Showing papers on "Dentate gyrus published in 1996"

Neurogenesis in the dentate gyrus of the adult rat: age-related decrease of neuronal progenitor proliferation

Abstract: The hippocampus is one of the few areas of the rodent brain that continues to produce neurons postnatally. Neurogenesis reportedly persists in rats up to 11 months of age. Using bromodeoxyuridine (BrdU) labeling, the present study confirms that in the adult rat brain, neuronal progenitor cells divide at the border between the hilus and the granule cell layer (GCL). In adult rats, the progeny of these cells migrate into the GCL and express the neuronal markers NeuN and calbindin-D28k. However, neurogenesis was drastically reduced in aged rats. Six-to 27-month-old Fischer rats were injected intraperitoneally with BrdU to detect newborn cells in vivo and to follow their fate in the dentate gyrus. When killed 4–6 weeks after BrdU labeling, 12- to 27- month-old rats exhibited a significant decline in the density of BrdU- positive cells in the granule cell layer compared with 6-month-old controls. Decreased neurogenesis in aging rats was accompanied by reduced immunoreactivity for poly-sialylated neural cell adhesion molecule, a molecule that is involved in migration and process elongation of developing neurons. When animals were killed immediately (12 hr) after BrdU injection, significantly fewer labeled cells were observed in the GCL and adjacent subgranular zone of aged rats, indicative of a decrease in mitotic activity of neuronal precursor cells. The reduced proliferation was not attributable to a general aged- related metabolic impairment, because the density of BrdU-positive cells was not altered in other brain regions with known mitotic activity (e.g., hilus and lateral ventricle wall). The decline in neurogenesis that occurs throughout the lifespan of an animal can thus be related to a decreasing proliferation of granule cell precursors.

Chronic antidepressant administration increases the expression of camp response element binding protein (creb) in rat hippocampus

Abstract: The present study demonstrates that chronic, but not acute, adminstration of several different classes of antidepressants, including serotonin- and norepinephrine-selective reuptake inhibitors, increases the expression of cAMP response element binding protein (CREB) mRNA in rat hippocampus. In contrast, chronic administration of several nonantidepressant psychotropic drugs did not influence expression of CREB mRNA, demonstrating the pharmacological specificity of this effect. In situ hybridization analysis demonstrates that antidepressant administration increases expression of CREB mRNA in CA1 and CA3 pyramidal and dentate gyrus granule cell layers of the hippocampus. In addition, levels of CRE immunoreactivity and of CRE binding activity were increased by chronic antidepressant administration, which indicates that expression and function of CREB protein are increased along with its mRNA. Chronic administration of the phosphodiesterase (PDE) inhibitors rolipram or papaverine also increased expression of CREB mRNA in hippocampus, demonstrating a role for the cAMP cascade. Moreover, coadministration of rolipram with imipramine resulted in a more rapid induction of CREB than with either treatment alone. Increased expression and function of CREB suggest that specific target genes may be regulated by these treatments. We have found that levels of brain-derived neurotrophic factor (BDNF) and trkB mRNA are also increased by administration of antidepressants or PDE inhibitors. These findings indicate that upregulation of CREB is a common action of chronic antidepressant treatments that may lead to regulation of specific target genes, such as BDNF and trkB, and to the long-term effects of these treatments on brain function.

Preserved neuron number in the hippocampus of aged rats with spatial learning deficits.

Abstract: Hippocampal neuron loss is widely viewed as a hallmark of normal aging. Moreover, neuronal degeneration is thought to contribute directly to age-related deficits in learning and memory supported by the hippocampus. By taking advantage of improved methods for quantifying neuron number, the present study reports evidence challenging these long-standing concepts. The status of hippocampal-dependent spatial learning was evaluated in young and aged Long-Evans rats using the Morris water maze, and the total number of neurons in the principal cell layers of the dentate gyrus and hippocampus was quantified according to the optical fractionator technique. For each of the hippocampal fields, neuron number was preserved in the aged subjects as a group and in aged individuals with documented learning and memory deficits indicative of hippocampal dysfunction. The findings demonstrate that hippocampal neuronal degeneration is not an inevitable consequence of normal aging and that a loss of principal neurons in the hippocampus fails to account for age-related learning and memory impairment. The observed preservation of neuron number represents an essential foundation for identifying the neurobiological effects of hippocampal aging that account for cognitive decline.

