Gestational protein restriction induces CA3 dendritic atrophy in dorsal hippocampal neurons but does not alter learning and memory performance in adult offspring

Studies have demonstrated that nutrient deficiency during pregnancy or in early postnatal life results in structural abnormalities in the offspring hippocampus and in cognitive impairment. In an attempt to analyze whether gestational protein restriction might induce learning and memory impairments associated with structural changes in the hippocampus, we carried out a detailed morphometric analysis of the hippocampus of male adult rats together with the behavioral characterization of these animals in the Morris water maze (MWM). Our results demonstrate that gestational protein restriction leads to a decrease in total basal dendritic length and in the number of intersections of CA3 pyramidal neurons whereas the cytoarchitecture of CA1 and dentate gyrus remained unchanged. Despite presenting significant structural rearrangements, we did not observe impairments in the MWM test. Considering the clear dissociation between the behavioral profile and the hippocampus neuronal changes, the functional significance of dendritic remodeling in fetal processing remains undisclosed. © 2012 ISDN.

A role for Src kinase in spontaneous epileptiform activity in the CA3 region of the hippocampus

Members of the Src family of nonreceptor protein tyrosine kinases (PTKs) have been implicated in the regulation of cellular excitability and synaptic plasticity. We have investigated the role of these PTKs in in vitro models of epileptiform activity. Spontaneous epileptiform discharges were induced in vitro in the CA3 region of rat hippocampal slices by superfusion with the potassium channel blocker 4-aminopyridine in Mg2+-free medium. In hippocampal slices treated in this fashion, Src kinase activity was increased and the frequency of epileptiform discharges could be greatly reduced by inhibitor of the Src family of PTKs, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), but not by the inactive structural analog 4-amino-7-phenylpyrazol[3,4-d]pyrimidine (PP3). 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine also reduced epileptiform activity induced by either 4-aminopyridine or Mg2+-free medium alone. These observations demonstrate a role for Src family PTKs in the pathophysiology of epilepsy and suggest potential therapeutic targets for antiepileptic therapy.

Rhythmic synaptic activity induced by mechanical injury of rat CA3 hippocampal area.

Mechanical injury of the CNS frequently results from accidents but also occurs in the course of neurosurgical interventions. A great variety of anatomical and physiological changes have been described to evolve after a brain trauma yet only little is known about processes that occur during a trauma. In the present study, I obtained whole-cell patch clamp recordings from pyramidal cells in hippocampal slice cultures while mechanically lesioning the CA3 area. Electrophysiological analysis revealed that traumatic injury massively increased excitatory and inhibitory synaptic activity in the entire CA3 region. Cutting the CA3 region induced highly rhythmic excitatory postsynaptic currents (EPSCs) that reached frequencies of around 70 Hz. Blocking voltage-dependent sodium channels with tetrodotoxin prevented the increase in synaptic activity and injury-induced neurotransmitter release in CA3 remote from the lesion site. With fast synaptic transmission blocked only neurons in the immediate vicinity of a lesion depolarized and fired action potentials upon mechanical damage. I hence suggest that mechanical injury damages the membrane and induces action potential firing in only a small population of neurons. This activity is then propagated throughout the undamaged CA3 network inducing highly rhythmic discharges. Thus mechanical brain injury initiates immediate functional changes that exceed the lesion site.

Large Scale Simulations of Hippocampal-Neocortical Interactions in a Parallel Version of Genesis

A hippocampal-CA3 memory model was constructed with PGENESIS, a recently developed version of GENESIS that allows for distributed processing of a neural network simulation. A number of neural models of the human memory system have identified the CA3 region of the hippocampus as storing the declarative memory trace. However, computational models designed to assess the viability of the putative mechanisms of storage and retrieval have generally been too abstract to allow comparison with empirical data. Recent experimental evidence has shown that selective knock-out of NMDA receptors in the CA1 of mice leads to reduced stability of firing specificity in place cells. Here a similar reduction of stability of input specificity is demonstrated in a biologically plausible neural network model of the CA3 region, under conditions of Hebbian synaptic plasticity versus an absence of plasticity. The CA3 region is also commonly associated with seizure activity. Further simulations of the same model tested the response to continuously repeating versus randomized nonrepeating input patterns. Each paradigm delivered input of equal intensity and duration. Non-repeating input patterns elicited a greater pyramidal cell spike count. This suggests that repetitive versus non-repeating neocortical inpus has a quantitatively different effect on the hippocampus. This may be relevant to the production of independent epileptogenic zones and the process of encoding new memories.

