Basal forebrain and prelimbic cortex connectivity is related to behavioral response in an attention task.

The basal forebrain (BF) is critical for the motivational recruitment of attention in response to reward-related cues. This finding is consistent with a role for the BF in encoding and transmitting motivational salience and readying prefrontal circuits for further attentional processing. We recorded local field potentials to determine connectivity between prelimbic cortex (PrL) and BF during the modulation of attention by reward-related cues. We find that theta and gamma power are robustly associated with behavior. Power in both bands is significantly lower during trials in which an incorrect behavioral response is made. We find strong coherence during responses that are significantly stronger when a correct response is made. We show that information flow is largely monodirectional from BF to and is strongest when correct responses are made. These experiments demonstrate that connectivity between BF and the PrL increases during periods of increased motivational recruitment of attentional resources.

Disrupted hippocampal synchrony following maternal immune activation in a rat model.

Maternal immune activation (MIA) is a risk factor for schizophrenia and other neurodevelopmental disorders. MIA in rats models a number of the brain and behavioral changes that are observed in schizophrenia, including impaired memory. Recent studies in the MIA model have shown that the firing of the hippocampal place cells that are involved in memory processes appear relatively normal, but with abnormalities in the temporal ordering of firing. In this study, we re-analyzed data from prior hippocampal electrophysiological recordings of MIA and control animals to determine whether temporal dysfunction was evident. We find that there is a decreased ratio of slow to fast gamma power, resulting from an increase in fast gamma power and a tendency toward reduced slow gamma power in MIA rats. Moreover, we observe a robust reduction in spectral coherence between hippocampal theta and both fast and slow gamma rhythms, as well as changes in the phase of theta at which fast gamma occurs. We also find the phasic organization of place cell phase precession on the theta wave to be abnormal in MIA rats. Lastly, we observe that the local field potential of MIA rats contains more frequent sharp-wave ripple events, and that place cells were more likely to fire spikes during ripples in these animals than control. These findings provide further evidence of desynchrony in MIA animals and may point to circuit-level changes that underlie failures to integrate and encode information in schizophrenia.

Non-Canonical Roles of Apoptotic Caspases in the Nervous System.

Caspases are a family of cysteine proteases that predominantly cleave their substrates after aspartic acid residues. Much of what we know of caspases emerged from investigation a highly conserved form of programmed cell death called apoptosis. This form of cell death is regulated by several caspases, including caspase-2, caspase-3, caspase-7, caspase-8 and caspase-9. However, these "killer" apoptotic caspases have emerged as versatile enzymes that play key roles in a wide range of non-apoptotic processes. Much of what we understand about these non-apoptotic roles is built on work investigating how "killer" caspases control a range of neuronal cell behaviors. This review will attempt to provide an up to date synopsis of these roles.

Hyper-Rigid Phasic Organization of Hippocampal Activity But Normal Spatial Properties of CA1 Place Cells in the Ts65Dn Mouse Model of Down Syndrome.

Down syndrome (DS) in humans is caused by trisomy of chromosome 21 and is marked by prominent difficulties in learning and memory. Decades of research have demonstrated that the hippocampus is a key structure in learning and memory, and recent work with mouse models of DS has suggested differences in hippocampal activity that may be the substrate of these differences. One of the primary functional differences in DS is thought to be an excess of GABAergic innervation from medial septum to the hippocampus. In these experiments, we probe in detail the activity of region CA1 of the hippocampus using in vivo electrophysiology in male Ts65Dn mice compared with their male nontrisomic 2N littermates. We find the spatial properties of place cells in CA1 are normal in Ts65Dn animals. However, we find that the phasic relationship of both CA1 place cells and gamma rhythms to theta rhythm in the hippocampus is profoundly altered in these mice. Since the phasic organization of place cell activity and gamma oscillations on the theta wave are thought to play a critical role in hippocampal function, the changes we observe agree with recent findings that organization of the hippocampal network is potentially of more relevance to its function than the spatial properties of place cells.SIGNIFICANCE STATEMENT Recent evidence has disrupted the view that spatial deficits are associated with place cell abnormalities. In these experiments, we record hippocampal place cells and local field potential from the Ts65Dn mouse model of Down syndrome, and find phenomenologically normal place cells, but profound changes in the association of place cells and gamma rhythms with theta rhythm, suggesting that the overall network state is critically important for hippocampal function. These findings also agree with evidence suggesting that excess inhibitory control is the cause of hippocampal dysfunction in Down syndrome. The findings also confirm new avenues for pharmacological treatment of Down syndrome.