Dexamethasone in the last week of pregnancy attenuates hippocampal glucocorticoid receptor gene expression and elevates blood pressure in the adult offspring in the rat

Abstract: Human epidemiological data show a strong association between low birth weight and hypertension in adulthood, an effect that has been ascribed to 'fetal programming'. In rats fetoplacental exposure to maternally administered dexamethasone throughout gestation reduces birth weight and produces hypertensive adult offspring, though the mechanism is unclear. Pre- and postnatal stress programmes hypothalamic-pituitary-adrenal (HPA) axis responses throughout the lifespan, an effect thought to be mediated via permanent effects on glucocorticoid receptor (GR) and/or mineralocorticoid receptor (MR) gene expression in the hippocampus. Corticosteroids also have specific central effects on blood pressure control mediated by GR and MR. This study investigated corticosterone (CORT) responses to restraint stress and GR and MR gene expression in areas of the brain postulated to mediate the central effects of corticosteroids on (i) HPA axis suppression (hippocampus), and (ii) blood pressure (organ vasculosum of the lamina terminalis (OVLT), sub-commissural organ, area postrema and nucleus tractus solitarius). Pregnant Wistar rats received dexamethasone (100 micrograms/kg.day-1) or vehicle on days 15-20 of gestation. This reduced birth weight by 11%. When the offspring were 16 weeks old, blood pressure was recorded directly and plasma CORT measured basally (AM) and after 30 min restraint. GR and MR mRNA expression were determined by in situ hybridization. Blood pressure was significantly elevated in the adult offspring of dexamethasone-treated pregnancies (dexamethasone 144 +/- 2/125 +/- 2 mm Hg vs. control 133 +/- 2.7/112 +/- 2.8 mm Hg; both p < 0.01). Offspring of dexamethasone-treated pregnancies had increased basal plasma CORT (155 +/- 29 nmol/l) compared to offspring of controls (79 +/-15 nmol/l, p < 0.05), but the CORT response to stress was similar. Hippocampal neuronal GR mRNA expression was significantly lower in the offspring of dexamethasone-treated pregnancies (dentate gyrus 20% lower, CA1 15% lower; p < 0.01). Similarly, hippocampal MR gene expression was decreased in CA1 and CA2 by 24 and 25%, respectively (p < 0.05). No differences in GR or MR mRNA expression were found in the OVLT, subcommissural organ, area postrema or nucleus tractus solitarius. These findings suggest that glucocorticoid excess in the last trimester of rat pregnancy (i) is sufficient to programme offspring hypertension; (ii) also increases basal plasma CORT levels, and (iii) permanently attenuates GR and MR mRNA expression in specific hippocampal subfields. Thus, if translated into protein, may reduce sensitivity to glucocorticoid feedback and thus contribute to the CORT excess. However, hypertension in this model is unlikely to be mediated by similar changes in GR or MR gene expression in the examined areas of the brain putatively involved in the more direct central regulation of blood pressure.

A theory of hippocampal function in memory

Abstract: First, what is computed by the hippocampus is considered. Based on the effects of damage to the hippocampus and neuronal activity recorded in the primate hippocampus, it is suggested that it is involved in associating together information usually originating from different cortical regions, for example, about objects and their place in a spatial environment. The rapid formation of such context-dependent memories is prototypical of memories of particular events or episodes. Second, a computational theory of how it performs this function, based on neuroanatomical and neurophysiological information about the different neuronal systems contained within the hippocampus, is described. Key hypotheses are that the CA3 pyramidal cells operate as a single autoassociation network to store new episodic information as it arrives via a number of specialized preprocessing stages from many different association areas of the cerebral cortex, and that the dentate granule cell/mossy fiber system is important particularly during learning to help to produce a new pattern of firing in the CA3 cells for each episode. The computational analysis shows how many memories could be stored in the hippocampus, and how quickly the CA3 autoassociation system would operate during recall. The analysis is then extended to show how the CA3 system could be used to recall the whole of an episodic memory when only a fragment of it is presented. It is shown how this retrieval within the hippocampus could lead to recall of neuronal activity in association areas of the cerebral neocortex similar to that present during the original episode, via modified synapses in backprojection pathways from the hippocampus to the cerebral neocortex. The recalled information in the cerebral neocortex could then by used by the neocortex in the formation of long-term memories and/or in the selection of appropriate actions.

IRK(1–3) and GIRK(1–4) Inwardly Rectifying K+Channel mRNAs Are Differentially Expressed in the Adult Rat Brain

Abstract: Molecular cloning together with functional characterization has shown that the newly identified family of inwardly rectifying K+ channels consists of several closely related members encoded by separate genes. In this report we demonstrate the differential mRNA expression and detailed cellular localization in the adult rat brain of seven members of the IRK and GIRK subfamilies. Using both radiolabeled cRNA riboprobes and specific oligonucleotide probes directed to nonconserved regions of both known and newly isolated rat brain cDNAs, in situ hybridization revealed wide distribution with partly overlapping expression of the mRNAs of IRK1–3 and GIRK1–4. Except for the low levels of GIRK4 transcripts observed, the overall distribution patterns of the other GIRK subunits were rather similar, with high levels of expression in the olfactory bulb, hippocampus, cortex, thalamus, and cerebellum. Marked differences in expression levels existed only in some thalamic, brainstem, and midbrain nuclei, e.g., the substantia nigra, superior colliculus, or inferior olive. In contrast, IRK subunits were expressed more differentially: all mRNAs were abundant in dentate gyrus, olfactory bulb, caudate putamen, and piriform cortex. IRK1 and IRK3 were restricted to these regions, but they were absent from most parts of the thalamus, cerebellum, and brainstem, where IRK2 was expressed predominantly. Because channel subunits may assemble as heteromultimers, additional functional characterization based on overlapping expression patterns may help to decipher the native K+ channels in neurons and glial cells.

