Cryopreservation, culture, and transplantation of human fetal mesencephalic tissue into monkeys.

Studies in animals suggest that fetal neural grafts might restore lost neurological function in Parkinson's disease. In monkeys, such grafts survive for many months and reverse signs of parkinsonism, without attendant graft rejection. The successful and reliable application of a similar transplantation procedure to human patients, however, will require neural tissue obtained from human fetal cadavers, with demonstrated cellular identity, viability, and biological safety. In this report, human fetal neural tissue was successfully grafted into the brains of monkeys. Neural tissue was collected from human fetal cadavers after 9 to 12 weeks of gestation and cryopreserved in liquid nitrogen. Viability after up to 2 months of storage was demonstrated by cell culture and by transplantation into monkeys. Cryopreservation and storage of human fetal neural tissue would allow formation of a tissue bank. The stored cells could then be specifically tested to assure their cellular identity, viability, and bacteriological and virological safety before clinical use. The capacity to collect and maintain viable human fetal neural tissue would also facilitate research efforts to understand the development and function of the human brain and provide opportunities to study neurological diseases.

Androgen modulation of hippocampal synaptic plasticity.

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.

Synaptic remodeling induced by gonadal hormones: neuronal plasticity as a mediator of neuroendocrine and behavioral responses to steroids.

During recent decades, it has become a generally accepted view that structural neuroplasticity is remarkably involved in the functional adaptation of the CNS. Thus, cellular morphology in the brain is in continuous transition throughout the life span, as a response to environmental stimuli. The effects of the environment on neuroplasticity are mediated by, to some extent, the changing levels of circulating gonadal steroid hormones. Today, it is clear that the function of gonadal steroids in the brain extends beyond simply regulating reproductive and/or neuroendocrine events. In addition, or even more importantly, gonadal steroids participate in the shaping of the developing brain, while their actions during adult life are implicated in higher brain functions such as cognition, mood and memory. A large body of evidence indicates that gonadal steroid-induced functional changes are accompanied by alterations in neuron and synapse numbers, as well as in dendritic and synaptic morphology. These structural modifications are believed to serve as a morphological basis for changes in behavior and cellular activity. Due to their growing functional and clinical significance, the specificity, timeframe, as well as the molecular and cellular mechanisms of hormone-induced neuroplasticity have become the focus of many studies. In this review, we briefly summarize current knowledge and the most significant recent discoveries from our laboratories on estrogen- and dehydroepiandrosterone-induced synaptic remodeling in the hypothalamus and hippocampus, two important brain areas heavily involved in autonomic and cognitive operations, respectively.

Cholinergic excitation of septohippocampal GABA but not cholinergic neurons: implications for learning and memory.

The medial septum/diagonal band (MSDB), which gives rise to the septohippocampal pathway, is a critical locus for the mnemonic effects of muscarinic drugs. Infusion of muscarinic cholinergic agonists into the MSDB enhance learning and memory processes both in young and aged rats and produce a continuous theta rhythm in the hippocampus. Intraseptal muscarinic agonists also alleviate the amnesic syndrome produced by systemic administration of muscarinic receptor antagonists. It has been presumed, but not proven, that the cellular mechanisms underlying the effects of muscarinic agonists in the MSDB involve an excitation of septohippocampal cholinergic neurons and a subsequent increase in acetylcholine (ACh) release in the hippocampus. Using a novel fluorescent labeling technique to selectively visualize live septohippocampal cholinergic neurons in rat brain slices, we have found that muscarinic agonists do not excite septohippocampal cholinergic neurons, instead they inhibit a subpopulation of cholinergic neurons. In contrast, unlabeled neurons, confirmed to be noncholinergic, septohippocampal GABA-type neurons using retrograde marking and double-labeling techniques, are profoundly excited by muscarine. Thus, the cognition-enhancing effects of muscarinic drugs in the MSDB cannot be attributed to an increase in hippocampal ACh release. Instead, disinhibitory mechanisms, caused by increased impulse flow in the septohippocampal GABAergic pathway, may underlie the cognition-enhancing effects of muscarinic agonists.

Opioids suppress IPSCs in neurons of the rat medial septum/diagonal band of Broca: involvement of mu-opioid receptors and septohippocampal GABAergic neurons.

