DREADD-inactivation of dorsal CA1 pyramidal neurons in mice impairs retrieval of object and spatial memories.

The hippocampus, a medial temporal lobe brain region, is critical for the consolidation of information from short-term memory into long-term episodic memory and for spatial memory that enables navigation. Hippocampal damage in humans has been linked to amnesia and memory loss, characteristic of Alzheimer's disease and other dementias. Numerous studies indicate that the rodent hippocampus contributes significantly to long-term memory for spatial and nonspatial information. For example, muscimol-induced depression of CA1 neuronal activity in the dorsal hippocampus impairs the encoding, consolidation, and retrieval of nonspatial object memory in mice. Here, a chemogenetic designer receptor exclusively activated by designer drugs (DREADDs) approach was used to test the selective involvement of CA1 pyramidal neurons in memory retrieval for objects and for spatial location in a cohort of male C57BL/6J mice. Activation of the inhibitory (hM4Di) DREADDs receptor expressed in CA1 neurons significantly impaired the retrieval of object memory in the spontaneous object recognition task and of spatial memory in the Morris water maze. Silencing of CA1 neuronal activity in hM4Di-expressing mice was confirmed by comparing Fos expression in vehicle- and clozapine-N-oxide-treated mice after exploration of a novel environment. Histological analyses revealed that expression of the hM4Di receptor was limited to CA1 neurons of the dorsal hippocampus. These results suggest that a common subset of CA1 neurons (i.e., those expressing hM4Di receptors) in mouse hippocampus contributed to the retrieval of long-term memory for nonspatial and spatial information. Our findings support the view that the contribution of the rodent hippocampus is like that of the primate hippocampus, specifically essential for global memory. Our results further validate mice as a suitable model system to study the neurobiological mechanisms of human episodic memory, but also in developing treatments and understanding the underlying causes of diseases affecting long-term memory, such as Alzheimer's disease.

Mice recognize 3D objects from recalled 2D pictures, support for picture-object equivalence.

Picture-object equivalence or recognizing a three-dimensional (3D) object after viewing a two-dimensional (2D) photograph of that object, is a higher-order form of visual cognition that may be beyond the perceptual ability of rodents. Behavioral and neurobiological mechanisms supporting picture-object equivalence are not well understood. We used a modified visual recognition memory task, reminiscent of those used for primates, to test whether picture-object equivalence extends to mice. Mice explored photographs of an object during a sample session, and 24 h later were presented with the actual 3D object from the photograph and a novel 3D object, or the stimuli were once again presented in 2D form. Mice preferentially explored the novel stimulus, indicating recognition of the "familiar" stimulus, regardless of whether the sample photographs depicted radially symmetric or asymmetric, similar, rotated, or abstract objects. Discrimination did not appear to be guided by individual object features or low-level visual stimuli. Inhibition of CA1 neuronal activity in dorsal hippocampus impaired discrimination, reflecting impaired memory of the 2D sample object. Collectively, results from a series of experiments provide strong evidence that picture-object equivalence extends to mice and is hippocampus-dependent, offering important support for the appropriateness of mice for investigating mechanisms of human cognition.

Object Recognition Memory: Distinct Yet Complementary Roles of the Mouse CA1 and Perirhinal Cortex.

While the essential contribution of the hippocampus to spatial memory is well established, object recognition memory has been traditionally attributed to the perirhinal cortex (PRh). However, the results of several studies indicate that under specific procedural conditions, temporary or permanent lesions of the hippocampus affect object memory processes as measured in the Spontaneous Object Recognition (SOR) task. The PRh and hippocampus are considered to contribute distinctly to object recognition memory based on memory strength. Allowing mice more, or less, exploration of novel objects during the encoding phase of the task (i.e., sample session), yields stronger, or weaker, object memory, respectively. The current studies employed temporary local inactivation and immunohistochemistry to determine the differential contributions of neuronal activity in PRh and the CA1 region of the hippocampus to strong and weak object memory. Temporary inactivation of the CA1 immediately after the SOR sample session impaired strong object memory but spared weak object memory; while temporary inactivation of PRh post-sample impaired weak object memory but spared strong object memory. Furthermore, mRNA transcription and de novo protein synthesis are required for the consolidation of episodic memory, and activation patterns of immediate early genes (IEGs), such as c-Fos and Arc, are linked to behaviorally triggered neuronal activation and synaptic plasticity. Analyses of c-Fos and Arc protein expression in PRh and CA1 neurons by immunohistochemistry, and of Arc mRNA by qPCR after distinct stages of SOR, provide additional support that strong object memory is dependent on CA1 neuronal activity, while weak object memory is dependent on PRh neuronal activity. Taken together, the results support the view that both PRh and CA1 are required for object memory under distinct conditions. Specifically, our results are consistent with a model that as the mouse begins to explore a novel object, information about it accumulates within PRh, and a weak memory of the object is encoded. If object exploration continues beyond some threshold, strong memory for the event of object exploration is encoded; the consolidation of which is CA1-dependent. These data serve to reconcile the dissension in the literature by demonstrating functional and complementary roles for CA1 and PRh neurons in rodent object memory.