Increased hippocampal excitability and impaired spatial memory function in mice lacking VGLUT2 selectively in neurons defined by tyrosine hydroxylase promoter activity

Three populations of neurons expressing the vesicular glutamate transporter 2 (Vglut2) were recently described in the A10 area of the mouse midbrain, of which two populations were shown to express the gene encoding, the rate-limiting enzyme for catecholamine synthesis, tyrosine hydroxylase (TH).One of these populations (‘‘TH– Vglut2 Class1’’) also expressed the dopamine transporter (DAT) gene while one did not ("TH–Vglut2 Class2"), and the remaining population did not express TH at all ("TH-Vglut2-only"). TH is known to be expressed by a promoter which shows two phases of activation, a transient one early during embryonal development, and a later one which gives rise to stable endogenous expression of the TH gene. The transient phase is, however, not specific to catecholaminergic neurons, a feature taken to advantage here as it enabled Vglut2 gene targeting within all three A10 populations expressing this gene, thus creating a new conditional knockout. These knockout mice showed impairment in spatial memory function. Electrophysiological analyses revealed a profound alteration of oscillatory activity in the CA3 region of the hippocampus. In addition to identifying a novel role for Vglut2 in hippocampus function, this study points to the need for improved genetic tools for targeting of the diversity of subpopulations of the A10 area

C-terminal processing of the teneurin proteins: Independent actions of a teneurin C-terminal associated peptide in hippocampal cells

Many neuropsychiatric conditions have a common set of neurological substrates associated with the integration of sensorimotor processing. The teneurins are a recently described family of proteins that play a significant role in visual and auditory development. Encoded on the terminal exon of the teneurin genes is a family of bioactive peptides, termed teneurin C-terminal associated peptides (TCAP), which regulate mood-disorder associated behaviors. Thus, the teneurin-TCAP system could represent a novel neurological system underlying the origins of a number of complex neuropsychiatric conditions. However, it is not known if TCAP-1 exerts its effects as part of a direct teneurin function, whereby TCAP represents a functional region of the larger teneurin protein, or if it has an independent role, either as a splice variant or post-translational proteolytic cleavage product of teneurin. In this study, we show that TCAP-1 can be transcribed as a smaller mRNA transcript. After translation, further processing yields a smaller 15. kDa protein containing the TCAP-1 region. In the mouse hippocampus, immunoreactive (ir) TCAP-1 is exclusively localized to the pyramidal layers of the CA1, CA2 and CA3 regions. Although the localization of TCAP and teneurin in hippocampal regions is similar, they are distinct within the cell as most ir-teneurin is found at the plasma membrane, whereas ir-TCAP-1 is predominantly found in the cytosol. Moreover, in mouse embryonic hippocampal cell culture, FITC-labeled TCAP-1 binds to the plasma membrane and is taken up into the cytosol via dynamin-dependent caveolae-mediated endocytosis. Our data provides novel evidence that TCAP-1 is structurally and functionally distinct from the larger teneurins. © 2012.

Fine structure of hippocampal mossy fiber synapses following rapid high-pressure freezing

Synapses of hippocampal neurons play important roles in learning and memory processes and are involved in aberrant hippocampal function in temporal lobe epilepsy. Major neuronal types in the hippocampus as well as their input and output synapses are well known, but it has remained an open question to what extent conventional electron microscopy (EM) has provided us with the real appearance of synaptic fine structure under in vivo conditions. There is reason to assume that conventional aldehyde fixation and dehydration lead to protein denaturation and tissue shrinkage, likely associated with the occurrence of artifacts. However, realistic fine-structural data of synapses are required for our understanding of the transmission process and for its simulation. Here, we used high-pressure freezing and cryosubstitution of hippocampal tissue that was not subjected to aldehyde fixation and dehydration in ethanol to monitor the fine structure of an identified synapse in the hippocampal CA3 region, that is, the synapse between granule cell axons, the mossy fibers, and the proximal dendrites of CA3 pyramidal neurons. Our results showed that high-pressure freezing nicely preserved ultrastructural detail of this particular synapse and allowed us to study rapid structural changes associated with synaptic plasticity.