The medial septum/diagonal band region (MSDB), which provides a major cholinergic and GABAergic input to the hippocampus, expresses a high density of opioid receptors. Behaviorally, intraseptal injections of opioids produce deficits in spatial memory, however, little is known about the electrophysiological effects of opioids on MSDB neurons. Therefore, we investigated the electrophysiological effects of opioids on neurons of the MSDB using rat brain slices. In voltage-clamp recordings with patch electrodes, bath-applied met-enkephalin, a nonselective opioid receptor agonist, decreased the number of tetrodotoxin and bicuculline-sensitive inhibitory synaptic currents in cholinergic- and GABA-type MSDB neurons. A similar effect occurred in brain slices containing only the MSDB, suggesting that opioids decrease GABA release primarily by inhibiting spontaneously firing GABAergic neurons located within the MSDB. Accordingly, in extracellular recordings, opioid-sensitive, spontaneously firing neurons could be found within the MSDB. Additionally, in intracellular recordings a subpopulation of GABA-type neurons were directly inhibited by opioids. All effects of met-enkephalin were mimicked by a mu receptor agonist, but not by delta or kappa agonists. In antidromic activation studies, mu-opioids inhibited a subpopulation of septohippocampal neurons with high conduction velocity fibers, suggestive of thickly myelinated GABAergic fibers. Consistent with the electrophysiological findings, in double-immunolabeling studies, 20% of parvalbumin-containing septohippocampal GABA neurons colocalized the mu receptor, which at the ultrastructural level, was found to be associated with the neuronal cell membrane. Thus, opioids, via mu receptors, inhibit a subpopulation of MSDB GABAergic neurons that not only make local connections with both cholinergic and noncholinergic-type MSDB neurons, but also project to the hippocampus.

Estrogen is essential for maintaining nigrostriatal dopamine neurons in primates: implications for Parkinson's disease and memory.

There are sexual differences in several parameters of the nigrostriatal dopamine neurons, as well as in the progression of diseases associated with this system, e.g., Parkinson's disease and dementia. These differences, as well as direct experimental data in rodents, suggest that gonadal hormones play a role in modulating this system. To determine whether circulating estrogen might have long-term effects by altering the number of dopamine neurons, the density of dopamine neurons was calculated in the compact zone of the substantia nigra of male and intact female short- (10 d) and longer-term (30 d) ovariectomized and short- and longer-term ovariectomized but estrogen-replaced nonhuman primates (African green monkeys). Furthermore, the number of tyrosine hydroxylase-expressing neurons, the total number of all types of neurons, and the volume of the compact zone of the substantia nigra were calculated in 30 d ovariectomized and in 30 d ovariectomized and estrogen-replaced monkeys. Unbiased stereological analyses demonstrated that a 30 d estrogen deprivation results in an apparently permanent loss of >30% of the total number of substantia nigra dopamine cells. Furthermore, the density calculations showed that brief estrogen replacement restores the density of tyrosine hydroxylase-immunoreactive cells after a 10 d, but not after a 30 d, ovariectomy. Moreover, the density of dopamine cells is higher in females than in males. These observations show the essential role of estrogen in maintaining the integrity of the nigral dopamine system, suggest a new treatment strategy for patients with Parkinson's disease and with certain forms of memory-impairing disorders, and provide another rationale for estrogen replacement therapy for postmenopausal women.

Muscarinic tone sustains impulse flow in the septohippocampal GABA but not cholinergic pathway: implications for learning and memory.

Systemic infusions of the muscarinic cholinergic receptor antagonists atropine and scopolamine (atr/scop) produce an amnesic syndrome in humans, subhuman primates, and rodents. In humans, this syndrome may resemble early symptoms of Alzheimer's disease. Behavioral studies in rats have demonstrated that the medial septum/diagonal band of Broca (MSDB), which sends cholinergic and GABAergic projections to the hippocampus, is a critical locus in mediating the amnesic effects of atr/scop. The amnesic effects of atr/scop in the MSDB have been presumed but not proven to be caused by a decrease in hippocampal acetylcholine (ACh) release after blockade of a muscarinic tone in the MSDB. Using electrophysiological recordings and fluorescent-labeling techniques to identify living septohippocampal neurons in rat brain slices, we now report that, contrary to current belief, a blockade of the muscarinic tone in the MSDB does not decrease impulse flow in the septohippocampal cholinergic pathway; instead, it decreases impulse flow in the septohippocampal GABAergic pathway via M(3) muscarinic receptors. We also report that the muscarinic tone in the MSDB is maintained by ACh that is released locally, presumably via axon collaterals of septohippocampal cholinergic neurons. As such, cognitive deficits that occur in various neurodegenerative disorders that are associated with a loss or atrophy of septohippocampal cholinergic neurons cannot be attributed solely to a decrease in hippocampal acetylcholine release. An additional, possibly more important mechanism may be the concomitant decrease in septohippocampal GABA release and a subsequent disruption in disinhibitory mechanisms in the hippocampus. Restoration of impulse flow in the septohippocampal GABA pathway, possibly via M(3) receptor agonists, may, therefore, be critical for successful treatment of cognitive deficits associated with neurodegenerative disorders such as Alzheimer's and Parkinson's disease.

