Systemic lipopolysaccharide administration impairs retrieval of context-object discrimination, but not spatial, memory: Evidence for selective disruption of specific hippocampus-dependent memory functions during acute neuroinflammation.

Neuroinflammation is implicated in impairments in neuronal function and cognition that arise with aging, trauma, and/or disease. Therefore, understanding the underlying basis of the effect of immune system activation on neural function could lead to therapies for treating cognitive decline. Although neuroinflammation is widely thought to preferentially impair hippocampus-dependent memory, data on the effects of cytokines on cognition are mixed. One possible explanation for these inconsistent results is that cytokines may disrupt specific neural processes underlying some forms of memory but not others. In an earlier study, we tested the effect of systemic administration of bacterial lipopolysaccharide (LPS) on retrieval of hippocampus-dependent context memory and neural circuit function in CA3 and CA1 (Czerniawski and Guzowski, 2014). Paralleling impairment in context discrimination memory, we observed changes in neural circuit function consistent with disrupted pattern separation function. In the current study we tested the hypothesis that acute neuroinflammation selectively disrupts memory retrieval in tasks requiring hippocampal pattern separation processes. Male Sprague-Dawley rats given LPS systemically prior to testing exhibited intact performance in tasks that do not require hippocampal pattern separation processes: novel object recognition and spatial memory in the water maze. By contrast, memory retrieval in a task thought to require hippocampal pattern separation, context-object discrimination, was strongly impaired in LPS-treated rats in the absence of any gross effects on exploratory activity or motivation. These data show that LPS administration does not impair memory retrieval in all hippocampus-dependent tasks, and support the hypothesis that acute neuroinflammation impairs context discrimination memory via disruption of pattern separation processes in hippocampus.

Temporal dynamics of Arc gene induction in hippocampus: relationship to context memory formation.

Past studies have proposed a role for the hippocampus in the rapid encoding of context memories. Despite this, there is little data regarding the molecular processes underlying the stable formation of a context representation that occurs in the time window established through such behavioral studies. One task that is useful for investigating the rapid encoding of context is contextual fear conditioning (CFC). Behavioral studies demonstrate that animals require approximately 30 s of exploration prior to a footshock to form a contextual representation supporting CFC. Thus, any potential molecular process required for the stabilization of the cellular representation for context must be activated within this narrow and behaviorally defined time window. Detection of the immediate-early gene Arc presents an ideal method to assess the activation of specific neuronal ensembles, given past studies showing the context specific expression of Arc in CA3 and CA1 subfields and the role of Arc in hippocampal long-term synaptic plasticity. Therefore, we examined the temporal dynamics of Arc induction within the hippocampus after brief context exposure to determine whether experience-dependent Arc expression could be involved in the rapid encoding of incidental context memories. We found that the duration of context exposure differentially activated Arc expression in hippocampal subfields, with CA3 showing rapid engagement within as little as 3 s of exposure. By contrast, Arc induction in CA1 required 30 s of context exposure to reach maximal levels. A parallel behavioral experiment revealed that 30 s, but not 3 s, exposure to a context resulted in strong conditioned freezing 24 h later, consistent with past studies from other laboratories. The current study is the first to examine the rapid temporal dynamics of Arc induction in hippocampus in a well-defined context memory paradigm. These studies demonstrate within 30 s of context exposure Arc is fully activated in CA3 and CA1, suggesting that the engagement of plastic processes requiring Arc function (such as long-term potentiation) occurs within the same temporal domain as that required for behavioral conditioning.

Loss of activity-dependent Arc gene expression in the retrosplenial cortex after hippocampal inactivation: interaction in a higher-order memory circuit.

The rodent hippocampus is well known for its role in spatial navigation and memory, and recent evidence points to the retrosplenial cortex (RSC) as another element of a higher order spatial and mnemonic circuit. However, the functional interplay between hippocampus and RSC during spatial navigation remains poorly understood. To investigate this interaction, we examined cell activity in the RSC during spatial navigation in the water maze before and after acute hippocampal inactivation using expression of two immediate-early genes (IEGs), Arc and Homer 1a (H1a). Adult male rats were trained in a spatial water maze task for 4 days. On day 5, the rats received two testing/training sessions separated by 20 min. Eight minutes before the second session, different groups of rats received bilateral intrahippocampal infusion of tetrodotoxin (TTX), muscimol (MUS), or vehicle. Another group of rats (uni-TTX) received infusion of TTX in one hippocampus and vehicle in the other. Signals from Arc and H1a RNA probes correspond to the post- and pre-infusion sessions, respectively. Bilateral TTX and MUS impaired spatial memory, as expected, and decreased Arc expression in CA1 of hippocampus. Importantly, bilateral inactivation of hippocampus resulted in loss of behavior-induced Arc expression in RSC. Despite a lateralized effect in CA1, Arc expression was equivalently and bilaterally decreased in RSC of uni-TTX rats, consistent with a network level interaction between hippocampus and RSC. We conclude that the loss of hippocampal input alters activity of RSC neurons and compromises their ability to engage plastic processes dependent on IEG expression.