Signalling pathways contributing to learning and memory deficits in the Ts65Dn mouse model of Down syndrome.

Down syndrome (DS) is a genetic trisomic disorder that produces life-long changes in physiology and cognition. Many of the changes in learning and memory seen in DS are reminiscent of disorders involving the hippocampal/entorhinal circuit. Mouse models of DS typically involve trisomy of murine chromosome 16 is homologous for many of the genes triplicated in human trisomy 21, and provide us with good models of changes in, and potential pharmacotherapy for, human DS. Recent careful dissection of the Ts65Dn mouse model of DS has revealed differences in key signalling pathways from the basal forebrain to the hippocampus and associated rhinal cortices, as well as changes in the microstructure of the hippocampus itself. In vivo behavioural and electrophysiological studies have shown that Ts65Dn animals have difficulties in spatial memory that mirror hippocampal deficits, and have changes in hippocampal electrophysiological phenomenology that may explain these differences, and align with expectations generated from in vitro exploration of this model. Finally, given the existing data, we will examine the possibility for pharmacotherapy for DS, and outline the work that remains to be done to fully understand this system.

Multiple head direction signals within entorhinal cortex: origin and function.

Sensory systems show hierarchical computation, starting from primary sensory receptors, with information transformed into multimodal representations as they move through subcortical and cortical brain regions. Here, we discuss recent evidence illustrating that the signaling of direction within the mammalian brain is likewise transformed and multiplexed as it progresses from subcortical regions that contain tightly direction-coupled neurons through thalamus to regions that support navigation, such as the subiculum, entorhinal cortex and hippocampus. Such transformations in the directional signal as it ascends from thalamus to higher-order regions may allow the directional system to support a repertoire of behaviors that go beyond an animal orienting in space.

Entorhinal velocity signals reflect environmental geometry.

The entorhinal cortex contains neurons that represent self-location, including grid cells that fire in periodic locations and velocity signals that encode running speed and head direction. Although the size and shape of the environment influence grid patterns, whether entorhinal velocity signals are equally influenced or provide a universal metric for self-motion across environments remains unknown. Here we report that speed cells rescale after changes to the size and shape of the environment. Moreover, head direction cells reorganize in an experience-dependent manner to align with the axis of environmental change. A knockout mouse model allows dissociation of the coordination between cell types, with grid and speed cells, but not head direction cells, responding in concert to environmental change. These results point to malleability in the coding features of multiple entorhinal cell types and have implications for which cell types contribute to the velocity signal used by computational models of grid cells.

Circadian-scale periodic bursts in theta and gamma-band coherence between hippocampus, cingulate and insular cortices.

Previous studies have demonstrated that mean activity levels in the hippocampus oscillate on a circadian timescale, both at the single neuron and EEG level. This oscillation is also entrained by the availability of food, suggesting that the circadian modulation of hippocampal activity might comprise part of the recently discovered food-entrainable circadian oscillator (FEO). In order to determine whether the circadian oscillation in hippocampal activity is linked to activity in other brain regions, we recorded field-potential EEG from hippocampus and two cortical regions known to connect to hippocampus; the anterior cingulate cortex and the agranular insular cortex. These latter regions are involved in executive control (cingulate) and gustatory feedback (insula) and so are in a position where they could usefully contribute to, or benefit from, hippocampal memorial information in order to undertake task-related processing. We recorded EEG from these three regions for 20 m every hour for 58 consecutive hours in one continuous exposure to the recording environment. We found that there are regular and distinct increases in magnitude coherence between hippocampus and both cortical regions for EEG in both theta (6-12 Hz) and gamma (30-48 Hz) bands. These periods of increased coherence are spaced approximately one solar day apart, appear not to be specifically light-entrained, and are most apparent for gamma frequency activity. The gamma association between the two cortical regions shows the same temporal pattern of coherence peaks as the hippocampal-cortical coherences. We propose that these peaks in coherence represent the transient synchronization of temporally tagged memorial information between the hippocampus and other brain regions for which this information may be relevant. These findings suggest that the FEO involves coordinated activity across a number of brain regions and may underlie a mechanism via which an organism can store and recall salient gustatory events on a circadian timescale.