Adenovirus-mediated transgene expression in nonhuman primate brain.

Transgene expression in the brain of St. Kitts green monkey, Cercopithecus aethiops sabeus, was studied following injection of a serotype 5 adenoviral vector deleted in E1 and E3. The vector harbored the transgene for Escherichia coli beta-galactosidase (beta-Gal) with the simian virus 40 (SV40) nuclear localization signal under control of the Rous sarcoma viral (RSV) long terminal repeat. Several titers ranging from 5 x 10(7) to 2 x 10(9) plaque-forming units (PFU) in volumes ranging from 5 to 250 microl were injected into the caudate nuclei of 18 monkeys. Monkeys were treated with dexamethasone for 9 days, beginning the day prior to surgery, and were sacrificed at 1 week or at 1, 2, or 3 months. At 1 week, beta-Gal was expressed in thousands of cells, including both neurons and astrocytes. In addition, some dopaminergic neurons in the substantia nigra expressed transgene, suggesting retrograde transport of the vector. At 1 month 162,000+/-68,000 (SEM) or 65,000+/-29,000 beta-Gal-expressing cells persisted in striatum injected with 6 x 10(8) PFU in 30 microl or 5 x 10(7) PFU in 5 microl, respectively. Transgene expression was also observed in one of two monkeys sacrificed at 2 months and in a single monkey sacrificed at 3 months. No transgene expression was observed at 1 month in striatum injected with a higher titer (2 x 10(9) PFU in 100 microl) or more dilute vector (5 x 10(7) PFU in 30 microl). Staining for the major histocompatibility complex II (MHC II) subtype DR showed intense staining in sites injected with a higher vector titer, in which no transgene persisted at 1 month, whereas low to moderate staining was present in sites with high transgene expression. These observations suggest that there is an optimal range of vector titers for obtaining persistent transgene expression from E1E3-deleted adenovirus in primate brain, above which host responses limit transgene stability.

Beta-endorphin innervation of dopamine neurons in the rat hypothalamus: a light and electron microscopic double immunostaining study.

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.

Insulin specific binding sites in the myometrium of pregnant rats.

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.

Neuropeptide-Y innervation of beta-endorphin-containing cells in the rat mediobasal hypothalamus: a light and electron microscopic double immunostaining analysis.

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.

Neuropeptide-Y innervation of estrogen-induced progesterone receptor-containing dopamine cells in the monkey hypothalamus: a triple labeling light and electron microscopic study.

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.

Role of neuropeptide-Y in episodic luteinizing hormone release in ovariectomized rats: an excitatory component and opioid involvement.

We tested the hypothesis that hypothalamic neuropeptide-Y (NPY) is an excitatory signal in the episodic secretion of LH in ovariectomized (ovx) rats and that the suppression of LH secretion that consistently follows intracerebroventricular administration of NPY is due to concurrent release of opioids or CRH, both previously shown to readily inhibit LH release. In the first experiment, ovx rats received continuous intraventricular infusion of either serum containing NPY antibodies (NPY-Ab) or normal rabbit serum (control) at dilutions of 1:5 or 1:1. NPY-Ab infusion at a 1:5 dilution significantly decreased mean plasma LH levels and LH pulse amplitude without affecting LH pulse frequency over a 3-h period of observation. However, infusion of relatively more concentrated NPY-Ab (1:1) markedly decreased not only mean plasma LH levels and LH pulse amplitude, but also the frequency of LH episodes. In the next experiment, we observed that intraventricular administration of NPY (0.2 nmol) suppressed LH release for 60 min. However, blockade of opiate receptors with iv infusion of naloxone (2 mg/h) before and after NPY injection completely counteracted the NPY-induced inhibition of LH release. On the other hand, prior blockade of the CRH receptors with alpha-helical CRH-(9-41) (25 or 100 micrograms/rat) was ineffective in reversing the inhibitory LH response of NPY (0.125 nmol). These results together with our previous demonstration of morphological communication between NPY and beta-endorphin neurons, show that suppression of LH by exogenous NPY in ovx rats may result from concurrent stimulation of opioids, primarily beta-endorphin. However, diminution of all parameters of episodic LH secretion by NPY-Ab affirms the notion that the NPY network is a physiologically important excitatory component of the hypothalamic pulse generator circuitry that regulates episodic LH secretion in rats.