Zinc-induced collapse of augmented inhibition by GABA in a temporal lobe epilepsy model.

Abstract: In the kindling model of temporal lobe epilepsy, several physiological indicators of inhibition by γ-aminobutyric acid (GABA) in the hippocampal dentate gyrus are consistent with an augmented, rather than a diminished, inhibition. In brain slices obtained from epileptic (kindled) rats, the excitatory drive onto inhibitory interneurons was increased and was paralleled by a reduction in the presynaptic autoinhibition of GABA release. This augmented inhibition was sensitive to zinc most likely after a molecular reorganization of GABA A receptor subunits. Consequently, during seizures, inhibition by GABA may be diminished by the zinc released from aberrantly sprouted mossy fiber terminals of granule cells, which are found in many experimental models of epilepsy and in human temporal lobe epilepsy.

Differential Regulation of NMDAR1 mRNA and Protein by Estradiol in the Rat Hippocampus

Abstract: Estradiol treatment increases the number of NMDA receptor binding sites, and changes evoked synaptic currents in a manner consistent with a steroid-induced functional enhancement of NMDA receptors in rat hippocampus. In this study, we investigate the cellular mechanisms of estradiol-induced NMDA receptor regulation at the protein and mRNA levels in ovariectomized rats treated with ovarian steroids using immunocytochemical and in situ hybridization techniques. Confocal laser scanning microscopy was used to quantify alterations in immunofluorescence intensity levels of NMDAR1 subunit proteins within neuronal somata and dendrites of discrete hippocampal fields, whereas in parallel, in situ hybridization was used to examine NMDAR1 mRNA levels in corresponding hippocampal regions. The data indicate that estradiol treatment in ovariectomized rats significantly increases immunofluorescence intensity levels in comparison with nonsteroid treated ovariectomized rats within the somata and dendrites of CA1 pyramidal cells and, to a lesser extent, within the granule cell somata of the dentate gyrus. In contrast, such alterations in immunofluorescence intensity occur without concomitant changes in mRNA hybridization levels. Thus, these data suggest that estradiol modulates NMDA receptor function via post-transcriptional regulation of the NMDAR1 subunit protein. The increase in immunofluorescence intensity may reflect an increase in the concentration of the subunit protein, which could account for estrogen-induced changes in pharmacological and physiological properties of the NMDA receptor.

Electrographic Seizures and New Recurrent Excitatory Circuits in the Dentate Gyrus of Hippocampal Slices from Kainate-Treated Epileptic Rats

Abstract: Mossy fiber sprouting has been proposed to lead to new excitatory connections between dentate granule cells, which in turn cause electrographic seizures. We tested this hypothesis in hippocampal slices from rats made epileptic-by kainate injections. The Timm's histological method revealed intense staining of the inner molecular layer in slices from all kainate-treated rats. In bicuculline (10 microM) and 6 mM [K +]o, antidromic stimulation of the granule cells evoked bursts of population spikes superimposed on long-lasting negative shifts in all slices tested from all kainate-treated rats. Long-duration (2-47 sec), seizure-like bursts with tonic and clonic components occurred spontaneously (53%) or in response to antidromic stimulation (81%). Under identical conditions, prolonged bursts were never seen in slices from controls or from kainate-injected rats 2-4 d after treatment. Glutamate microdrops applied in the granule cell layer evoked abrupt increases in the frequency of excitatory postsynaptic potentials (EPSPs) in two thirds of the cells tested. Glutamate microstimulation was effective at several sites in the granule cell layer but ineffective in the hilus. The proportion of granule cells responding to local application of glutamate by an increase in EPSPs was higher in slices with long bursts (80% with bursts of > 3 sec) than in slices with shorter bursts (33% with bursts of < 3 sec). Glutamate microstimulation did not affect EPSPs in granule cells from control preparations. These results support the hypothesis that kainate-induced mossy fiber sprouting forms new excitatory connections between granule cells and can lead to increased seizure susceptibility in the dentate gyrus.

