Cell formation in the human hippocampal formation from mid-gestation to the late postnatal period

In the present study cell formation was studied in the human hippocampal formation from the 24th gestational week until the end of the first postnatal year. Proliferating cells were detected with the monoclonal antibody MIB-1.The cytoarchitectonic layers of Ammon's horn are formed before the 24th gestational week. In harmony with this observation, cell proliferation in the hippocampal ventricular zone is minimal after the 24th week. In addition, local cell multiplication in Ammon's horn is occasional and the proliferating cells are glial or endothelial cells. In contrast, cell formation continues in the hilar region of the dentate gyrus even after birth. Immature cells accumulate in the hilus, and at the border between the hilus and the granule cell layer throughout the first eight postnatal months. The subgranular zone of the dentate gyrus becomes a cell sparse area at about the 11th postnatal month, indicating that immature cells from the hilus have already migrated to the granule cell layer and differentiated into granule cells. There is an increase in glial cell proliferation both in Ammon's horn and the dentate gyrus at the 11.5th postnatal month suggesting the onset of myelination by the end of the first year.Our findings indicate that most pyramidal neurons of Ammon's horn are generated in the first half of pregnancy and no pyramidal neurons are formed after the 24th gestational week. In contrast, granule cells of the dentate gyrus proliferate in a decreasing rate during the second half of pregnancy and after birth. Proliferating neuronal precursors occur in a low percentage in the dentate gyrus of 3-, 5- and 11.5-month-old children.     

Local circuit neurons in both the dentate gyrus and Ammon's horn establish synaptic connections with principal neurons in five day old rats: a morphological basis for inhibition in early development

Summary Glutamate decarboxylase (GAD)-positive and Golgi impregnated local circuit neurons of the hippocampal formation of five day old rats were examined in light and electron microscopic preparations. The ultrastructural features of these neurons were similar in both the dentate gyrus and CA1 area of Ammon's horn. Somata displayed a perikaryal cytoplasm rich in organelles but lacked organized Nissl bodies. Most nuclei showed intranuclear infoldings of varying degrees but no intranuclear sheets or rods were found. Somata and dendrites were contacted by relatively immature axon terminals that formed mainly symmetric synapses. The axons of local circuit neurons in both the dentate gyrus and Ammon's horn formed symmetric synapses with somata and dendrites of the principal neurons in these regions. Thus, both GAD-positive and Golgi-impregnated terminals of local circuit neurons were observed to form synapses with pyramidal and granule cells. These terminals were usually small and contained relatively few pleomorphic synaptic vesicles. The results show that a circuitry for inhibition is established in the 5 day old dentate gyrus and Ammon's horn, even though the local circuit neurons lack some of the typical adult ultrastructural features at this age.     

Learning-induced afterhyperpolarization reductions in hippocampus are specific for cell type and potassium conductance

Summary Hippocampal slices were prepared from rabbits trained in a trace eye-blink conditioning task and from naive and pseudoconditioned controls. Measurements of the post-burst afterhyperpolarization (AHP), action potential, and other cellular properties were obtained from intracellular recordings of CA1 pyramidal (N=49) and dentate gyrus granule cells (N=52). A conditioning-specific reduction in the amplitude of the AHP was found in CA1 cells but not in dentate granule cells. This reduction in the AHP was apparent at 50 ms after the end of a depolarizing current pulse, and was maintained for at least 650 ms. Other measured cell characteristics (input resistance, resting membrane potential, action potential shape, inward rectification, spike threshold) were not affected by training, in either CA1 pyramidal or dentate granule cells. Time-course measures indicate that both the medium, Ca²⁺-independent AHP and the slow, Ca²⁺-dependent AHP are reduced by conditioning. The slow AHP largely reflects the Ca²⁺-dependent K⁺ current, I_AHP Rising and falling slopes, peak amplitude, and width of individual action potentials were not changed by learning. This contrasts with observations from invertebrates in which action potential broadening was reported following learning. We conclude that the reduction in AHP that follows hippocampally-dependent associative learning occurs in specific hippocampal cell types and not others, and is mediated by changes in a Ca²-independent AHP and a particular Ca²⁺-dependent K⁺ current, I_AHP.     

