in primate hippocampus provides evidence that the same neuropeptides can be found in both symmetric and asymmetric contacts and may be added to other evidence challenging the traditional concept that symmetric synapses mediate exclusively inhibitory and asymmetric exclusively excitatory transmission. Furthermore, although our comparative analysis confirms considerable similarities in the distribution of CCK-containing elements in primate and rodent hippocampus it also revealed a potentially important difference in synaptoarchitecture that should be taken into account in extrapolations from one species to the other.

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.

A pre-embedding immunostaining procedure was developed using ferritin and peroxidase to enable simultaneous electron microscopic localization of two antigens in the same tissue section. This method was used to study the anatomic relationship between glutamic acid decarboxylase (GAD) immunoreactive axons and tyrosine hydroxylase (TH) — containing neurons of the rat arcuate nucleus. The findings provide ultrastructural evidence that GAD-immunoreactive terminals establish symmetric (Gray II) synapses on TH-reactive neurons.

Amygdalopetal cholinergic neurons in the ventral pallidum were identified by combining choline acetyltransferase (ChAT) immunohistochemistry with retrograde tracing of horseradish peroxidase (HRP) following injections of the tracer in the basolateral amygdaloid nucleus. Although ChAT-positive terminals were identified in the ventral pallidum, they were never seen in contact with either immunonegative or ChAT-positive amygdalopetal neurons. In material, in which immunostaining against glutamic acid decarboxylase (GAD), the synthesizing enzyme for GABA was combined with retrograde tracing of HRP from the basolateral amygdaloid nucleus, GAD-positive terminals were seen to contact immunonegative amygdalopetal neurons. In addition, when sections of the rostral forebrain were processed, first to preserve and identify the transported HRP, and then were sequentially tested for both ChAT and GAD immunohistochemistry with the immunoperoxidase reaction for both tissue antigens, GAD-immunopositive terminals were seen to make synaptic contacts with cholinergic amygdalopetal neurons. These results suggest that amygdalopetal, presumably cholinergic, neurons receive GAD-positive terminals. In separate experiments using immunoperoxidase for ChAT and ferritin-avidin for GAD labeling, we confirmed the presence of GAD-containing terminals on cholinergic neurons. In addition, cholinergic terminals were seen in synaptic contact with GAD-positive cell bodies. These morphological studies suggest that direct GABAergic-cholinergic and cholinergic-GABAergic interactions take place in the rostral forebrain.

In spite of partial success in treating Parkinson's disease by using ectopically placed grafts of dopamine-producing cells, restoration of the original neuroanatomical circuits, if possible, might work better. Previous evidence of normal anatomic projections from ventral mesencephalic (VM) grafts placed in the substantia nigra (SN) has been limited to neonatal rodents and double grafting or bridging procedures. This study attempted to determine whether injection of a potent growth-promoting factor, glial cell line-derived neurotrophic factor (GDNF), into the target regions or placement of fetal striatal co-grafts in the nigrostriatal pathway might elicit neuritic outgrowth to the caudate nucleus. Four adult St. Kitts green monkeys received embryonic VM grafts into the rostral mesencephalon near the host SN, and injections of adeno-associated virus 2 (AAV2)/GDNF or equine infectious anemia virus (EIAV)/GDNF into the caudate. Three adult monkeys were co-grafted with fetal VM tissue near the SN and fetal striatal grafts (STR) 2.5 mm rostral in the nigrostriatal pathway. Before sacrifice, the striatal target regions were injected with the retrograde tracer Fluoro-Gold (FG). FG label was found in tyrosine hydroxylase-labeled neurons in VM grafts in the SN of only those monkeys that received AAV2/GDNF vector injections into the ipsilateral striatum. All monkeys showed FG labeling in the host SN when FG labeling was injected on the same side. These data show that grafted dopaminergic neurons can extend neurites to a distant target releasing an elevated concentration of GDNF, and suggest that grafted neurons can be placed into appropriate loci for potential tract reconstruction. Copyright 2009 Wiley-Liss, Inc.