Rapid activation of plasticity-associated gene transcription in hippocampal neurons provides a mechanism for encoding of one-trial experience.

The hippocampus is hypothesized to support rapid encoding of ongoing experience. A critical prerequisite for such function is the ability to readily recruit enduring synaptic plasticity in hippocampal neurons. Hippocampal long-term potentiation (LTP) and memory consolidation require expression of the immediate-early gene (IEG) Arc. To determine whether Arc transcription could be driven by limited and controlled behavioral experience, we used a rectangular track paradigm. In past electrophysiological studies, pyramidal neurons recorded from rats running in one direction on similar tracks typically exhibited a single firing field. Using fluorescence in situ hybridization, we show that the behavioral activity associated with a single lap around the track was sufficient to trigger Arc transcription in complete CA3 neuronal ensembles, as predicted given the role of CA3 in one-trial learning. In contrast, Arc transcription in CA1 ensembles was recruited incrementally, with maximal activation achieved after four laps a day for 4 consecutive days. To test whether Arc transcription is linked to learning and plasticity, or merely elicited by location-specific firing, we inactivated the medial septum, a treatment that compromises hippocampus-dependent learning and LTP but spares location-specific firing in CA1 neurons. Septal inactivation abolished track training-induced Arc transcription in CA1 and CA3 neurons, showing that Arc transcription requires plasticity-inducing stimuli. Accordingly, LTP induction activated Arc transcription in CA1 neurons in vivo. These findings demonstrate for the first time that a single brief experience, equivalent to a single crossing of a firing field, can trigger IEG expression required for long-term plasticity in the hippocampus.

Networks of neurons, networks of genes: an integrated view of memory consolidation.

Investigations into the mechanisms of memory formation have abided by the central tenet of the consolidation theory-that memory formation occurs in stages which differ in their requirement for protein synthesis. The current most widely accepted hypothesis posits that new memories are encoded as neural activity-induced changes in synaptic efficacy, and stabilization of these changes requires de novo protein synthesis. However, the basic assumptions of this view have been challenged by concerns regarding the specificity of the effects of the protein synthesis inhibitors used to support the claim. Studies on immediate-early genes (IEGs), in particular Arc, provide a distinct and independent perspective on the issue of the requirement of new protein synthesis in synaptic plasticity and memory consolidation. The IEG Arc and its protein are dynamically induced in response to neuronal activity, and are directly involved in synaptic plasticity and memory consolidation. Although we provide extensive data on Arc's properties to address the requirement of genomic and proteomic responses in memory formation, Arc is merely one element in a network of genes that interact in a coordinated fashion to serve memory consolidation. From gene expression and other studies, we propose the view that the stabilization of a memory trace is a continuous and ongoing process, which does not have a discrete endpoint and cannot be reduced to a single deterministic "molecular cascade". Rather, memory traces are maintained within metastable networks, which must integrate and update past traces with new ones. Such an updating process may well recruit and use many of the plasticity mechanisms necessary for the initial encoding of memory.

Using immediate-early genes to map hippocampal subregional functions.

Different functions have been suggested for the hippocampus and its subdivisions along both transversal and longitudinal axes. Expression of immediate-early genes (IEGs) has been used to map specific functions onto neuronal activity in different areas of the brain including the hippocampus (IEG imaging). Here we review IEG studies on hippocampal functional dissociations with a particular focus on the CA3 subregion. We first discuss the cellular functions of IEGs and the brain system interactions that govern their dynamic expression in hippocampal neurons to provide a more solid framework for interpreting the findings from IEG studies. We show the pitfalls and shortcomings of conventional IEG imaging studies and describe advanced methods using IEGs for imaging of neuronal activity or functional intervention. We review the current IEG evidence of hippocampal function, subregional-specific contribution to different stages of memory formation, systems consolidation, functional dissociation between memory and anxiety/behavioral inhibition along the septotemporal axis, and different neural network properties of hippocampal subregions. In total, IEG studies provide support for (1) the role of the hippocampus in spatial and contextual learning and memory, (2) its role in continuous encoding of ongoing experience, (3) septotemporal dissociations between memory and anxiety, and (4) a dynamic relationship between pattern separation and pattern completion in the CA3 subregion. In closing, we provide a framework for how cutting-edge IEG imaging and intervention techniques will likely contribute to better understanding of the specific functions of CA3 and other hippocampal subregions.