Induction of somatostatin by kainic acid in pyramidal and granule cells of the rat hippocampus.

Seizures were induced in rats by systemic administration of kainic acid and, 1.5-12 h after, expression of preprosomatostatin and c-fos mRNAs in 9 hippocampal areas and in the cerebral perirhinal cortex was investigated using in situ hybridization histochemistry. Immunohistochemistry was also performed to study somatostatin peptide. In the control animals preprosomatostatin mRNA was expressed in some cells in the dentate hilus, the stratum oriens and the stratum radiatum of Ammon's horn, the subiculum and the cortex. Starting 3 h after kainic acid administration preprosomatostatin mRNA was expressed in a subpopulation of granule and pyramidal cells which did not normally express it. Preprosomatostatin mRNA-positive cells were markedly increased in the subiculum. Immunohistochemical examination confirmed that preprosomatostatin mRNA in granule and pyramidal cells was translated into peptide. In contrast, c-fos mRNA was induced in most hippocampal and cortical neurons starting 1.5 h after the kainic acid injection. When diazepam was injected to suppress the generalized seizures, preprosomatostatin mRNA was still expressed in pyramidal and subicular cells but not in granule cells.     

Timing and efficacy of transmitter release at mossy fiber synapses in the hippocampal network

It is widely accepted that the hippocampus plays a major role in learning and memory. The mossy fiber synapse between granule cells in the dentate gyrus and pyramidal neurons in the CA3 region is a key component of the hippocampal trisynaptic circuit. Recent work, partially based on direct presynaptic patch-clamp recordings from hippocampal mossy fiber boutons, sheds light on the mechanisms of synaptic transmission and plasticity at mossy fiber synapses. A high Na⁺ channel density in mossy fiber boutons leads to a large amplitude of the presynaptic action potential. Together with the fast gating of presynaptic Ca²⁺ channels, this generates a large and brief presynaptic Ca²⁺ influx, which can trigger transmitter release with high efficiency and temporal precision. The large number of release sites, the large size of the releasable pool of vesicles, and the huge extent of presynaptic plasticity confer unique strength to this synapse, suggesting a large impact onto the CA3 pyramidal cell network under specific behavioral conditions. The characteristic properties of the hippocampal mossy fiber synapse may be important for pattern separation and information storage in the dentate gyrus-CA3 cell network.     

Immunohistochemical mapping of total and phosphorylated eukaryotic initiation factor 4G in rat hippocampus following global brain ischemia and reperfusion

Partial proteolysis and phosphorylation of the translation initiation factor eukaryotic initiation factor 4G (eIF4G) occur in reperfused brain, but the contribution of eIF4G alterations to brain injury has not been established. A component of the complex delivering mRNA to the small ribosomal subunit, eIF4G is also found in stress granules. Stress granules sequester inactive 48S preinitiation complexes during stress-induced translation arrest. We performed double-labeling immunofluorescence histochemistry for total or ser 1108 phosphorylated eIF4G and the stress granule component T-cell internal antigen following normothermic, 10 min cardiac arrest-induced global brain ischemia and up to 4 h reperfusion in the rat. In cornu ammonis (Ammon's horn; CA) 1 at 90 min and 4 h reperfusion, eIF4G staining transformed from a homogeneous to an aggregated distribution. The number of eIF4G-containing stress granules differed between CA1 and CA3 during reperfusion. In hippocampal pyramidal neurons, phosphorylated eIF4G appeared exclusively in stress granules. Supragranular interneurons of the dentate gyrus showed a large increase in cytoplasmic eIF4G(P) following reperfusion. Immunoblot analysis with antisera against different portions of eIF4G showed a large increase in phosphorylated C-terminal eIF4G fragments, suggesting these accumulate in the cytoplasm of dentate gyrus interneurons. Thus, altered eIF4G subcellular compartmentalization may contribute to prolonged translation arrest in CA1 pyramidal neurons. Accumulation of phosphorylated eIF4G fragments may contribute to the vulnerability of dentate interneurons. Ischemia and reperfusion invoke different translational control responses in distinct hippocampal neuron populations, which may contribute to the differential ischemic vulnerabilities of these cells.     