Overexpression of bcl-2 with herpes simplex virus vectors protects cns neurons against neurological insults in vitro and in vivo

Abstract: Previous studies have demonstrated that overexpression of the proto-oncogene bcl-2 can protect neuron and neuron-like cell lines from growth factor deprivation, calcium ionophores, glutamate excitotoxicity, hypoglycemia, free radicals, and lipid peroxidation. To determine whether Bcl-2 exhibits a similar protective effect in CNS neurons, we generated defective herpes simplex virus (HSV) vectors capable of overexpressing Bcl-2 in primary cultures and in the intact brain. Infection of hippocampal cultures with Bcl-2 vectors enhanced neuron survivorship after exposure to adriamycin, a potent oxygen radical generator. Furthermore, dichlorofluorescein measurements indicated that there was a significant reduction in the accumulation of oxygen radicals associated with this insult. Bcl-2 vectors also enhanced survival in cultured neurons after exposure to glutamate and hypoglycemia. Most significantly, the in vivo delivery of the vector protected neurons against adriamycin toxicity in the dorsal horn of the dentate gyrus and focal ischemia in the striatum.

Basal expression and induction of glutamate decarboxylase GABA in excitatory granule cells of the rat and monkey hippocampal dentate gyrus

Abstract: The excitatory, glutamatergic granule cells of the hippocampal dentate gyrus are presumed to play central roles in normal learning and memory, and in the genesis of spontaneous seizure discharges that originate within the temporal lobe. In localizing the two GABA producing forms of glutamate decarboxylase (GAD65 and GAD67) in the normal hippocampus as a prelude to experimental epilepsy studies, we unexpectedly discovered that, in addition to its presence in hippocampal nonprincipal cells, GAD67-like immunoreactivity (LI) was present in the excitatory axons (the mossy fibers) of normal dentate granule cells of rats, mice, and the monkey Macaca nemestrina. Using improved immunocytochemical methods, we were also able to detect GABA-LI in normal granule cell somata and processes. Conversely, GAD65-LI was undetectable in normal granule cells. Perforant pathway stimulation for 24 hours, which evoked population spikes and epileptiform discharges in both dentate granule cells and hippocampal pyramidal neurons, induced GAD65-, GAD67-, and GABA-LI only in granule cells. Despite prolonged excitation, normally GAD- and GABA-negative dentate hilar neurons and hippocampal pyramidal cells remained immunonegative. Induced granule cell GAD65-, GAD67-, and GABA-LI remained elevated above control immunoreactivity for at least 4 days after the end of stimulation. Pre-embedding immunocytochemical electron microscopy confirmed that GAD67- and GABA-LI were induced selectively within granule cells; granule cell layer glia and endothelial cells were GAD- and GABA-immunonegative. In situ hybridization after stimulation revealed a similarly selective induction of GAD65 and GAD67 mRNA in dentate granule cells. Neurochemical analysis of the microdissected dentate gyrus and area CA1 determined whether changes in GAD- and GABA-LI reflect changes in the concentrations of chemically identified GAD and GABA. Stimulation for 24 hours increased GAD67 and GABA concentrations sixfold in the dentate gyrus, and decreased the concentrations of the GABA precursors glutamate and glutamine. No significant change in GAD65 concentration was detected in the microdissected dentate gyrus despite the induction of GAD65-LI. The concentrations of GAD65, GAD67, GABA, glutamate and glutamine in area CA1 were not significantly different from control concentrations. These results indicate that dentate granule cells normally contain two “fast-acting” amino acid neurotransmitters, one excitatory and one inhibitory, and may therefore produce both excitatory and inhibitory effects. Although the physiological role of granule cell GABA is unknown, the discovery of both basal and activity-dependent GAD and GABA expression in glutamatergic dentate granule cells may have fundamental implications for physiological plasticity presumed to underlie normal learning and memory. Furthermore, the induction of granule cell GAD and GABA by afferent excitation may constitute a mechanism by which epileptic seizures trigger compensatory interictal network inhibition or GABA-mediated neurotrophic effects. © 1996 Wiley-Liss, Inc.

Immunohistochemical localization of ORL‐1 in the central nervous system of the rat

Abstract: A novel member of the opioid receptor family (ORL-1) has been cloned from a variety of vertebrates. ORL-1 does not bind any of the classical opioids, although a high affinity endogenous agonist with close homology to dynorphin has recently been identified. We have generated a monoclonal antibody to the N-terminus of ORL-1 to map areas of receptor expression in rat central nervous system (CNS). Intense and specific immunolabeling was observed in multiple areas in the diencephalon, mesencephalon, pons/medulla, and spinal cord. In the telencephalon, intense labeling was observed in the neuropil throughout layers II–V in the neocortex, the anterior olfactory nuclear complex, the pyriform cortex, the CA1–CA4 fields and dentate gyrus of the hippocampus, and in many of the septal and basal forebrain areas. In contrast to other members of the opioid receptor family, light labeling for ORL-1 was observed in telencephalic areas such as caudate-putamen. In the cerebellum, ORL-1 immunoreactivity was only observed in the deep nuclei. Throughout the CNS the majority of labelling was localized to fiber processes and fine puncta, although labeled scattered perikarya were observed in a few brain areas such as the hilus dentate in the hippocampus and some nuclei in the brainstem and spinal cord. The present mapping study is consistent with the reported distribution of ORL-1 mRNA and provides the first immunohistochemical report on anatomical and cellular distribution of ORL-1 receptor in the rat CNS. © 1996 Wiley-Liss, Inc.