Object and place information processing by CA1 hippocampal neurons of C57BL/6J mice.

Medial and lateral entorhinal cortices convey spatial/contextual and item/object information to the hippocampus, respectively. Whether the distinct inputs are integrated as one cognitive map by hippocampal neurons to represent location and the objects therein, or whether they remain as parallel outputs, to be integrated in a downstream region, remains unclear. Principal, or complex spike bursting, neurons of hippocampus exhibit location-specific firing, and it is likely that the activity of "place cells" supports spatial memory/navigation in rodents. Consistent with cognitive map theory, the activity of CA1 hippocampal neurons is also critical for nonspatial memory, such as object recognition. However, the degree to which CA1 neuronal activity represents the associations of object-context or object-in-place memory is not well understood. Here, the contributions of mouse CA1 neuronal activity to object recognition memory and the emergence of object-place conjunctive representations were tested using in vivo recordings and functional inactivation. Independent of arena configuration, CA1 place fields were stable throughout testing and object-place representations were not identified in CA1, although the number of fields per cell increased during object sessions, and few object-related firing CA1 neurons (nonplace) were recorded. The results of the inactivation studies confirmed the significant contribution of CA1 neuronal activity to object recognition memory when a delay of 20 min, but not 5 min, was imposed between encoding and retrieval. Together, our results confirm the delay-dependent contribution of the CA1 region to object memory and suggest that object information is processed in parallel with the ongoing spatial mapping function that is a hallmark of hippocampal memory.NEW & NOTEWORTHY We developed variations of the object recognition task to examine the contribution of mouse CA1 neuronal activity to object memory and the degree to which object-context conjunctive representations are formed during object training. Our results indicate that, within the CA1 region, object information is processed in a parallel but delay-dependent manner, with ongoing spatial mapping.

Temporary inactivation reveals that the CA1 region of the mouse dorsal hippocampus plays an equivalent role in the retrieval of long-term object memory and spatial memory.

Recognition of a previously experienced item or object depends upon the successful retrieval of memory for the object. The neural mechanisms that support object recognition memory in the mammalian brain are not well understood. The rodent hippocampus plays a well-established role in spatial memory, and we previously demonstrated that temporary inactivation of the mouse hippocampus impairs object memory, as assessed with a novel object preference (NOP) test. The present studies were designed to test some remaining issues regarding the contribution of the CA1 sub-region of the mouse dorsal hippocampus to long-term object memory. Specifically, we examined whether the retrieval of spatial memory (as assessed by the Morris water maze; MWM) and object recognition memory are differentially sensitive to inactivation of the CA1 region. The current study used pre-test local microinfusion of muscimol directly into the CA1 region of dorsal hippocampus to temporarily interrupt its function during the respective retrieval phases of both behavioral tasks, in order to compare the contribution of the CA1 to object memory and spatial memory. Histological analyses revealed that local intra-CA1 injection of muscimol diffused within, and not beyond, the CA1 region of dorsal hippocampus. The degree of memory retrieval impairment induced by muscimol was comparable in the two tasks, supporting the view that object memory and spatial memory depend similarly on the CA1 region of rodent hippocampus. Further, we confirmed that the muscimol-induced impairment of CA1 function is temporary. First, mice that exhibited impaired object memory retrieval immediately after intra-CA1 muscimol, subsequently exhibited unimpaired retrieval of object memory when tested 24h later. Secondly, a cohort of mice that exhibited impaired object memory retrieval after intra-CA1 muscimol later acquired spatial memory in the MWM comparable to that of control mice. Together, these results offer further support for the involvement of the CA1 region of mouse hippocampus in object recognition memory, and provide evidence to suggest that the NOP task is as much a test of hippocampal function as the classic MWM test.

Examination of the hippocampal contribution to serotonin 5-HT2A receptor-mediated facilitation of object memory in C57BL/6J mice.