Loss of dynorphin-mediated inhibition of voltage-dependent Ca2+ currents in hippocampal granule cells isolated from epilepsy patients is associated with mossy fiber sprouting

The endogenous kappa receptor selective opioid peptide dynorphin has been shown to inhibit glutamate receptor-mediated neurotransmission and voltage-dependent Ca2+ channels. It is thought that dynorphin can be released from hippocampal dentate granule cells in an activity-dependent manner. Since actions of dynorphin may be important in limiting excitability in human epilepsy, we have investigated its effects on voltage-dependent Ca2+ channels in dentate granule cells isolated from hippocampi removed during epilepsy surgery. One group of patients showed classical Ammon's horn sclerosis characterized by segmental neuronal cell loss and astrogliosis. Prominent dynorphin-immunoreactive axon terminals were present in the inner molecular layer of the dentate gyrus, indicating pronounced recurrent mossy fiber sprouting. A second group displayed lesions in the temporal lobe that did not involve the hippocampus proper. All except one of these specimens showed a normal pattern of dynorphin immunoreactivity confined to dentate granule cell somata and their mossy fiber terminals in the hilus and CA3 region. In patients without mossy fiber sprouting the application of the kappa receptor selective opioid agonist dynorphin A ([D-Arg6]1-13, 1 microM) caused a reversible and dose-dependent depression of voltage-dependent Ca2+ channels in most granule cells. These effects could be antagonized by the non-selective opioid antagonist naloxone (1 microM). In contrast, significantly less dentate granule cells displayed inhibition of Ca2+ channels by dynorphin A in patients with mossy fiber sprouting (Chi-square test, P < 0.0005). The lack of dynorphin A effects in patients showing mossy fiber sprouting compares well to the loss of kappa receptors on granule cells in Ammon's horn sclerosis but not lesion-associated epilepsy. Our data suggest that a protective mechanism exerted by dynorphin release and activation of kappa receptors may be lost in hippocampi with recurrent mossy fiber sprouting.     

Granule cells are the main source of excitatory input to a subpopulation of GABAergic hippocampal neurons as revealed by electron microscopic double staining for zinc histochemistry and parvalbumin immunocytochemistry

Immunocytochemistry was combined with a recent modification of Timm's method to evaluate semiquantitatively the mossy fiber innervation of dendrites and somata of parvalbumin-containing neurons of the hilus of the dentate gyrus and the CA3 area of Ammon's horn. Using this electron microscopic double staining technique, it was found that (1) the overwhelming majority (95%) of terminals forming asymmetric synapses with parvalbumin-positive dendrites in the dentate hilus, and the strata pyramidale and lucidum of the CA3 area of Ammon's horn, originated from granule cells; (2) two-thirds of the asymmetric axosomatic terminals of parvalbumin-positive neurons contained zinc; and (3) no zinc-containing axon terminals formed synapses with somata or main dendritic shafts of the granule cells.     

The Role of High-Conductivity Ca<Superscript>2+</Superscript>-Activated Potassium Channels in the Preconditioning Effects of Hypoxia and Posthypoxic Hyperexcitability of Neurons in Hippocampal Field CA1 in Vitro

The effects of the specific high-conductivity Ca²⁺-activated potassium channel blocker iberiotoxin on processes induced in rat hippocampal field CA1 slices by transient episodes of hypoxia were studied, i.e., 1) the suppressing effect of hypoxia on pyramidal neuron activity during hypoxic episodes, 2) the preconditioning action of hypoxia, and 3) posthypoxic neuron hyperexcitability. These experiments showed that iberiotoxin (10–20 nM) produced a tendency to decreases in the effectiveness of hypoxic episodes in depressing the amplitudes of population spikes recorded in hippocampal field CA1 during hypoxia. The high-conductivity Ca²⁺-activated potassium channel blocker significantly decreased the preconditioning effects of the first two episodes of hypoxia on the ability of the third episode of hypoxia to suppress neuron activity. Iberiotoxin also suppressed the posthypoxic hyperexcitability of pyramidal neurons. It is suggested that high-conductivity Ca²⁺-activated potassium channels play an important role in the mechanisms of development of types of neuroplasticity induced by transient episodes of hypoxia such as rapid hypoxic preconditioning and posthypoxic hyperexcitability.     

