Functional organization of the hippocampal memory system

In humans declarative or explicit memory is supported by the hippocampus and related structures of the medial temporal lobe working in concert with the cerebral cortex. This paper reviews our progress in developing an animal model for studies of cortical–hippocampal interactions in memory processing. Our findings support the view that the cortex maintains various forms of memory representation and that hippocampal structures extend the persistence and mediate the organization of these codings. Specifically, the parahippocampal region, through direct and reciprocal interconnections with the cortex, is sufficient to support the convergence and extended persistence of cortical codings. The hippocampus itself is critical to the organization cortical representations in terms of relationships among items in memory and in the flexible memory expression that is the hallmark of declarative memory.

Sorting and directed transport of membrane proteins during development of hippocampal neurons in culture

Hippocampal neurons in culture develop morphological polarity in a sequential pattern; axons form before dendrites. Molecular differences, particularly those of membrane proteins, underlie the functional polarity of these domains, yet little is known about the temporal relationship between membrane protein polarization and morphological polarization. We took advantage of viral expression systems to determine when during development the polarization of membrane proteins arises. All markers were unpolarized in neurons before axonogenesis. In neurons with a morphologically distinguishable axon, even on the first day in culture, both axonal and dendritic proteins were polarized. The degree of polarization at these early stages was somewhat less than in mature cells and varied from cell to cell. The cellular mechanism responsible for the polarization of the dendritic marker protein transferrin receptor (TfR) in mature cells centers on directed transport to the dendritic domain. To examine the relationship between cell surface polarization and transport, we assessed the selectivity of transport by live cell imaging. TfR-green fluorescent protein-containing vesicles were already preferentially transported into dendrites at 2 days, the earliest time point we could measure. The selectivity of transport also varied somewhat among cells, and the amount of TfR-green fluorescent protein fluorescence on intracellular structures within the axon correlated with the amount of cell surface expression. This observation implies that selective microtubule-based transport is the primary mechanism that underlies the polarization of TfR on the cell surface. By 5 days in culture, the extent of polarization on the cell surface and the selectivity of transport reached mature levels.

Synaptic sprouting increases the uptake capacities of motoneurons in amyotrophic lateral sclerosis mice

Using adenoviruses encoding reporter genes as retrograde tracers, we assessed the capacity of motoneurons to take up and retrogradely transport adenoviral particles injected into the muscles of transgenic mice expressing the G93A human superoxide dismutase mutation, a model of amyotrophic lateral sclerosis. Surprisingly, transgene expression in the motoneurons was significantly higher in symptomatic mice than in control or presymptomatic mice. Using botulinum toxin to induce nerve sprouting at neuromuscular junctions, we showed that the unexpectedly high level of motoneurons retrograde transduction results, at least in part, from newly acquired uptake properties of the sprouts. These findings demonstrate the remarkable uptake properties of amyotrophic lateral sclerosis motoneurons in response to denervation and the rationale of using intramuscular injections of adenoviruses to overexpress therapeutic proteins in motor neuron diseases.

Different modes of hippocampal plasticity in response to estrogen in young and aged female rats

Estrogen regulates hippocampal dendritic spine density and synapse number in an N-methyl-d-aspartate (NMDA) receptor-dependent manner, and these effects may be of particular importance in the context of age-related changes in endocrine status. We investigated estrogen's effects on axospinous synapse density and the synaptic distribution of the NMDA receptor subunit, NR1, within the context of aging. Although estrogen induced an increase in axospinous synapse density in young animals, it did not alter the synaptic representation of NR1, in that the amount of NR1 per synapse was equivalent across groups. Estrogen replacement in aged female rats failed to increase axospinous synapse density; however, estrogen up-regulated synaptic NR1 compared with aged animals with no estrogen. Therefore, the young and aged hippocampi react differently to estrogen replacement, with the aged animals unable to mount a plasticity response generating additional synapses, yet responsive to estrogen with respect to additional NMDA receptor content per synapse. These findings have important implications for estrogen replacement therapy in the context of aging.

Brain activity evidence for recognition without recollection after early hippocampal damage

Amnesic patients with early and seemingly isolated hippocampal injury show relatively normal recognition memory scores. The cognitive profile of these patients raises the possibility that this recognition performance is maintained mainly by stimulus familiarity in the absence of recollection of contextual information. Here we report electrophysiological data on the status of recognition memory in one of the patients, Jon. Jon's recognition of studied words lacks the event-related potential (ERP) index of recollection, viz., an increase in the late positive component (500–700 ms), under conditions that elicit it reliably in normal subjects. On the other hand, a decrease of the ERP amplitude between 300 and 500 ms, also reliably found in normal subjects, is well preserved. This so-called N400 effect has been linked to stimulus familiarity in previous ERP studies of recognition memory. In Jon, this link is supported by the finding that his recognized and unrecognized studied words evoked topographically distinct ERP effects in the N400 time window. These data suggest that recollection is more dependent on the hippocampal formation than is familiarity, consistent with the view that the hippocampal formation plays a special role in episodic memory, for which recollection is so critical.