The rodent hippocampus supports non-spatial object memory. Serotonin 5-HT2A receptors (5-HT2AR) are widely expressed throughout the hippocampus. We previously demonstrated that the activation of 5-HT2ARs enhanced the strength of object memory assessed 24 h after a limited (i.e., weak memory) training procedure. Here, we examined the subcellular distribution of 5-HT2ARs in the hippocampal CA1 region and underlying mechanisms of 5-HT2AR-mediated object memory consolidation. Analyses with immuno-electron microscopy revealed the presence of 5-HT2ARs on the dendritic spines and shafts of hippocampal CA1 neurons, and presynaptic terminals in the CA1 region. In an object recognition memory procedure that places higher demand on the hippocampus, only post-training systemic or intrahippocampal administration of the 5-HT2AR agonist TCB-2 enhanced object memory. Object memory enhancement by TCB-2 was blocked by the 5-HT2AR antagonist, MDL 11,937. The memory-enhancing dose of systemic TCB-2 increased extracellular glutamate levels in hippocampal dialysate samples, and increased the mean in vivo firing rate of hippocampal CA1 neurons. In summary, these data indicate a pre- and post-synaptic distribution of 5-HT2ARs, and activation of 5-HT2ARs selectively enhanced the consolidation of object memory, without affecting encoding or retrieval. The 5-HT2AR-mediated facilitation of hippocampal memory may be associated with an increase in hippocampal neuronal firing and glutamate efflux during a post-training time window in which recently encoded memories undergo consolidation.

Assessing rodent hippocampal involvement in the novel object recognition task. A review.

The novel object recognition (NOR) task has emerged as a popular method for testing the neurobiology of nonspatial memory in rodents. This task exploits the natural tendency of rodents to explore novel items and depending on the amount of time that rodents spend exploring the presented objects, inferences about memory can be established. Despite its wide use, the underlying neural circuitry and mechanisms supporting NOR have not been clearly defined. In particular, considerable debate has focused on whether the hippocampus plays a significant role in the object memory that is encoded, consolidated and then retrieved during discrete stages of the NOR task. Here we analyzed the results of all published reports in which the role of the rodent hippocampus in object memory was inferred from performance in the task with restricted parameters. We note that the remarkable variability in NOR methods across studies complicates the ability to draw meaningful conclusions from the work. Focusing on 12 reports in which a minimum criterion of sample session object exploration was imposed, we find that temporary or permanent lesion of the hippocampus consistently disrupts object memory when a delay of 10 min or greater is imposed between the sample and test sessions. We discuss the significance of a delay-dependent role of the hippocampus in NOR within the framework of the medial temporal lobe. We assert that standardization of the NOR protocol is essential for obtaining reliable data that can then be compared across studies to build consensus as to the specific contribution of the rodent hippocampus to object memory.

The neurosteroid allopregnanolone impairs object memory and contextual fear memory in male C57BL/6J mice.

Allopregnanolone (ALLO, or 3α-hydroxy-5α-pregnan-20-one) is a steroid metabolite of progesterone and a potent endogenous positive allosteric modulator of GABA-A receptors. Systemic ALLO has been reported to impair spatial, but not nonspatial learning in the Morris water maze (MWM) and contextual memory in rodents. These cognitive effects suggest an influence of ALLO on hippocampal-dependent memory, although the specific nature of the neurosteroid's effects on learning, memory or performance is unclear. The present studies aimed to determine: (i) the memory process(es) affected by systemic ALLO using a nonspatial object memory task; and (ii) whether ALLO affects object memory via an influence within the dorsal hippocampus. Male C57BL/6J mice received systemic ALLO either before or immediately after the sample session of a novel object recognition (NOR) task. Results demonstrated that systemic ALLO impaired the encoding and consolidation of object memory. A subsequent study revealed that bilateral microinfusion of ALLO into the CA1 region of dorsal hippocampus immediately following the NOR sample session also impaired object memory consolidation. In light of debate over the hippocampal-dependence of object recognition memory, we also tested systemic ALLO-treated mice on a contextual and cued fear-conditioning task. Systemic ALLO impaired the encoding of contextual memory when administered prior to the context pre-exposure session. Together, these results indicate that ALLO exhibits primary effects on memory encoding and consolidation, and extend previous findings by demonstrating a sensitivity of nonspatial memory to ALLO, likely by disrupting dorsal hippocampal function.

The rodent hippocampus is essential for nonspatial object memory.

Elucidating the role of the rodent hippocampus in object recognition memory is critical for establishing the appropriateness of rodents as models of human memory and for their use in the development of memory disorder treatments. In mammals, spatial memory and nonspatial memory depend upon the hippocampus and associated medial temporal lobe (MTL) structures. Although well established in humans, the role of the rodent hippocampus in object memory remains highly debated due to conflicting findings across temporary and permanent hippocampal lesion studies and evidence that the perirhinal cortex may support object memory. In the current studies, we used intrahippocampal muscimol microinfusions to transiently inactivate the male C57BL/6J mouse hippocampus at distinct stages during the novel object recognition (NOR) task: during object memory encoding and consolidation, just consolidation, and/or retrieval. We also assessed the effect of temporary hippocampal inactivation when objects were presented in different contexts, thus eliminating the spatial or contextual components of the task. Lastly, we assessed extracellular dorsal hippocampal glutamate efflux and firing properties of hippocampal neurons while mice performed the NOR task. Our results reveal a clear and compelling role of the rodent hippocampus in nonspatial object memory.

