Embryonic substantia nigra grafts in the mesencephalon send neurites to the host striatum in non-human primate after overexpression of GDNF.

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.

AAV2-mediated gene transfer of GDNF to the striatum of MPTP monkeys enhances the survival and outgrowth of co-implanted fetal dopamine neurons.

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 overexpression 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.

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.

Effects of testosterone on hippocampal CA1 spine synaptic density in the male rat are inhibited by fimbria/fornix transection.

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.

Gonadal hormones act extrinsic to the hippocampus to influence the density of hippocampal astroglial processes.

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.

Supramammillary area mediates subcortical estrogenic action on hippocampal synaptic plasticity.

It is well established that systemically administered estrogen to ovariectomized rats positively affects the density of pyramidal cell spines in the hippocampal CA1 subfield and intact subcortical connections of the hippocampus are essential in this hormonal action. This study explored whether local estrogen administration into the supramammillary area influences the density of CA1 pyramidal cell spine synapses in ovariectomized rats. The first group of experiments using a combination of retrograde tracer technique and immunostaining for estrogen receptor-alpha demonstrated that a large population of supramammillary area estrogen receptor-alpha-containing neurons projects to the hippocampus. Animals belonging to the second experimental group were ovariectomized and received cannulae filled with 0.4% 17 beta-estradiol placed unilaterally into the supramammillary area. Control animals received a cholesterol-containing cannula into the supramammillary area or an estrogen-filled cannula implanted into the head of the caudate nucleus. One week later, rats were killed and CA1 pyramidal cell spine synapse density was determined using electron microscopic unbiased stereological procedures. Animals that received an estrogen-filled cannula into the supramammillary area exhibited a significantly higher (37%) density of CA1 pyramidal cell spine synapses than both other control groups. These observations indicate that the supramammillary area is involved in mediating synaptoplastic, estrogenic effects 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.

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.

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.

The transentorhinal cortex of the African green monkey: a combined light- and electron-microscopic study of calcium-binding protein containing neurons.

The transentorhinal cortex (TEC) is a primate-specific transition zone between the entorhinal allocortex and the temporal isocortex. Neurons in the lamina pre-alpha of TEC are known to be the first to develop intraneuronal changes in the course of Alzheimer's disease. In order to shed light on this important feature, we studied as yet unknown morphological and neurochemical characteristics of the TEC of the African green monkey (Cercopithecus aethiops sabaeus). Using light- and electron-microscopic immunocytochemistry, the distribution and morphology of neurons containing calcium-binding proteins were described and compared with those in the adjacent cortices. Light-microscopic analysis revealed that parvalbumin-containing neurons were distributed in all cortical layers. Calbindin-containing cells were fewer but also present in each layer. Calretinin-containing neurons were largely confined to the upper layers of the TEC. All three types of neuron showed pyramidal-like, multipolar and bipolar shapes; their dendrites were smooth or beaded. Ultrastructural studies revealed immunopositive somata with infolded nuclei and large amounts of cytoplasm. The somata were only sparsely innervated by symmetric synapses. Immunopositive dendrites were almost exclusively covered with immunonegative axon terminals establishing symmetric and asymmetric synapses. Immunopositive terminals established symmetric contacts with immunonegative dendrites and somata. Only occasionally, could synaptic contacts between immunopositive pre- and postsynaptic structures be observed. The comparison of neurons in the TEC and adjacent cortices revealed no striking differences. In summary, the morphological and neurochemical characteristics of TEC neurons as analyzed in our study do not provide an explanation for the early onset of neurodegenerative changes in the TEC.

AMPA receptors colocalize with neuropeptide-Y- and galanin-containing, but not with dopamine, neurons of the female rat arcuate nucleus: a semiquantitative immunohistochemical colocalization study.

It is well established that excitatory amino acid (EAA) neurotransmission is an essential component in the regulation of the gonadotropin-releasing hormone (GnRH) delivery system. However, the morphological interconnection of these systems is not fully understood. The objective of the present study was to determine whether or not alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors--as indicators of aspartate/glutamatergic innervation--are present in the major neuronal populations, such as the neuropeptide-Y-(NPY), galanin- (GAL) and tyrosine-hydroxylase- (TH) containing neurons of the arcuate nucleus (AN) of the female rat. Colocalization experiments using the "mirror" technique demonstrated that: (1) AN neurons containing GluR1 are also immunoreactive (IR) for GluR2/3; (2) 38.32% of AMPA-IR cells contain NPY and 31.72% of AMPA-containing neurons are also IR for GAL; in turn, 79.41% of NPY- and 56.19% of GAL-containing neurons are IR for AMPA receptors; none of the neurons are IR for both AMPA receptors and TH. These data suggest that an excitatory aspartate/glutamatergic input is implicated in the regulation of the examined neuropeptide-containing AN neurons but not in that of TH-IR cells of the same area.