L-type Ca currents at CA1 synapses,but not CA3 or dentate granule neuron synapses,are increased in 3xTgAD mice in an age-dependent manner

Abnormal neuronal excitability and impaired synaptic plasticity might occur before the degeneration and death of neurons in Alzheimer's disease (AD). To elucidate potential biophysical alterations underlying aberrant neuronal network activity in AD, we performed whole-cell patch clamp analyses of L-type (nifedipine-sensitive) Ca²⁺ currents (L-VGCC), 4–aminopyridine-sensitive K⁺ currents, and AMPA (2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid) and NMDA (N-methyl-D-aspartate) currents in CA1, CA3, and dentate granule neurons in hippocampal slices from young, middle-age, and old 3xTgAD mice and age-matched wild type mice. 3xTgAD mice develop progressive widespread accumulation of amyloid β-peptide, and selective hyperphosphorylated tau pathology in hippocampal CA1 neurons, which are associated with cognitive deficits, but independent of overt neuronal degeneration. An age-related elevation of L-type Ca²⁺ channel current density occurred in CA1 neurons in 3xTgAD mice, but not in wild type mice, with the magnitude being significantly greater in older 3xTgAD mice. The NMDA current was also significantly elevated in CA1 neurons of old 3xTgAD mice compared with in old wild type mice. There were no differences in the amplitude of K⁺ or AMPA currents in CA1 neurons of 3xTgAD mice compared with wild type mice at any age. There were no significant differences in Ca²⁺, K⁺, AMPA, or NMDA currents in CA3 and dentate neurons from 3xTgAD mice compared with wild type mice at any age. Our results reveal an age-related increase of L-VGCC density in CA1 neurons, but not in CA3 or dentate granule neurons, of 3xTgAD mice. These findings suggest a potential contribution of altered L-VGCC to the selective vulnerability of CA1 neurons to tau pathology in the 3xTgAD mice and to their degeneration in AD patients.     

Mossy fibres form synapses with identified pyramidal basket cells in the CA3 region of the guinea-pig hippocampus: a combined Golgi-electron microscope study

Summary Mossy fibres, i.e. the axons of dentate granule cells, terminate with characteristic giant boutons on large spines or excrescences of the pyramidal cells in regio inferior of the hippocampus. In addition to pyramidal cells there are several types of non-pyramidal neuron which extend their dendrites into the termination zone of mossy fibres. By using the combined Golgi-electron microscope technique mossy fibre terminals were found, which established asymmetric synaptic contacts with both spines of pyramidal cells and smooth dendrites of identified (Golgi-stained) pyramidal basket cells in the CA3 region of the guinea-pig hippocampus. The observed synaptic connection with pyramidal basket cells suggests an involvement of the mossy fibre system in feed-forward inhibition of the hippocampal pyramidal neurons.     

Whole-cell voltage-clamp recordings in granule cells acutely isolated from hippocampal slices of adult or aged rats

Whole-cell voltage-clamp recordings were undertaken in granule cells acutely dissociated by a simple enzymatic procedure from the dentate gyri of hippocampal slices obtained from adult or aged rats. This dissociation procedure also allowed the concomitant isolation of other neuron types from diverse hippocampal subfields for the study of ionic currents through voltage- and neurotransmitter-gated channels. For example, in isolated granule cells of the dentate gyrus both transient and sustained Ca2+ currents could be observed in the presence of extracellular tetrodotoxin (TTX) and intracellular Cs+. In addition, ionic currents mediated by activation of excitatory amino acid receptors of the N-methyl-D-aspartate (NMDA) type were present. A technique for the rapid dissociation of neurons from brain slices of adult or aged rats is described in detail.     