Morphological and pharmacological evidence for neuropeptide Y-galanin interaction in the rat hypothalamus.

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.

Serotonergic innervation of the rat pituitary intermediate lobe: decrease after stalk section.

Serotonin (5-HT)-containing nerve fibers and terminals, but not cell bodies, have been demonstrated by light and electron microscopic immunocytochemistry in the intermediate lobe of the rat pituitary. Seven days after pituitary stalk transection, 5-HT immunoreactive fibers disappeared almost entirely from the intermediate lobe. Intermediate lobe 5-HT and 5-hydroxyindoleacetic acid concentrations, measured by high pressure liquid chromatography coupled to electrochemical detection, fell to 56% and 62% of control, respectively. There were no changes in 5-HT concentrations in anterior or posterior lobes of the pituitary or in the median eminence. These findings, and the failure of sympathectomy to cause a drop in pars intermedia 5-HT, indicate that in the intermediate lobe of the pituitary neuronal 5-HT originates in cells situated in the central nervous system.

Immunohistochemical localization of aromatase cytochrome P-450 and estradiol dehydrogenase in the syncytiotrophoblast of the human placenta.

Immunohistochemistry employing immunoglobulin G fractions raised against aromatase cytochrome P-450 and antiserum against 17 beta-estradiol dehydrogenase was used to localize these two steroid-converting enzymes in the human placenta. Immunostaining for both enzymes was found exclusively in the syncytiotrophoblast, while the underlying cytotrophoblast and the villus core did not stain. Ultrastructural examination of aromatase cytochrome P-450- and 17 beta-estradiol dehydrogenase-labeled sections disclosed immunoreactive product in the membranes of the endoplasmic reticulum; the nucleus, mitochondria, Golgi apparatus, and secretory granules were free of staining. These findings suggest that the syncytiotrophoblast is actively involved in the synthesis and metabolism of estrogens and in their role in placental endocrine function.

Psychiatric status after human fetal mesencephalic tissue transplantation in Parkinson's disease.

This report describes the prospective and systematic psychiatric assessment of nine patients who received transplantation of human fetal mesencephalic tissue into the caudate nucleus for treatment of Parkinson's disease. Unlike adrenal medullary transplantation, which often causes psychosis or delirium, this procedure appeared to have few perioperative sequelae. On longer-term follow-up, there was some statistical evidence of deterioration in psychiatric status, as manifested primarily in depressive and nonspecific emotional and behavioral symptoms. This group effect was partly attributable to the occurrence of discrete episodes of illness (major depression and panic disorder with agoraphobia) in some patients, but it was unclear whether such episodes occurred more often than would ordinarily be expected in Parkinson's disease. Differences in the neurobiological effects of fetal mesencephalic and adrenal medullary grafts may account for differences in the psychiatric sequelae of patients receiving these procedures.

Neuropeptide Y (NPY)-immunoreactive neurons in the primate fascia dentata; occasional coexistence with calcium-binding proteins: a light and electron microscopic study.

Neuropeptide Y (NPY)-containing neurons are known to be highly vulnerable following sustained electrical stimulation in rats and in humans suffering from temporal lobe epilepsy. This has been related to a strong excitatory input. In contrast, there is evidence that neurons containing calcium-binding proteins exhibit a high resistance under experimental seizure and hypoxia conditions. The aim of this study was to determine the coexistence of NPY and calcium-binding proteins in inhibitory neurons of the primate fascia dentata and their synaptic connections. Vibratome sections of hippocampi of African green monkeys (Cercopithecus aethiops) were immunostained with antibodies against NPY, PARV, and CB. A quantitative coexistence study was performed for NPY and PARV on consecutive semithin sections. In contrast to the rodent hippocampus, NPY-immunoreactive neurons were found exclusively in the hilus of fascia dentata with horizontally oriented dendrites which did not extend into the granular and molecular layer. Conversely, PARV-immunoreactive neurons were also present in the granular and inner molecular layer and extended their dendrites far out in the molecular layer and the hilus. Axon terminals immunoreactive for NPY were mostly concentrated in the middle and outer molecular layer and the hilar region and were rare in the granular layer. PARV-immunoreactive boutons were basically restricted to the granular layer where they formed typical baskets. The antibody against calbindin stained almost exclusively granule cells. Coexistence of NPY- and PARV-immunoreactivity was found only in hilar neurons and was rare (9 out of 152 cells analyzed). These results suggest that most NPY-immunoreactive neurons do not contain calcium-binding proteins. NPY-containing neurons exhibited ultrastructural characteristics as described for inhibitory neurons. Their dendrites were only sparsely contacted by mostly asymmetric synaptic terminals, including a very small number of mossy fiber axon terminals. In turn, numerous NPY-immunoreactive axon terminals formed symmetric synapses with spines and dendritic shafts of unlabeled neurons in the middle and outer molecular layer, whereas no contact with granule cell bodies was evident. Thus, we conclude that the vulnerability of NPY-containing inhibitory neurons may be due more to the lack of calcium-binding proteins than to a strong excitatory innervation. As their axons may contribute to the inhibitory control of the major excitatory input from the entorhinal cortex, their loss following overstimulation may play a role in perpetuating hippocampal seizure activity.