Anxiolytic-like effects of leptin on fixed interval responding.

Leptin has been shown to affect energy homeostasis, learning and memory, and some models of anxiolytic action. However, leptin has produced inconsistent results in previous non-operant behavioural tests of anxiety. Here, we test the anxiolytic potential of leptin in an operant paradigm that has produced positive results across all classes of anxiolytic so far tested. Rats were tested in the Fixed Interval 60 Seconds (FI60) task following administration of 0/0.5/1.0mg/kg (i.p.) leptin or an active anxiolytic control of 5mg/kg (i.p.) chlordiazepoxide (CDP). By the end of the 14days of testing in the FI60 task, 0.5mg/kg leptin released suppressed responding in a manner similar to CDP, and 1.0mg/kg leptin produced a relative depression in responding, a similar outcome pattern to previously tested 5HT-agonist anxiolytics. This suggests that leptin behaves similarly to established serotonergic anxiolytics such as buspirone and fluoxetine; with the delay in development of effect during testing, and the inverted-U dose-response curve explaining the inconsistent behaviour of leptin in behavioural tests of anxiety, as this type of pattern is common to serotonergic anxiolytics.

The frequency of hippocampal theta rhythm is modulated on a circadian period and is entrained by food availability.

The hippocampal formation plays a critical role in the generation of episodic memory. While the encoding of the spatial and contextual components of memory have been extensively studied, how the hippocampus encodes temporal information, especially at long time intervals, is less well understood. The activity of place cells in hippocampus has previously been shown to be modulated at a circadian time-scale, entrained by a behavioral stimulus, but not entrained by light. The experimental procedures used in the previous study of this phenomenon, however, necessarily conflated two alternative entraining stimuli, the exposure to the recording environment and the availability of food, making it impossible to distinguish between these possibilities. Here we demonstrate that the frequency of theta-band hippocampal EEG varies with a circadian period in freely moving animals and that this periodicity mirrors changes in the firing rate of hippocampal neurons. Theta activity serves, therefore, as a proxy of circadian-modulated hippocampal neuronal activity. We then demonstrate that the frequency of hippocampal theta driven by stimulation of the reticular formation also varies with a circadian period. Because this effect can be observed without having to feed the animal to encourage movement we were able to identify what stimulus entrains the circadian oscillation. We show that with reticular-activated recordings started at various times of the day the frequency of theta varies quasi-sinusoidally with a 25 h period and phase-aligned when referenced to the animal's regular feeding time, but not the recording start time. Furthermore, we show that theta frequency consistently varied with a circadian period when the data obtained from repeated recordings started at various times of the day were referenced to the start of food availability in the recording chamber. This pattern did not occur when data were referenced to the start of the recording session or to the actual time of day when this was not also related to feeding time. This double dissociation demonstrates that hippocampal theta is modulated with a circadian timescale, and that this modulation is strongly entrained by food. One interpretation of this finding is that the hippocampus is responsive to a food entrainable oscillator (FEO) that might modulate foraging behavior over circadian periods.

Manual hematoxylin and eosin staining of mouse tissue sections.