Comparative evaluation of synaptophysin-based methods for quantification of synapses.

Abstract: Development, ageing, and a variety of neurological disorders are characterized by selective alterations in specific populations of nerve cells which are, in turn, associated with changes in the numbers of synapses in the target fields of these neurons. To begin to delineate the significance of changes in synapses in development, ageing, and disease, it is first essential to quantify the number of synapses in defined regions of the CNS. In the past, investigators have used EM methods to assess synapse numbers or density, but these approaches are costly, labour intensive, and technically difficult, particularly in autopsy material. To begin to define reliable strategies useful for studies of both animals and humans, we used three techniques to measure synaptophysin-immunoreactivity in rat brain. The levels of synaptophysin protein were determined by Western blots of five hippocampal subregions; the intensity of synaptophysin-immunoreactivity in dentate gyrus and stratum oriens was determined by optical densitometry of immunocytochemically stained sections; and the total number of synaptophysin-immunoreactivity presynaptic boutons in dentate gyrus and stratum oriens was assessed by unbiased stereology. Each approach has advantages and disadvantages. Western blotting is the least time-consuming of the three methods and allows simultaneous processing of multiple samples. In systematically sampled histological sections, both densitometry and stereology allow precise definition of the region of interest, and the sterological optical disector method allows quantitation of the numbers of synaptophysin-immunoreactive boutons. Stereology was the only method that clearly demonstrated greater synaptophysin-immunoreactivity in the dentate gyrus as compared to stratum oriens. The use of systematic sampling and the disector technique offer a high degree of anatomical resolution (lacking in Western blot methods) and has quantitative advantage over the greyscale-based density approach. Thus, at present, stereology is the most useful method for estimating synaptic numbers in defined regions of the brain.

Influence of actinomycin d, a rna synthesis inhibitor, on long-term potentiation in rat hippocampal neurons in vivo and in vitro

Abstract: 1. Hippocampal long-term potentiation (LTP) may serve as an elementary process underlying certain forms of learning and memory in vertebrates. As is the case with behavioural memory, hippocampal LTP in the rat CA1 region and in the dentate gyrus occurs in stages, which can be separated by an inhibitor of RNA synthesis. 2. Experiments have been performed in two brain regions, in the hippocampal CA1 region in vitro and in the dentate gyrus in vivo. 3. Maintenance of hippocampal LTP in the CA1 region in vitro was influenced by the RNA synthesis inhibitor actinomycin D from 4 h onwards. 4. The effect of actinomycin D on the time course of the population spike potentiation was more pronounced than the effect on the time course of the EPSP component, suggesting different mechanisms for the two forms of potentiation. 5. Intrahippocampal and intracerebroventricular injection of actinomycin D into rats prevented a late stage of LTP in the dentate gyrus in vivo measured as the population spike amplitude. 6. Since actinomycin D was only effective in influencing the maintenance of LTP when applied before tetanization, the requirement for transcription during LTP may have a critical time window. 7. Actinomycin D influenced the maintenance of LTP specifically, since the drug did not alter any potentials in control experiments after its removal or when it was administered shortly after tetanization. A second, structurally different RNA synthesis inhibitor, 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole, mimicked the effect of actinomycin D in vitro.

Considerations arising from a complementary learning systems perspective on hippocampus and neocortex.

Abstract: We discuss a framework for the organization of learning systems in the mammalian brain, in which the hippocampus and related areas form a memory system complementary to learning mechanisms in neocortex and other areas. The hippocampal system stores new episodes and "replays" them to the neocortical system, interleaved with ongoing experience, allowing generalization as cortical memories form. The data to account for include: 1) neurophysiological findings concerning representations in hippocampal areas, 2) behavioral evidence demonstrating a spatial role for hippocampus, 3) and effects of surgical and pharmacological manipulations on neuronal firing in hippocampal regions in behaving animals. We hypothesize that the hippocampal memory system consists of three major modules: 1) an invertible encoder subsystem supported by the pathways between neocortex and entorhinal cortex, which provides a stable, compressed, invertible encoding in entorhinal cortex (EC) of cortical activity patterns, 2) a memory separation, storage, and retrieval subsystem, supported by pathways between EC, dentate gyrus and area CA3, including the CA3 recurrent collaterals, which facilitates encoding and storage in CA3 of individual EC patterns, and retrieval of those CA3 encodings, in a manner that minimizes interference, and 3) a memory decoding subsystem, supported by the Shaffer collaterals from area CA1 to area CA3 and the bi-directional pathways between EC and CA3, which provides the means by which a retrieved CA3 coding of an EC pattern can reinstate that pattern on EC. This model has shown that 1) there is a trade-off between the need for information-preserving, structure-extracting encoding of cortical traces and the need for effective storage and recall of arbitrary traces, 2) long-term depression of synaptic strength in the pathways subject to long-term potentiation is crucial in preserving information, 3) area CA1 must be able to exploit correlations in EC patterns in the direct perforant path synapses.