Expression and function of SNAP-25 as a universal SNARE component in GABAergic neurons.

Intracellular vesicular trafficking and membrane fusion are important processes for nervous system development and for the function of neural circuits. Synaptosomal-associated protein 25 kDa (SNAP-25) is a component of neural soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) core complexes that mediate the exocytotic release of neurotransmitters at chemical synapses. Previous results from mouse mutant models and pharmacological/neurotoxin blockades have demonstrated a critical role for SNAP-25-containing SNARE complexes in action potential (AP)-dependent release at cholinergic and glutamatergic synapses and for calcium-triggered catecholamine release from chromaffin cells. To examine whether SNAP-25 participates in the evoked release of other neurotransmitters, we investigated the expression and function of SNAP-25 in GABAergic terminals. Patch-clamp recordings in fetal Snap25-null mutant cortex demonstrated that ablation of SNAP-25 eliminated evoked GABA(A) receptor-mediated postsynaptic responses while leaving a low level of spontaneous AP-independent events intact, supporting the involvement of SNAP-25 in the regulated synaptic transmission of early developing GABAergic neurons. In hippocampal cell cultures of wild-type mice, punctate staining of SNAP-25 colocalized with both GABAergic and glutamatergic synaptic markers, whereas stimulus-evoked vesicular recycling was abolished at terminals of both transmitter phenotypes in Snap25-/- neurons. Moreover, immunohistochemistry and fluorescence in situ hybridization revealed coexpression of SNAP-25, VGAT (vesicular GABA transporter), and GAD65/67 (glutamic acid decarboxylase 65/67) in interneurons within several regions of the adult brain. Our results thus provide evidence that SNAP-25 is critical for evoked GABA release during development and is expressed in the presynaptic terminals of mature GABAergic neurons, consistent with its function as a component of a fundamental core SNARE complex required for stimulus-driven neurotransmission.

Recent behavioral history modifies coupling between cell activity and Arc gene transcription in hippocampal CA1 neurons.

The ability of neurons to alter their transcriptional programs in response to synaptic input is of fundamental importance to the neuroplastic mechanisms underlying learning and memory. Because of technical limitations of conventional gene detection methods, the current view of activity-dependent neural transcription derives from experiments in which neurons are assumed quiescent until a signaling stimulus is given. The present study was designed to move beyond this static model by examining how earlier episodes of neural activity influence transcription of the immediate-early gene Arc. Using a sensitive FISH method that detects primary transcript at genomic alleles, the proportion of hippocampal CA1 neurons that activate transcription of Arc RNA was constant at approximately 40% in response to both a single novel exploration session and daily sessions repeated over 9 days. This proportion is similar to the percentage of active neurons defined electrophysiologically. However, this close correspondence was disrupted in rats exposed briefly, but repeatedly, to the same environment within a single day. Arc transcription in CA1 neurons declined dramatically after as few as four 5-min sessions, despite stable electrophysiological activity during all sessions. Additional experiments indicate that the decrement in Arc transcription occurred at the cellular, rather than synaptic level, and was not simply linked to habituation to novelty. Thus, the neural genomic response is governed by recent, but not remote, cell firing history in the behaving animal. This state-dependence of neuronal transcriptional coupling provides a mechanism of metaplasticity and may regulate capacity for synaptic modification in neural networks.

Memory-influencing intra-basolateral amygdala drug infusions modulate expression of Arc protein in the hippocampus.