Morphologic abnormalities in the postnatal differentiation of CA1 pyramidal cells and granule cells in the hippocampal formation of the Ataxic mouse

The postnatal differentiation of the hippocampal formation of the ataxic mouse was studied. Brains from ataxic mice (ax^J/ax^J) and littermate controls (+/?), from 19 to 51 days of age, were either impregnated according to a Golgi-Cox procedure or sectioned and stained with Weil-hematoxylin and Darrow Red. The hippocampus and dentate gyrus, although somewhat reduced in cross-sectional area in the ataxic brain, appeared to have a normal complement of both pyramidal cells and granule cells, respectively. Examination of Golgi-Cox material showed significant differences in the differentiation of the dendritic tree of both pyramidal and granule cells. At 41 days the height of the apical dendrite of CA1 pyramidal cells in the ataxic brain was 76% of the control value, and the width was only 35%. Similarly, the basal dendritic tree was narrower in the ataxic mouse. In the dentate gyrus of the 41-day ataxic brain, the height of the granule cell dendritic tree was only 74% of the control value. These and other alterations in the dendritic morphology of both CA1 pyramidal cells and granule cells can be explained by a lack of growth of the dendritic tree during the developmental period studied. These findings are discussed in relation to other studies on intrinsic and extrinsic factors and their effect on normal hippocampal differentiation.     

Glial reaction after seizure induced hippocampal lesion: immunohistochemical characterization of proliferating glial cells

Summary Kainic acid treatment (a model of temporal lobe epilepsy) induces Ammon's horn sclerosis, which is characterized by degeneration of CA3 pyramidal neurons and reactive gliosis. In the present study we have combined autoradiographic analysis of³H-thymidine incorporation and immunocytochemistry to investigate this glial scarring phenomenon. The present results demonstrate that in the fields showing neuronal degeneration (i.e. CA3–CA4 fields of Ammon's horn and dentate hilus) the glial reaction consists of a proliferation and hypertrophy of astrocytes and microglia-macrophages. In the regions showing exclusively terminal axonal degeneration (i.e. the molecular layer of kainate-treated rats), glial cells do not proliferate but astrocytes show a transient hypertrophy. These results also demonstrate that oligodendrocytes do not proliferate in the hippocampus of kainate-treated rats. In agreement with our previous report we find that hippocampal astrocytes from kainate-treated rats express A2B5 immunoreactivity, a marker of rype-2 astrocytes. A2B5 immunoreactivity was expressed by astrocytes not only in areas showing glial proliferation such as CA3–CA4 fields, but also in the molecular layer, where astrocytes do not proliferate. This suggests that in the CNS, normal resident astrocytes acquire the phenotypic properties of type-2 astrocytes.     

The phosphodiesterase III inhibitor olprinone inhibits hippocampal glutamate release via a cGMP/PKG pathway

Olprinone, an inhibitor of cyclic nucleotide phosphodiesterase III, inhibited an increase in intracellular Ca²⁺ concentrations for acutely dissociated rat hippocampal pyramidal neurons induced by extracellular high K⁺ (35 mM) depolarization. Olprinone (100 μM) significantly reduced spontaneous glutamate release from rat hippocampal slices. Furthermore, olprinone significantly decreased the rate of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor-mediated miniature excitatory postsynaptic currents (AMPA-mEPSCs) monitored from CA1 pyramidal neurons of rat hippocampal slices, and the effect was blocked by KT5823, an inhibitor of protein kinase G (PKG), but not by H-89, an inhibitor of protein kinase A (PKA). In the PKA assay using PC-12 cells, olprinone did not activate PKA. Taken together, the results of the present study show that olprinone attenuates intracellular Ca²⁺ rise through voltage-sensitive Ca²⁺ channels and inhibits presynaptic glutamate release via a cGMP/PKG pathway.     