Dentate gyrus formation requires Emx2.

Abstract: Emx 1 and 2 are the murine homologues of the Drosophila empty spiracles gene and based on their expression pattern may be involved in the regional specification of the mammalian forebrain. During early embryogenesis, Emx2 is expressed in the presumptive cerebral cortex and olfactory bulbs and later, in the hippocampus proper and dentate gyrus. The latter are involved in memory processes. To understand the role of Emx2 in vivo, we have mutated the gene in mice. Homozygous embryos die postnatally because of severe urogenital alterations. These mice present cerebral hemispheres with a reduced size and exhibit specific morphological alterations in allocortical structures of the medial wall of the brain. The dentate gyrus is missing and the hippocampus proper is reduced. The medial limbic cortex is also severely shortened. The development of the dentate gyrus is affected at the onset of its formation with defects in the neuroepithelium from which it originates. These findings demonstrate that Emx2 is required for the development of several forebrain structures.

TrkB and TrkC neurotrophin receptors cooperate in promoting survival of hippocampal and cerebellar granule neurons.

Abstract: The Trk family of protein tyrosine kinases (TrkA/B/C) are receptors for neurotrophins, a family of closely related proteins that are important physiological regulators of the survival of specific neurons within the peripheral nervous system (PNS) of vertebrates. In contrast to the PNS, brains of mutant mice deficient in a single neurotrophin or Trk receptor species do not show signs of major cell loss. However, in double mutant mice, we now show that reducing the expression of both TrkB and TrkC causes massive cell death of postnatal hippocampal and cerebellar granule neurons. Kinetic analysis of neuronal death in the hippocampus showed that dentate gyrus granule neurons become dependent on TrkB and TrkC after the first postnatal week, shortly after the period of naturally occurring cell death, indicating a role of these receptors in supporting postmitotic neurons. Correlating with the loss of granule cells, the number of mossy fibers projecting to CA3 pyramidal neurons was markedly reduced in mice carrying mutant trkB/trkC alleles, demonstrating impairment of excitatory pathways in the hippocampus. In the cerebellum, TrkB and TrkC receptors were specifically required for premigratory granule neurons located in the external granule layer. In contrast, cerebellar Purkinje cells were found to be poorly differentiated, but showed no signs of increased cell death. These results provide in vivo evidence that neurotrophins are essential physiological survival factors for specific central neurons. Moreover, they suggest that central, in contrast to peripheral, neurons are capable of using more than one neurotrophin/Trk receptor signaling pathway to stay alive.

Apoptosis and necrosis induced in different hippocampal neuron populations by repetitive perforant path stimulation in the rat

Abstract: Patients experiencing spontaneous seizures of temporal lobe origin often exhibit a shrunken hippocampus, which results from the loss of dentate granule cells, hilar neurons, and hippocampal pyramidal cells. Although experimental attempts to replicate the human pattern of hippocampal sclerosis in animals indicate that prolonged seizures cause prominent injury to dentate hilar neurons and hippocampal pyramidal cells, dentate granule cells of animals are generally regarded as relatively resistant to seizure-induced injury. By evaluating pathology shortly after hippocampal seizure discharges were induced electrically, we discovered that some granule cells are highly vulnerable to prolonged excitation and that they exhibit acute degenerative features distinct from those of other vulnerable cell populations. Intermittent perforant path stimulation for 24 hours induced acute degeneration of dentate granule cells, dentate hilar neurons, and hippocampal pyramidal cells. However, stimulation for 8 hours, which was insufficient to injure hilar neurons and hippocampal pyramidal cells, was nonetheless sufficient to induce bilateral granule cell degeneration. Degenerating granule cells were consistently more numerous in the infrapyramidal than the suprapyramidal blade, and were consistently more numerous in the rostral than caudal dentate gyrus. Depending on the nature of the insult, acutely degenerating neurons exhibit distinct morphological features that are classifiable as either apoptosis or necrosis, although the degree of possible overlap is unknown. Light and electron microscopic analysis of the acute pathology caused by prolonged afferent stimulation revealed that degenerating hilar neurons and pyramidal cells exhibited the morphological features of necrosis, which is characterized in part by early cytoplasmic vacuolization before nuclear changes occur. However, acutely degenerating granule cells exhibited the clearly distinct morphological features of apoptosis, which include an early coalescence of nuclear chromatin into multiple nuclear bodies, compaction of the cytoplasm, cell shrinkage, and budding-off of 'apoptotic bodies' that are engulfed by glia. Whereas pyramidal cell debris persisted for months, granule cell debris disappeared rapidly. This observation may explain why significant granule cell vulnerability has not been described previously. These data document for the first time that dentate granule cells are among the cell types most vulnerable to seizure-induced injury, and demonstrate that whereas hilar neurons and pyramidal cells undergo a typically necrotic degenerative process, granule cells simultaneously exhibit morphological features that more closely resemble the degenerative process of apoptosis. This finding implies that the type of cell death induced by excessive excitation may be determined postsynaptically by the way in which different target cells 'interpret' an excitatory insult. This experimental model may be useful for identifying the biochemical mechanisms that initiate and mediate neuronal apoptosis and necrosis, and for developing strategies to prevent or induce these presumably distinct forms of neuronal death.