Tracking the Flow of Information through the Hippocampal Formation in the Rat

The hippocampus receives input from upper levels of the association cortex and is implicated in many mnemonic processes, but the exact mechanisms by which it codes and stores information is an unresolved topic. This work examines the flow of information through the hippocampal formation while attempting to determine the computations that each of the hippocampal subfields performs in learning and memory. The formation, storage, and recall of hippocampal-dependent memories theoretically utilize an autoassociative attractor network that functions by implementing two competitive, yet complementary, processes. Pattern separation, hypothesized to occur in the dentate gyrus (DG), refers to the ability to decrease the similarity among incoming information by producing output patterns that overlap less than the inputs. In contrast, pattern completion, hypothesized to occur in the CA3 region, refers to the ability to reproduce a previously stored output pattern from a partial or degraded input pattern. Prior to addressing the functional role of the DG and CA3 subfields, the spatial firing properties of neurons in the dentate gyrus were examined. The principal cell of the dentate gyrus, the granule cell, has spatially selective place fields; however, the behavioral correlates of another excitatory cell, the mossy cell of the dentate polymorphic layer, are unknown. This report shows that putative mossy cells have spatially selective firing that consists of multiple fields similar to previously reported properties of granule cells. Other cells recorded from the DG had single place fields. Compared to cells with multiple fields, cells with single fields fired at a lower rate during sleep, were less likely to burst, and were more likely to be recorded simultaneously with a large population of neurons that were active during sleep and silent during behavior. These data suggest that single-field and multiple-field cells constitute at least two distinct cell classes in the DG. Based on these characteristics, we propose that putative mossy cells tend to fire in multiple, distinct locations in an environment, whereas putative granule cells tend to fire in single locations, similar to place fields of the CA1 and CA3 regions. Experimental evidence supporting the theories of pattern separation and pattern completion comes from both behavioral and electrophysiological tests. These studies specifically focused on the function of each subregion and made implicit assumptions about how environmental manipulations changed the representations encoded by the hippocampal inputs. However, the cell populations that provided these inputs were in most cases not directly examined. We conducted a series of studies to investigate the neural activity in the entorhinal cortex, dentate gyrus, and CA3 in the same experimental conditions, which allowed a direct comparison between the input and output representations. The results show that the dentate gyrus representation changes between the familiar and cue altered environments more than its input representations, whereas the CA3 representation changes less than its input representations. These findings are consistent with longstanding computational models proposing that (1) CA3 is an associative memory system performing pattern completion in order to recall previous memories from partial inputs, and (2) the dentate gyrus performs pattern separation to help store different memories in ways that reduce interference when the memories are subsequently recalled.

Activation of metabotropic glutamate 5 and NMDA receptors underlies the induction of persistent bursting and associated long-lasting changes in CA3 recurrent connections.

The aim of this study was to describe the induction and expression mechanisms of a persistent bursting activity in a horizontal slice preparation of the rat limbic system that includes the ventral part of the hippocampus and the entorhinal cortex. Disinhibition of this preparation by bicuculline led to interictal-like bursts in the CA3 region that triggered synchronous activity in the entorhinal cortex. Washout of bicuculline after a 1 hr application resulted in a maintained production of hippocampal bursts that continued to spread to the entorhinal cortex. Separation of CA3 from the entorhinal cortex caused the activity in the latter to become asynchronous with CA3 activity in the presence of bicuculline and disappear after washout; however, in CA3, neither the induction of bursting nor its persistence were affected. Associated with the CA3 persistent bursting, a strengthening of recurrent collateral excitatory input to CA3 pyramidal cells and a decreased input to CA3 interneurons was found. Both the induction of the persistent bursting and the changes in synaptic strength were prevented by antagonists of metabotropic glutamate 5 (mGlu5) or NMDA receptors or protein synthesis inhibitors and did not occur in slices from mGlu5 receptor knock-out mice. The above findings suggest potential synaptic mechanisms by which the hippocampus switches to a persistent interictal bursting mode that may support a spread of interictal-like bursting to surrounding temporal lobe regions.

Increased hippocampal excitability and impaired spatial memory function in mice lacking VGLUT2 selectively in neurons defined by tyrosine hydroxylase promoter activity

Three populations of neurons expressing the vesicular glutamate transporter 2 (Vglut2) were recently described in the A10 area of the mouse midbrain, of which two populations were shown to express the gene encoding, the rate-limiting enzyme for catecholamine synthesis, tyrosine hydroxylase (TH).One of these populations (‘‘TH– Vglut2 Class1’’) also expressed the dopamine transporter (DAT) gene while one did not ("TH–Vglut2 Class2"), and the remaining population did not express TH at all ("TH-Vglut2-only"). TH is known to be expressed by a promoter which shows two phases of activation, a transient one early during embryonal development, and a later one which gives rise to stable endogenous expression of the TH gene. The transient phase is, however, not specific to catecholaminergic neurons, a feature taken to advantage here as it enabled Vglut2 gene targeting within all three A10 populations expressing this gene, thus creating a new conditional knockout. These knockout mice showed impairment in spatial memory function. Electrophysiological analyses revealed a profound alteration of oscillatory activity in the CA3 region of the hippocampus. In addition to identifying a novel role for Vglut2 in hippocampus function, this study points to the need for improved genetic tools for targeting of the diversity of subpopulations of the A10 area