Long-term, progressive hippocampal cell loss and dysfunction induced by early-life administration of corticotropin-releasing hormone reproduce the effects of early-life stress

Stress early in postnatal life may result in long-term memory deficits and selective loss of hippocampal neurons. The mechanisms involved are poorly understood, but they may involve molecules and processes in the immature limbic system that are activated by stressful challenges. We report that administration of corticotropin-releasing hormone (CRH), the key limbic stress modulator, to the brains of immature rats reproduced the consequences of early-life stress, reducing memory functions throughout life. These deficits were associated with progressive loss of hippocampal CA3 neurons and chronic up-regulation of hippocampal CRH expression. Importantly, they did not require the presence of stress levels of glucocorticoids. These findings indicate a critical role for CRH in the mechanisms underlying the long-term effects of early-life stress on hippocampal integrity and function.

Mutations in copper-zinc superoxide dismutase that cause amyotrophic lateral sclerosis alter the zinc binding site and the redox behavior of the protein.

A series of mutant human and yeast copper-zinc superoxide dismutases has been prepared, with mutations corresponding to those found in familial amyotrophic lateral sclerosis (ALS; also known as Lou Gehrig's disease). These proteins have been characterized with respect to their metal-binding characteristics and their redox reactivities. Replacement of Zn2+ ion in the zinc sites of several of these proteins with either Cu2+ or Co2+ gave metal-substituted derivatives with spectroscopic properties different from those of the analogous derivative of the wild-type proteins, indicating that the geometries of binding of these metal ions to the zinc site were affected by the mutations. Several of the ALS-associated mutant copper-zinc superoxide dismutases were also found to be reduced by ascorbate at significantly greater rate than the wild-type proteins. We conclude that similar alterations in the properties of the zinc binding site can be caused by mutations scattered throughout the protein structure. This finding may help to explain what is perhaps the most perplexing question in copper-zinc superoxide dismutase-associated familial ALS-i.e., how such a diverse set of mutations can result in the same gain of function that causes the disease.

Expansion of a recurrent V beta 5.3+ T-cell population in newly diagnosed and untreated HLA-DR2 multiple sclerosis patients.

We have used a PCR-based technology to study the V beta 5 and V beta 17 repertoire of T-cell populations in HLA-DR2 multiple sclerosis (MS) patients. We have found that the five MS DR2 patients studied present, at the moment of diagnosis and prior to any treatment, a marked expansion of a CD4+ T-cell population bearing V beta 5-J beta 1.4 beta chains. The sequences of the complementarity-determining region 3 of the expanded T cells are highly homologous. One shares structural features with that of the T cells infiltrating the central nervous system and of myelin basic protein-reactive T cells found in HLA-DR2 MS patients. An homologous sequence was not detectable in MS patients expressing DR alleles other than DR2. However, it is detectable but not expanded in healthy DR2 individuals. The possible mechanisms leading to its in vivo proliferation at the onset of MS are discussed.

Virus-mediated gene transfer into hippocampal CA1 region restores long-term potentiation in brain-derived neurotrophic factor mutant mice.

Long-term potentiation (LTP) has been shown to be impaired in mice deficient in the brain-derived neurotrophic factor (BDNF) gene, as well as in a number of other knockout animals. Despite its power the gene-targeting approach is always fraught with the danger of looking at the cumulative direct and indirect effects of the absence of a particular gene rather than its immediate function. The re-expression of a specific gene at a selective time point and at a specific site in gene-defective mutants presents a potent procedure to overcome this limitation and to evaluate the causal relationship between the absence of a particular gene and the impairment of a function in gene-defective animals. Here we demonstrate that the re-expression of the BDNF gene in the CA1 region almost completely restores the severely impaired LTP in hippocampal slices of BDNF-deficient mice. The results therefore provide strong evidence for the direct involvement of BDNF in the process of LTP.

Differential synaptic localization of two major gamma-aminobutyric acid type A receptor alpha subunits on hippocampal pyramidal cells.