Regional specificity of alterations in NGF, BDNF and NT-3 levels in Alzheimer's disease

Abstract: Using two-site enzyme immunoassays (EIAs), we measured the levels of neurotrophins, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) simultaneously in three brain regions (motor cortex, dentate gyrus and entorhinal cortex) of patients with Alzheimer's disease (AD) and control individuals. Significant differences between the neurotrophin levels of these two groups were found in the different brain regions depending on the neurotrophin. The NGF level in the dentate gyrus of AD patients was higher, whereas the BDNF level in the entorhinal cortex and the NT-3 level in the motor cortex were lower than the corresponding control levels. These results indicate that protein levels of individual neurotrophins in different brain regions are affected differently by AD, and such differential changes may contribute to the complex pathology of AD.

Normal spatial learning despite regional inhibition of LTP in mice lacking Thy-1

Abstract: The process of learning involves stable changes in synaptic efficacy for which long-term potentiation (LTP) provides a widely adopted mammalian model. Synaptic modification induced by learning or LTP may involve the action of cell adhesion molecules. One such candidate is the ubiquitous neuronal glycoprotein Thy-1. In mice in which the gene encoding Thy-1 has been inactivated, we find a regionally selective impairment of LTP in vivo in the hippocampal formation: LTP is normal in area CA1 but strongly inhibited in the dentate gyrus. Spatial learning by Thy-1-deficient mice, as assessed in the watermaze, is unimpaired. Thus LTP in the cortical input to the dentate gyrus seems not to be required for spatial learning.

GABA plasma membrane transporters, GAT-1 and GAT-3, display different distributions in the rat hippocampus.

Abstract: This study evaluates the distribution of two high affinity gamma-aminobutyric acid (GABA) transporters (GAT-1 and GAT-3) in the rat hippocampus using immunocytochemistry and affinity purified antibodies. GAT-1 immunoreactivity was prominent in punctate structures and axons in all layers of the dentate gyrus. In Ammon's horn, immunoreactive processes were concentrated around the somata of pyramidal cells, particularly at their basal regions. The apical and basal dendritic fields of pyramidal cells also displayed numerous GAT-1 immunoreactive punctate structures and axons. The zone of termination of the mossy fibers that includes both the hilus of the dentate gyrus and stratum lucidum of the CA3 area was the lightest immunolabeled region of the hippocampal complex. Electron microscopic preparations demonstrated that GAT-1 immunoreactive axon terminals form symmetric synapses with somata, axon initial segments, and dendrites of granule and pyramidal cells in the dentate gyrus and Ammon's horn, respectively. Immunoreactivity was localized to the plasma membrane and the cytoplasm of axon terminals. The somata of previously described local circuit neurons in the dentate gyrus and Ammon's horn contained GAT-1 immunoreactivity associated with the Golgi complex. Light, diffuse GAT-3 immunoreactivity was present throughout the hippocampal formation. Thin, astrocytic glial processes displayed GAT-1 and GAT-3 immunoreactivity. This localization of GAT-1 and GAT-3 indicates that they are involved in the uptake of GABA from the extracellular space into GABAergic axon terminals and astrocytes.

Distribution of the mRNA for a pituitary adenylate cyclase-activating polypeptide receptor in the rat brain: An in situ hybridization study