Increased hippocampal excitability and impaired spatial memory function in mice lacking VGLUT2 selectively in neurons defined by tyrosine hydroxylase promoter activity

Three populations of neurons expressing the vesicular glutamate transporter 2 (Vglut2) were recently described in the A10 area of the mouse midbrain, of which two populations were shown to express the gene encoding, the rate-limiting enzyme for catecholamine synthesis, tyrosine hydroxylase (TH).One of these populations (‘‘TH– Vglut2 Class1’’) also expressed the dopamine transporter (DAT) gene while one did not ("TH–Vglut2 Class2"), and the remaining population did not express TH at all ("TH-Vglut2-only"). TH is known to be expressed by a promoter which shows two phases of activation, a transient one early during embryonal development, and a later one which gives rise to stable endogenous expression of the TH gene. The transient phase is, however, not specific to catecholaminergic neurons, a feature taken to advantage here as it enabled Vglut2 gene targeting within all three A10 populations expressing this gene, thus creating a new conditional knockout. These knockout mice showed impairment in spatial memory function. Electrophysiological analyses revealed a profound alteration of oscillatory activity in the CA3 region of the hippocampus. In addition to identifying a novel role for Vglut2 in hippocampus function, this study points to the need for improved genetic tools for targeting of the diversity of subpopulations of the A10 area

Modulation of network excitability and epileptiform activity in the hippocampus of immature rats by the activation of GlyRs

Epileptic seizures are the manifestations of epilepsy, which is a major neurological disorder and occurs with a high incidence during early childhood. A fundamental mechanism underlying epileptic seizures is loss of balance between neural excitation and inhibition toward overexcitation. Glycine receptor (GlyR) is ionotropic neurotransmitter receptor that upon binding of glycine opens an anion pore and mediates in the adult nervous system a consistent inhibitory action. While previously it was assumed that GlyRs mediate inhibition mainly in the brain stem and spinal cord, recent studies reported the abundant expression of GlyRs throughout the brain, in particular during neuronal development. But no information is available regarding whether activation of GlyRs modulates neural network excitability and epileptiform activities in the immature central nervous system (CNS). Therefore the study in this thesis addresses the role of GlyRs in the modulation of neuronal excitability and epileptiform activity in the immature rat brain. By using in vitro intact corticohippocampal formation (CHF) of rats at postnatal days 4-7 and electrophysiological methods, a series of pharmacological examinations reveal that GlyRs are directly implicated in the control of hippocampal excitation levels at this age. In this thesis I am able to show that GlyRs are functionally expressed in the immature hippocampus and exhibit the classical pharmacology of GlyR, which can be activated by both glycine and the presumed endogenous agonist taurine. This study also reveals that high concentration of taurine is anticonvulsive, but lower concentration of taurine is proconvulsive. A substantial fraction of both the pro- and anticonvulsive effects of taurine is mediated via GlyRs, although activation of GABAA receptors also considerably contributes to the taurine effects. Similarly, glycine exerts both pro- and anticonvulsive effects at low and high concentrations, respectively. The proconvulsive effects of taurine and glycine depend on NKCC1-mediated Cl- accumulation, as bath application of NKCC1 inhibitor bumetanide completely abolishes proconvulsive effects of low taurine and glycine concentrations. Inhibition of GlyRs with low concentration of strychnine triggers epileptiform activity in the CA3 region of immature CHF, indicating that intrinsically an inhibitory action of GlyRs overwhelms its depolarizing action in the immature hippocampus. Additionally, my study indicates that blocking taurine transporters to accumulate endogenous taurine reduces epileptiform activity via activation of GABAA receptors, but not GlyRs, while blocking glycine transporters has no observable effect on epileptiform activity. From the main results of this study it can be concluded that in the immature rat hippocampus, activation of GlyRs mediates both pro- and anticonvulsive effects, but that a persistent activation of GlyRs is required to prevent intrinic neuronal overexcitability. In summary, this study uncovers an important role of GlyRs in the modulation of neuronal excitability and epileptiform activity in the immature rat hippocampus, and indicates that glycinergic system can potentially be a new therapeutic target against epileptic seizures of children.