Activation of beta-adrenoceptors in the basolateral complex of the amygdala (BLA) modulates memory storage processes and long-term potentiation in downstream targets of BLA efferents, including the hippocampus. Here, we show that this activation also increases hippocampal levels of activity-regulated cytoskeletal protein (Arc), an immediate-early gene (also termed Arg 3.1) implicated in hippocampal synaptic plasticity and memory consolidation processes. Infusions of the beta-adrenoreceptor agonist, clenbuterol, into the BLA immediately after training on an inhibitory avoidance task enhanced memory tested 48 h later. The same dose of clenbuterol significantly increased Arc protein levels in the dorsal hippocampus. Additionally, posttraining intra-BLA infusions of a memory-impairing dose of lidocaine significantly reduced Arc protein levels in the dorsal hippocampus. Increases in Arc protein levels were not accompanied by increases in Arc mRNA, suggesting that amygdala modulation of Arc protein and synaptic plasticity in efferent brain regions occurs at a posttranscriptional level. Finally, infusions of Arc antisense oligodeoxynucleotides into the dorsal hippocampus impaired performance of an inhibitory avoidance task, indicating that the changes in Arc protein expression are related to the observed changes in memory performance.

Peripheral arousal-related hormones modulate norepinephrine release in the hippocampus via influences on brainstem nuclei.

The peripheral hormone epinephrine (EPI) is known to modulate memory for arousing experiences. The mnemonic effects of EPI are attributed almost exclusively to actions on amygdala noradrenergic (NE) systems. EPI also increases neuronal activity in the locus coeruleus (LC), the primary source of NE to other limbic structures that process memory such as the hippocampus (HIPP). The actions of EPI on the LC suggest that its mnemonic properties may also be mediated by influencing NE output in the HIPP. To test this hypothesis, dialysate levels of NE were collected from the HIPP of male rats given an i.p. injection of saline that was followed 100 min later by i.p. EPI (0.3 mg/kg). NE levels sampled 20 min after EPI injection were significantly larger than baseline and continued to show significant peaks for 60 min. Experiment 2 examined whether peripheral signals initiated by EPI influence the HIPP via the nucleus of the solitary tract (NTS) by inactivating this nucleus with lidocaine prior to EPI injection. EPI injection did not increase NE levels sampled from the HIPP of rats given lidocaine into the NTS. EPI injection did produce significant elevations in HIPP NE levels in animals given a control solution into the NTS prior to the EPI injection. These findings indicate that the mnemonic effects of EPI reported in a wide range of learning conditions may not be mediated solely by NE release in the amygdala, but may also involve coactivation of the HIPP NE system.

Enhancement of noradrenergic neurotransmission in the nucleus of the solitary tract modulates memory storage processes.

These studies examined whether posttraining activation of alpha1-noradrenergic receptors in the nucleus tractus solitarius (NTS) influences neural processes that are involved in encoding information into memory. Different groups of male Sprague-Dawley rats were trained in two separate learning tasks. In experiment 1, rats were given either a control solution or the alpha1-noradrenergic agonist phenylephrine (0.5, 1.0, 5.0, or 10 microg/0.5 microl) directly into the NTS immediately after they were given a footshock (0.35 mA, 0.5 s) in the dark compartment of an inhibitory apparatus. In a retention test given 48 h later, groups that received either 5.0 or 10.0 microg of phenylephrine avoided the dark compartment for a significantly longer period of time than the PBS control group (P<0.05 and P<0.01, respectively). In experiment 2, identical doses of phenylephrine were infused in the NTS following footshock delivery in one alley of a Y-maze. Animals given either 1.0 or 5.0 microg of phenylephrine performed significantly better than PBS controls on several different measures that served as indices of retention. The results indicate that activation of alpha1-noradrenergic receptors in the NTS plays a critical role in the transmission of signals from the periphery to brain systems that process memory for emotionally significant experiences.

Glutamatergic transmission in the nucleus of the solitary tract modulates memory through influences on amygdala noradrenergic systems.

The authors examined whether glutamate release from the vagus nerve onto the nucleus of the solitary tract (NTS) is one mechanism by which the vagus influences memory and neural activity in limbic structures. Rats trained to drink from a spout were given a footshock (0.35 mA) on Day 5 after approaching the spout. Phosphate-buffered saline or 5.0, 50.0, or 100.0 nmol/0.5 microl glutamate was then infused into the NTS. Glutamate (5.0 or 50.0 nmol) significantly enhanced memory on the retention test. In Experiment 2, this effect was attenuated by blocking noradrenergic receptors in the amygdala with propranolol (0.3 microg/0.5 microl). Experiment 3 used in vivo microdialysis to determine whether footshock plus glutamate (50.0 nmol) alters noradrenergic output in the amygdala. These treatments caused a significant and long-lasting increase in amygdala noradrenergic concentrations. The results indicate that glutamate may be one transmitter that conveys the effects of vagal activation on brain systems that process memory.