Cholinergic innervation of hippocampal GAD- and somatostatin-immunoreactive commissural neurons.

This study describes the cholinergic innervation of chemically defined nonpyramidal neurons in the hilar region of the rat hippocampus. Cholinergic terminals were identified by immunocytochemistry employing a monoclonal antibody against choline acetyltransferase (ChAT), the acetylcholine-synthesizing enzyme, and the avidin-biotin-peroxidase (ABC) technique. Nonpyramidal neurons in the hilar region were characterized by immunostaining with antibodies against glutamate decarboxylase (GAD), the gamma aminobutyric acid (GABA)-synthesizing enzyme, and somatostatin (SS). The immunoreactivity to these antibodies was detected by using biotinylated secondary antibodies and avidinated ferritin as an electron-dense marker. This electron microscopic double immunostaining procedure enabled us to demonstrate that immunoperoxidase-labeled ChAT-immunoreactive terminals established symmetric synaptic contacts on the ferritin-labeled GAD- and SS-immunoreactive hilar cells. In additional experiments at least some of the GAD- and SS-immunoreactive hilar neurons were further characterized as commissural neurons by retrograde filling with horseradish peroxidase (HRP) following an injection of the tracer into the contralateral hilus. From these triple labeling experiments, we concluded that at least some GABAergic and somatostatin-containing neurons in the hilar region, which are postsynaptic to cholinergic terminals, project to the contralateral hippocampus. Together with previous studies on the cholinergic innervation of the hippocampus and fascia dentata, our present results thus demonstrate that different types of hippocampal cells, including GABAergic and peptidergic commissural neurons in the hilar region, receive a cholinergic input.

Substance P innervation of the rat hippocampal formation.

Light and electron microscopic substance P (SP) immunostaining was performed on hippocampal sections of colchicine-pretreated, control, untreated fimbria-fornix-transected (5 days), as well as perforant path-stimulated Sprague-Dawley rats fixed in 5% acrolein. Numerous SP-immunoreactive neurons could be observed in the stratum oriens of the Ammon's horn and subiculum, fewer were seen in the dentate hilar area and stratum radiatum of CA2 and CA3, and even fewer were seen at the border between the CA1 strata radiatum and the lacunosum moleculare of CA1 subfield. A higher dose of colchicine resulted in SP immunoreactivity in a large population of granule cells and mossy axon terminals. The entire CA2 region, the stratum oriens of CA1, CA3, and the subiculum were densely innervated by SP-containing axon terminals. A homogeneous SP innervation was found in the stratum radiatum of CA1. Only a few SP fibers were seen adjacent to the granule cell layer. Symmetric axosomatic contacts were seen between SP-containing boutons and somata in the stratum oriens of the Ammon's horn. However, throughout the hippocampal formation, the majority of SP-containing axons formed axodendritic symmetric synapses. A dense population of SP-immunoreactive boutons that formed axodendritic asymmetric synapses was observed in the strata oriens and radiatum of the CA3a and CA2 regions, and a few were found in the supragranular and subgranular layers of the dentate gyrus. Fimbria-fornix transection resulted in a marked loss of SP fibers in the strata oriens, pyramidale, and radiatum of the CA3a and CA2 subfields. In perforant pathway-stimulated animals, a population of granule cells and a large number of mossy axon terminals were immunoreactive for SP. These observations suggest two sources of SP innervation to the hippocampal formation: one arising from intrinsic sources (interneurons and granule cells) and one arising from extrinsic sources, most likely the supramammillary region.