Abstract: The distribution of the mRNA for a pituitary adenylate cyclase-activating polypeptide (PACAP) receptor (PACAP-R) was examined in the rat brain, and also in the hypophysis and pineal gland, by in situ hybridization with a specific 35S-labeled riboprobe which was generated from a rat PACAP-R cDNA clone. In the brain, expression of PACAP-R mRNA was most prominent in the periglomerular and granule cells of the olfactory bulb, granule cells of the dentate gyrus, supraoptic nucleus, and area postrema. The expression was also intense in the piriform, cingulate, and retrosplenial cortices, pyramidal cells in CA2, non-pyramidal cells in CA1-CA3, neuronal cells in the hilus of the dentate gyrus, lateral septal nucleus, intercalated amygdaloid nucleus, anterodorsal thalamic nucleus, most of the midline and intralaminar thalamic nuclei, many regions of the hypothalamus, dorsal motor nucleus of the vagus nerve, hypoglossal nucleus, and lateral reticular nucleus. No significant expression was detected in the mitral and tufted cells in the olfactory bulb, pyramidal cells in CA1 and CA3, posterior nuclear group of the thalamus, dorsal lateral geniculate nucleus, and Purkinje, Golgi, and granule cells in the cerebellar cortex. Moderate-to-weak expression was further observed in many other regions of the brain. In the cerebellar cortex, presumed Bergmann glia cells showed moderate expression. In the hypophysis, the expression was moderate in the anterior lobe, and weak to moderate in the posterior lobe; no significant expression was observed in the intermediate lobe. In the pineal gland, the expression was very weak, if any. Thus, the expression of PACAP-R was detected not only on neuronal cells but also on some particular glial cells. The present study has shown, for the first time, the exact site of PACAP-R expression in the brain and hypophysis. Although the functional significance of PACAP and PACAP-R in the brain still remains to be clarified, the present results are considered to provide some direction for future functional studies.

Age-Dependent Neuronal and Synaptic Degeneration in Mice Transgenic for the C Terminus of the Amyloid Precursor Protein

Abstract: The molecular basis for the degeneration of neurons and the deposition of amyloid in plaques and in the cerebrovasculature in Alzheimer’s disease (AD) is incompletely understood. We have proposed that one molecule common to these abnormal processes is a fragment of the Alzheimer amyloid precursor protein (APP) comprising the C-terminal 100 amino acids of this molecule (APP-C100). We tested this hypothesis by creating transgenic mice expressing APP-C100 in the brain. We report here that aging (18–28 month) APP-C100 transgenic mice exhibit profound degeneration of neurons and synapses in Ammon’s horn and the dentate gyrus of the hippocampal formation. Of the 106 transgenic mice between 8 and 28 months of age that were examined, all of those older than 18 months displayed severe hippocampal degeneration. The numerous degenerating axonal profiles contained increased numbers of neurofilaments, whorls of membrane, and accumulations of debris resembling secondary lysosomes near the cell body. The dendrites of degenerating granule and pyramidal cells contained disorganized, wavy microtubules. Cerebral blood vessels had thickened refractile basal laminae, and microglia laden with debris lay adjacent to larger venous vessels. Mice transgenic for Flag-APP-C100 (in which the hydrophilic Flag tag was fused to the N terminus of APP-C100) showed a similar degree of neurodegeneration in the hippocampal formation as early as 12 months of age. The 45 control mice displayed only occasional necrotic cells and no extensive cell degeneration in the same brain regions. These findings show that APP-C100 is capable of causing some of the neuropathological features of AD.

Human fetal hippocampal development: I. Cytoarchitecture, myeloarchitecture, and neuronal morphologic features.

Abstract: To characterize better the process of anatomic development of the human hippocampus, we studied the cytoarchitecture, myeloarchitecture, and neuronal morphology in human fetal and postnatal hippocampi. Twenty cases were studied in which the ages ranged from 9 weeks gestation through 62 years. Fixed, paraffin-embedded, hippocampal sections were stained with cresyl violet for Nissl substance and immunolabeled for myelin basic protein. The hippocampal region at 9 weeks contains 4 layers: a ventricular zone, an intermediate zone, a homogeneous-appearing hippocampal plate comprised of bipolar-shaped neurons, and a wide marginal zone. At 15-19 weeks, individual subfields can be distinguished. A distal-to-proximal gradient of cytoarchitectural and neuronal morphologic maturity is seen, with the subiculum appearing more developed than the ammonic subfields and the dentate gyrus appearing least mature. Within each subfield, an "inside-out" gradient of maturity is also evident. By 32-34 weeks gestational age, neurons in CA2 and CA3 have undergone rapid enlargement and morphologic maturation, surpassing CA1, which still contains some immature neurons. The dentate gyrus is the latest area to develop, only assuming a mature cytoarchitecture after 34 weeks. The essential cytoarchitectural appearance of the hippocampal subfields is stable after birth, although there is progressive neuronal enlargement and a decrease in neuronal density throughout childhood into adulthood. Myelination is first evident near term, with strong myelin basic protein immunoreactivity present in the angular bundle, alveus, and fimbria and relatively scant immunoreactivity in the nascent perforant pathway. Myelination in the hippocampus increases in childhood until adolescence, after which the pattern remains unchanged. These studies delineate normal neuroanatomic development and can be used to understand better the mechanisms underlying human neurodevelopmental and neurodegenerative disorders of the hippocampal formation.