The hematoxylin and eosin (H&E) stain is the standard used for microscopic examination of tissues that have been fixed, processed, embedded, and sectioned. It can be performed manually or by automation. For economic reasons, the manual technique is generally the method of choice for facilities with a low sample volume. This protocol describes manual H&E staining of fixed, processed, paraffin-embedded, and sectioned mouse tissues. In H&E-stained tissues, the nucleic acids stain dark blue and the proteins stain red to pink or orange. For accurate phenotyping and delineation of tissue detail, the protocol must be adhered to rigorously. This includes frequent reagent changes as well as the use of "in-date" reagents. Appropriate color in a good H&E stain allows for identification of many tissue subtleties that are necessary for accurate diagnosis.

Manual immunohistochemistry staining of mouse tissues using the avidin-biotin complex (ABC) technique.

There are many variations on the immunohistochemistry (IHC) procedure, but all are based on attachment of a primary antibody to a unique epitope on or within the cell. This step is followed by incubation of the cell/primary antibody complex with another, secondary antibody that recognizes the species in which the primary antibody was produced. The secondary antibody has an indicator molecule attached to it. The indicator produces a colored reaction product at the site of original epitope, allowing visualization. This basic two-antibody "sandwich" procedure has many modifications that include other layers of antibodies and numerous indicators, but all variations depend upon the unique ability of antibodies to recognize specific epitopes or antigenic determinants. The procedure described here is called the ABC (avidin-biotin complex) technique. The method utilizes the high avidity of biotin for avidin, which allows formation of a strong bond. The reagents described in this technique produce a gold/brown reaction product that identifies the epitope of interest.

Analysis of mouse model pathology: a primer for studying the anatomic pathology of genetically engineered mice.

This primer of pathology is intended to introduce investigators to the structure (morphology) of cancer with an emphasis on genetically engineered mouse (GEM) models (GEMMs). We emphasize the necessity of using the entire biological context for the interpretation of anatomic pathology. Because the primary investigator is responsible for almost all of the information and procedures leading up to microscopic examination, they should also be responsible for documentation of experiments so that the microscopic interpretation can be rendered in context of the biology. The steps involved in this process are outlined, discussed, and illustrated. Because GEMMs are unique experimental subjects, some of the more common pitfalls are discussed. Many of these errors can be avoided with attention to detail and continuous quality assurance.

Limited mouse necropsy.

Procurement of mouse tissues or organs is essential for complete verification of almost any phenotype. A proper necropsy can yield information that is difficult to obtain by limited biopsy or surgical intervention. The protocol described here is for a limited autopsy involving the thorax and abdomen only, and does not include all organs.

Mouse tissue fixation.

One of the primary goals of fixation is to stop postmortem changes that degrade the tissue and allow optimal preservation of morphologic and cytological detail as well as nucleic acid integrity. Following death, tissues soon undergo autolysis, and if organisms from the gastrointestinal, urinary, or respiratory tracts are present, their colonization can soon cause putrefaction. Time is of the essence because warmer temperatures accelerate both types of degradation. Placing the tissue into a fixative stops the postmortem changes. Fixatives have their effect on tissue by cross-linking, coagulation, or a combination of both. This article outlines the basic tissue fixation procedure and offers guidance on choosing an appropriate fixative, the timing and duration of fixation, sample storage, and quality issues.

Structured reporting in anatomic pathology for coclinical trials: the caELMIR model.

Electronic media, with their tremendous potential for storing, retrieving, and integrating data, are an essential part of modern collaborative multidisciplinary science. Structured reporting is a fundamental aspect of keeping accurate, searchable electronic records. This discussion on structured reporting in anatomic pathology for pre- and coclinical trials in animal models provides background information for scientists who are not familiar with structured reporting. Practical examples are provided using a working database system for preclinical research-caELMIR (Cancer Electronic Laboratory Management Information and Retrieval)-developed by the U.S. National Cancer Institute's (NCI's) Mouse Models of Human Cancers Consortium (MMHCC).

Quantitation of fixative-induced morphologic and antigenic variation in mouse and human breast cancers.