Hippocampal pyramidal cells, receiving domain specific GABAergic inputs, express up to 10 different subunits of the gamma-aminobutyric acid type A (GABAA) receptor, but only 3 different subunits are needed to form a functional pentameric channel. We have tested the hypothesis that some subunits are selectively located at subsets of GABAergic synapses. The alpha 1 subunit has been found in most GABAergic synapses on all postsynaptic domains of pyramidal cells. In contrast, the alpha 2 subunit was located only in a subset of synapses on the somata and dendrites, but in most synapses on axon initial segments innervated by axo-axonic cells. The results demonstrate that molecular specialization in the composition of postsynaptic GABAA receptor subunits parallels GABAergic cell specialization in targeting synapses to a specific domain of postsynaptic cortical neurons.

Identification of peptides specific for cerebrospinal fluid antibodies in multiple sclerosis by using phage libraries.

The study of the origin and pathogenetic relevance of the oligoclonal antibodies present in the cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients has been hampered by a lack of specific ligands. We recently reported a general strategy, based on phage-displayed random peptide libraries, to identify ligands for disease-specific antibodies even in the absence of any information on the nature of the pathologic antigen. With this procedure, we identified several peptides specifically recognized by antibodies present in the CSF of MS patients. Using these peptides as reagents, we demonstrated that they mimic different natural epitopes and react with antibodies enriched in the CSF of MS patients. Antibodies recognizing the selected peptides are commonly found with equal frequency in the sera of MS patients and of normal individuals. In contrast, the repertoire of CSF antibodies appears to be individual-specific and is probably the result of a nonspecific immunodysregulation rather than a stereotyped response to a single antigen/agent.

Cooperative interactions in the induction of long-term potentiation and depression of synaptic excitation between hippocampal CA3-CA1 cell pairs in vitro.

The requirement for cooperative interactions between multiple synaptic inputs in the induction of long-term potentiation (LTP) and long-term depression (LTD) has been tested at Schaffer collateral synapses with paired recordings from monosynaptically coupled CA3-CA1 cell pairs in rat hippocampal slice cultures. Tetanization of single presynaptic neurons at 50 Hz (repeated 5-7 times for 300-500 ms each) induced only a transient potentiation (< 3 min) of excitatory postsynaptic potentials (EPSPs). Persistent potentiation (> 15 min) was induced only when single presynaptic action potentials were synchronously paired with directly induced postsynaptic depolarizing pulses (repeated 50-100 times). Tetanus-induced potentiation of extracellularly evoked EPSPs lasting > 4 min could only be obtained if the EPSP was > 4 mV. Because unitary EPSP amplitudes average approximately 1 mV, we conclude that high-frequency discharge must occur synchronously] in 4-5 CA3 cells for LTP to be induced in a common postsynaptic CA1 cell. Asynchronous pairing of presynaptic action potentials with postsynaptic depolarizing current pulses (preceding each EPSP by 800 ms) depressed both naive and previously potentiated unitary EPSPs. Likewise, homosynaptic LTD of unitary EPSPs was induced when the presynaptic cell was tetanized at 3 Hz for 3 min, regardless of their amplitude (0.3-3.2 mV). Homosynaptic LTD of extracellularly evoked Schaffer collateral EPSPs < 4 mV could be induced if no inhibitory postsynaptic potential was apparent, but was prevented by eliciting a large inhibitory postsynaptic potential or by injection of hyperpolarizing current in the postsynaptic cell. We conclude that cooperative interactions among multiple excitatory inputs are not required for induction of homosynaptic LTD of unitary EPSPs.

Rat hippocampal neurons express genes for both rod retinal and olfactory cyclic nucleotide-gated channels: novel targets for cAMP/cGMP function.

Cyclic nucleotide-gated (CNG) channels are Ca(2+)-permeable, nonspecific cation channels that can be activated through direct interaction with cAMP and/or cGMP. Recent electrophysiological evidence for these channels in cultured hippocampal neurons prompted us to investigate the expression of CNG channel genes in hippocampus. PCR amplification detected the expression of transcripts for subunit 1 of both the rod photoreceptor (RCNGC1) and the olfactory receptor cell (OCNGC1) subtype of CNG channel in adult rat hippocampus. In situ hybridization detected expression of both channel subtypes in most principal neurons, including pyramidal cells of the CA1 through CA3 regions and granule cells of the dentate gyrus. From the hybridization patterns, we conclude that the two genes are colocalized in individual neurons. Comparison of the patterns of expression of type 1 cGMP-dependent protein kinase and the CNG channels suggests that hippocampal neurons can respond to changes in cGMP levels with both rapid changes in CNG channel activity and slower changes induced by phosphorylation. Future models of hippocampal function should include CNG channels and their effects on both electrical responses and intracellular Ca2+ levels.