Slow synaptic inhibition in relation to frequency habituation in dentate granule cells of rat hippocampal slices

Summary In paired pulse stimulation experiments the mechanism underlying frequency habituation of postsynaptic potentials in dentate granule cells of rat hippocampal slices was studied by measuring extra and intracellular potentials as well as changes in extracellular calcium ([Ca²⁺]₀) and potassium concentrations ([K⁺]₀). Orthodromic stimulation of the perforant path induced in most granule cells a late, slow hyperpolarization (SH), lasting for up to 1.2 s. During the SH the membrane conductance was increased by up to 40%. The reversal potential of the SH was around -90 mV and varied with the [K⁺]₀. Frequency habituation was seen in all cells with the SH, whereas cells which display frequency potentiation had no SH. Lowering of [Ca²⁺]₀ reversed paired pulse induced frequency habituation into frequency potentiation at [Ca²⁺]₀ levels where the SH disappeared. Phaclofen blocked the SH and reversed frequency habituation into frequency potentiation. Elevating [Mg²⁺]₀ also reversed frequency habituation into frequency potentiation and reduced the SH. We conclude that the SH represents a late, slow IPSP which is responsible for frequency habituation in dentate granule cells. We noted that during repetitive stimulation the SH soon started to fade. This effect can in part be attributed to extracellular K⁺-accumulation as suggested by the K⁺-dependence of the slow IPSP and the observations of changes in [K⁺]₀ during repetitive stimulation. This could explain why frequency habituation reverses into frequency potentiation during repetitive stimulation.On leave from the Department of Pharmacology, Uniformed Services, University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814-4799, USA     

Atrophy of pyramidal neurons and increased stress-induced glutamate levels in CA3 following chronic suppression of adult neurogenesis

Following their birth in the adult hippocampal dentate gyrus, newborn progenitor cells migrate into the granule cell layer where they differentiate, mature, and functionally integrate into existing circuitry. The hypothesis that adult hippocampal neurogenesis is physiologically important has gained traction, but the precise role of newborn neurons in hippocampal function remains unclear. We investigated whether loss of new neurons impacts dendrite morphology and glutamate levels in area CA3 of the hippocampus by utilizing a human GFAP promoter-driven thymidine kinase genetic mouse model to conditionally suppress adult neurogenesis. We found that chronic ablation of new neurons induces remodeling in CA3 pyramidal cells and increases stress-induced release of the neurotransmitter glutamate. The ability of persistent impairment of adult neurogenesis to influence hippocampal dendrite morphology and excitatory amino acid neurotransmission has important implications for elucidating newborn neuron function, and in particular, understanding the role of these cells in stress-related excitoxicity.     

Elicitation of penile erection following activation of the hippocampal formation in the rat.

We explore the possible involvement of the hippocampal formation in penile erection, using male, adult Sprague-Dawley rats that were anesthetized with pentobarbital sodium. The intracavernous pressure (ICP) was used as the experimental index for penile erection. Electrical activation of the hippocampal formation resulted in two patterns, viz, multiple and single episodes of elevation in ICP, along with visible penile erection and ejaculation. The former pattern exhibited an increase in ICP that was more sustained, with higher peak amplitude and longer latency. Furthermore, they originated respectively from the granule cells of the dentate gyrus and pyramidal cells of the CA1 and CA3 fields of the Ammon's horn. Chemical stimulation of the hippocampus with glutamate also elicited significant increase in ICP. These results thus provided direct evidence to establish that the hippocampal formation may be involved in central neural regulation of the erectile process.     

Calcium-binding proteins are concentrated in the CA2 field of the monkey hippocampus: a possible key to this region's resistance to epileptic damage

Summary Previous immunocytochemical studies have shown a heterogeneous distribution of parvalbumin (PA) and calbindin (CB) in the rat hippocampal formation. The results of the present study showed a heterogeneous distribution of PA and CB in primate Ammon's horn. The density and intensity of immunoreactivity for both of these calcium-binding proteins was greatest in CA2 as compared to CA1 and CA3. CB-immunoreactivity was localized to the cell bodies, dendrites, and axon initial segments of pyramidal cells whereas PA-immunostaining was found in the axon terminals, dendrites and cell bodies of interneurons that have features similar to GABAergic inhibitory neurons. Based on previous studies that have shown a protective role of calcium-binding proteins in neurons exposed to hyperstimulation, these results suggest that the resistance of CA2 pyramidal cells in temporal lobe epilepsy is due to the high concentration of CB and PA in this region of Ammon's horn.