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

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

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.

Inflammatory responses and their impact on beta-galactosidase transgene expression following adenovirus vector delivery to the primate caudate nucleus.

An E1, E3 deleted adenovirus vector, serotype 5, carrying the marker gene LacZ was bilaterally microinfused into the caudate nuclei of 10 St Kitts green monkeys. The location and number of cells expressing transgene and host immunologic response were evaluated at 1 week (n = 2) and 1 month (n = 8) following vector infusion. A large number of cells expressed beta-galactosidase in some monkeys, exceeding 600000 in one monkey, but no expression was seen in three of 10. All monkeys had positive adenoviral antibody titers before vector infusion, indicating the possibility of previous exposure to some adenovirus, but only one showed a significant increase in titer afterwards. Inflammatory cell markers revealed an inverse correlation between transgene expression and the extent of inflammatory response. Dexamethasone administered immediately before and for 8 days following vector delivery, however, had no effect on transgene expression. The demonstration of significant inflammatory responses in the brain of some individual primates, including demyelination, indicates the need for new generations of adenovirus vectors, or the successful suppression of inflammatory responses, before this vector is suitable for non-cytotoxic clinical applications in the CNS.

Estrogen receptor-alpha in the raphe serotonergic and supramammillary area calretinin-containing neurons of the female rat.

It is well established that estrogen affects hippocampal long-term potentiation and hippocampus-related memory processes. Furthermore, theta rhythm, in conjunction with long-term potentiation, influences memory and is regulated by subcortical structures, including the median raphe and supramammillary area. To test the validity of the hypothesis that the effects of estrogen on the hippocampus are mediated, at least partly, via these subcortical structures, it must first be determined whether the neurons of the median raphe and supramammillary area contain estrogen receptors. Light and electron microscopic double immunostaining for estrogen receptor-alpha plus serotonin and estrogen receptor-alpha plus calretinin on vibratome sections of the median raphe and supramammillary area, respectively, demonstrated that large populations of the median raphe serotonin and supramammillary area calretinin neurons exhibit estrogen receptor-immunoreactive nuclei. These observations indicate that circulating gonadal hormones can affect hippocampal electric activity indirectly, via those subcortical structures that are involved in theta rhythm regulation.

Median raphe serotonergic innervation of medial septum/diagonal band of broca (MSDB) parvalbumin-containing neurons: possible involvement of the MSDB in the desynchronization of the hippocampal EEG.

Activation of median raphe serotonergic neurons results in the desynchronization of hippocampal electroencephalographic (EEG) activity. This could be a direct effect, because serotonin (5-HT) fibers terminate on a specific population of hippocampal interneurons. On the other hand, it could be an indirect action through the medial septum/diagonal band of Broca (MSDB) pacemaker cells, because, in addition to previously described inhibitory effects, excitatory actions of 5-HT have been demonstrated on MSDB gamma-aminobutyric acid (GABA)-containing neurons through 5-HT2A receptors. Electron microscopic double immunostaining for Phaseolus vulgaris-leucoagglutinin (PHA-L) injected into the median raphe (MR) and parvalbumin, choline acetyltransferase, or calretinin as well as double immunostaining for 5-HT and parvalbumin, and colocalization for parvalbumin and 5-HT2A receptors were done in rats. The results demonstrated that: 1) MR axons form perisomatic and peridendritic baskets and asymmetric synaptic contacts on MSDB parvalbumin neurons; 2) these fibers do not terminate on septal cholinergic and calretinin neurons; 3) 5-HT fibers form synapses identical to those formed by PHA-L-immunolabeled axons with parvalbumin neurons; and 4) MSDB parvalbumin cells contain 5-HT2A receptors. These observations indicate that 5-HT has a dual action on the activity of hippocampal principal cells: 1) an inhibition of the input sector by activation of hippocampal GABA neurons that terminate exclusively on apical dendrites of pyramidal cells, and 2) a disinhibition of the output sector of principal neurons. MSDB parvalbumin-containing GABAergic neurons specifically innervate hippocampal basket and chandelier cells. Thus, 5-HT-elicited activation of MSDB GABAergic neurons will result in a powerful inhibition of these GABA neurons.

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.

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.