Quantitative Image Analysis (QIA) of digitized whole slide images for morphometric parameters and immunohistochemistry of breast cancer antigens was used to evaluate the technical reproducibility, biological variability, and intratumoral heterogeneity in three transplantable mouse mammary tumor models of human breast cancer. The relative preservation of structure and immunogenicity of the three mouse models and three human breast cancers was also compared when fixed with representatives of four distinct classes of fixatives. The three mouse mammary tumor cell models were an ER+/PR+ model (SSM2), a Her2+ model (NDL), and a triple negative model (MET1). The four breast cancer antigens were ER, PR, Her2, and Ki67. The fixatives included examples of (1) strong cross-linkers, (2) weak cross-linkers, (3) coagulants, and (4) combination fixatives. Each parameter was quantitatively analyzed using modified Aperio Technologies ImageScope algorithms. Careful pre-analytical adjustments to the algorithms were required to provide accurate results. The QIA permitted rigorous statistical analysis of results and grading by rank order. The analyses suggested excellent technical reproducibility and confirmed biological heterogeneity within each tumor. The strong cross-linker fixatives, such as formalin, consistently ranked higher than weak cross-linker, coagulant and combination fixatives in both the morphometric and immunohistochemical parameters.

The firing rate of hippocampal CA1 place cells is modulated with a circadian period.

The accurate recall of an event is usually dependent on a memory trace that encodes three pieces of information; what happened, when the event happened, and where. The established phenomenology of hippocampal CA1 pyramidal neurons could reflect mechanisms via which some of this information (where and what) is encoded; but so far there has been little evidence for a mechanism by which these cells might represent "when." It was therefore of interest to examine the activity of CA1 neurons over a substantial temporal duration. Forty-eight CA1 neurons were recorded once an hour during long (24-48 h) exposures to a single, stable environment where minimal time-of-day cues were available. Only data from the first 25 h of recording was analyzed quantitatively. We found that the mean ensemble firing rate of these cells changed predictably such that it was closely correlated (r = 0.707) to a reference sine wave with a 25-h period and a positive peak at recording start. This relationship was not explained by changes in the animal's running speed or amount of the recording environment covered in each recording session. When data were referenced to the onset or offset of the normal light-on period, the correlation with the sinusoid was abolished. At an individual cell level, the majority of neurons (n = 31) had significant correlations (P < 0.05) with the reference sine. We conclude that the firing rate of a large proportion of cells in area CA1 of the hippocampus are modulated over a circadian period but that this modulation is not entrained to light. Rather, entry into the environment and the associated food availability appear to be the entraining factors. We hypothesize that these neurons may be part of the putative food-entrainable oscillator. Such a system could enable an animal to discriminate between spatial representations on a temporal dimension with reference to the time of food availability.

Effects of fluoxetine on hippocampal rhythmic slow activity and behavioural inhibition.

Anxiolytics that act as GABAA agonists and those that act as 5-HT1A receptor agonists all reduce the frequency of hippocampal rhythmic slow activity (RSA). Changes in RSA have been linked to changes in behavioural inhibition and therefore anxiety - but this has not been tested with specific serotonin reuptake inhibitors, which are antidepressant and anxiolytic; therefore we tested the effects of fluoxetine on RSA and behavioural inhibition. Fluoxetine (FLU; 10 and 20 mg/kg, intraperitoneally) produced a dose-related reduction in the frequency of reticular-elicited RSA. Groups of rats received, intraperitoneally, either (i) saline, or 5 mg/kg fluoxetine, or 10 mg/kg fluoxetine; or (ii) saline, or 20 mg/kg fluoxetine, or 6.6 mg/kg of the 5-HT1A agonist buspirone (BUS) and were tested on a fixed interval 60-s schedule and a differential reinforcement of low rates 15-s schedule. FLU at 5 mg/kg produced effects similar to low doses of BUS and other anxiolytics. FLU (10 and 20 mg/kg) produced effects more like those reported earlier for higher doses of BUS. These results continue to link anxiolysis, RSA and behavioural inhibition, and suggest that serotonergic anxiolytics share some of the central actions of GABAergic anxiolytics, but at higher doses, administered acutely, have distinct side effects that can obscure their anxiolytic action in behavioural tasks.