A presynaptic locus for long-term potentiation of elementary synaptic transmission at mossy fiber synapses in culture.

The complex circuitry of the CA3 region and the abundance of collateral connections has made it difficult to study the mossy fiber pathway in hippocampal slices and therefore to establish the site of expression of long-term potentiation at these synapses. Using a novel cell culture system, we have produced long-term potentiation of the elementary synaptic connections on single CA3 pyramidal neurons following tetanic stimulation of individual dentate gyrus granule cells. As is the case for the hippocampal slice, this potentiation was independent of N-methyl-D-aspartate receptor activation, was simulated by application of forskolin, and its induction did not require any modulatory input. The increase in synaptic strength was accompanied by a reduction in the number of failures of transmission and by an increase in the coefficient of variation of the responses and was prevented by presynaptic injection of an inhibitor of protein kinase A. These findings show that mossy fiber long-term potentiation has a presynaptic locus and that its expression is dependent on protein kinase A.

Lobe specific Ca2+-calmodulin nano-domain in neuronal spines: a single molecule level analysis.

Calmodulin (CaM) is a ubiquitous Ca(2+) buffer and second messenger that affects cellular function as diverse as cardiac excitability, synaptic plasticity, and gene transcription. In CA1 pyramidal neurons, CaM regulates two opposing Ca(2+)-dependent processes that underlie memory formation: long-term potentiation (LTP) and long-term depression (LTD). Induction of LTP and LTD require activation of Ca(2+)-CaM-dependent enzymes: Ca(2+)/CaM-dependent kinase II (CaMKII) and calcineurin, respectively. Yet, it remains unclear as to how Ca(2+) and CaM produce these two opposing effects, LTP and LTD. CaM binds 4 Ca(2+) ions: two in its N-terminal lobe and two in its C-terminal lobe. Experimental studies have shown that the N- and C-terminal lobes of CaM have different binding kinetics toward Ca(2+) and its downstream targets. This may suggest that each lobe of CaM differentially responds to Ca(2+) signal patterns. Here, we use a novel event-driven particle-based Monte Carlo simulation and statistical point pattern analysis to explore the spatial and temporal dynamics of lobe-specific Ca(2+)-CaM interaction at the single molecule level. We show that the N-lobe of CaM, but not the C-lobe, exhibits a nano-scale domain of activation that is highly sensitive to the location of Ca(2+) channels, and to the microscopic injection rate of Ca(2+) ions. We also demonstrate that Ca(2+) saturation takes place via two different pathways depending on the Ca(2+) injection rate, one dominated by the N-terminal lobe, and the other one by the C-terminal lobe. Taken together, these results suggest that the two lobes of CaM function as distinct Ca(2+) sensors that can differentially transduce Ca(2+) influx to downstream targets. We discuss a possible role of the N-terminal lobe-specific Ca(2+)-CaM nano-domain in CaMKII activation required for the induction of synaptic plasticity.

Distinct short-term plasticity at two excitatory synapses in the hippocampus

A single mossy fiber input contains several release sites and is located on the proximal portion of the apical dendrite of CA3 neurons. It is, therefore, well suited to exert a strong influence on pyramidal cell excitability. Accordingly, the mossy fiber synapse has been referred to as a detonator or teacher synapse in autoassociative network models of the hippocampus. The very low firing rates of granule cells [Jung, M. W. & McNaughton, B. L. (1993) Hippocampus 3, 165–182], which give rise to the mossy fibers, raise the question of how the mossy fiber synapse temporally integrates synaptic activity. We have therefore addressed the frequency dependence of mossy fiber transmission and compared it to associational/commissural synapses in the CA3 region of the hippocampus. Paired pulse facilitation had a similar time course, but was 2-fold greater for mossy fiber synapses. Frequency facilitation, during which repetitive stimulation causes a reversible growth in synaptic transmission, was markedly different at the two synapses. At associational/commissural synapses facilitation occurred only at frequencies greater than once every 10 s and reached a magnitude of about 125% of control. At mossy fiber synapses, facilitation occurred at frequencies as low as once every 40 s and reached a magnitude of 6-fold. Frequency facilitation was dependent on a rise in intraterminal Ca2+ and activation of Ca2+/calmodulin-dependent kinase II, and was greatly reduced at synapses expressing mossy fiber long-term potentiation. These results indicate that the mossy fiber synapse is able to integrate granule cell spiking activity over a broad range of frequencies, and this dynamic range is substantially reduced by long-term potentiation.