Stimulation of serotonin 2A receptors facilitates consolidation and extinction of fear memory in C57BL/6J mice.

Excessive fear is a hallmark of several emotional and mental disorders such as phobias and panic disorders. Considerable attention is focused on defining the neurobiological mechanisms of the extinction of conditioned fear memory in an effort to identify mechanisms that may hold clinical significance for remediating aberrant fear memory. Serotonin modulates the acquisition and retention of conditioned emotional memory, and the serotonin 2A receptor (5HT2AR) may be one of the postsynaptic targets mediating such effects. Here we tested the hypothesis that the 5HT2AR regulates the consolidation and extinction of fear memory in male C57BL/6J mice. The influence of 5HT2ARs on memory consolidation was further confirmed with a novel object recognition task. With a trace fear conditioning paradigm, administration of the 5HT2AR agonist TCB-2 (1.0 mg/kg, i.p.) before the extinction test facilitated the acquisition of extinction of fear memory as compared to vehicle treatment. In contrast, administration of the 5HT2AR antagonist MDL 11,939 (0.5 mg/kg, i.p.) delayed the acquisition of extinction of fear memory. Further, the post-conditioning administration of TCB-2 enhanced contextual and cued fear memory, possibly by facilitating the consolidation of fear memory. Administration of TCB-2 also facilitated the acquisition of extinction of fear memory in delay fear conditioned mice. Stimulation or blockade of 5HT2ARs did not affect the encoding or retrieval of conditioned fear memory. Finally, administration of TCB-2 right after training in an object recognition task enhanced the consolidation of object memory. These results suggest that stimulation of 5HT2ARs facilitates the consolidation and extinction of trace and delay cued fear memory and the consolidation of object memory. Blocking the 5HT2AR impairs the acquisition of fear memory extinction. The results support the view that serotonergic activation of the 5HT2AR provides an important modulatory influence on circuits engaged during extinction learning. Taken together these results suggest that the 5HT2AR may be a potential therapeutic target for enhancing hippocampal and amygdala-dependent memory. This article is part of a Special Issue entitled 'Cognitive Enhancers'.

Evaluation of tissue interactions with mechanical elements of a transscleral drug delivery device.

The goal of this work was to evaluate tissue-device interactions due to implantation of a mechanically operated drug delivery system onto the posterior sclera. Two test devices were designed and fabricated to model elements of the drug delivery device-one containing a free-spinning ball bearing and the other encasing two articulating gears. Openings in the base of test devices modeled ports for drug passage from device to sclera. Porous poly(tetrafluoroethylene) (PTFE) membranes were attached to half of the gear devices to minimize tissue ingrowth through these ports. Test devices were sutured onto rabbit eyes for 10 weeks. Tissue-device interactions were evaluated histologically and mechanically after removal to determine effects on device function and changes in surrounding tissue. Test devices were generally well-tolerated during residence in the animal. All devices encouraged fibrous tissue formation between the sclera and the device, fibrous tissue encapsulation and invasion around the device, and inflammation of the conjunctiva. Gear devices encouraged significantly greater inflammation in all cases and a larger rate of tissue ingrowth. PTFE membranes prevented tissue invasion through the covered drug ports, though tissue migrated in through other smaller openings. The torque required to turn the mechanical elements increased over 1000 times for gear devices, but only on the order of 100 times for membrane-covered gear devices and less than 100 times for ball bearing devices. Maintaining a lower device profile, minimizing microscale motion on the eye surface and covering drug ports with a porous membrane may minimize inflammation, decreasing the risk of damage to surrounding tissues and minimizing disruption of device operation.

Novel technology for rapid species-specific detection of Bacillus spores.

There is an urgent need for a small, inexpensive sensor that can rapidly detect bio-warfare agents with high specificity. Bacillus anthracis, the causative agent of anthrax, would be a perilous disease-causing organism in the event of a release. Currently, most anthrax detection research is based on nucleic acid detection, immunoassays and mass spectrometry, with few detection levels reported below 10(5) spores. Here, we show the ability to distinguish Bacillus spores to a level approaching 10(3) spores, below the reported median infectious dose of B. anthracis, using pyrolysis--micromachined differential mobility spectrometry and novel pattern recognition algorithms that combine lead cluster mapping with genetic algorithms.

Species-specific bacteria identification using differential mobility spectrometry and bioinformatics pattern recognition.

As bacteria grow and proliferate, they release a variety of volatile compounds that can be profiled and used for speciation, providing an approach amenable to disease diagnosis through quick analysis of clinical cultures as well as patient breath analysis. As a practical alternative to mass spectrometry detection and whole cell pyrolysis approaches, we have developed methodology that involves detection via a sensitive, micromachined differential mobility spectrometer (microDMx), for sampling headspace gases produced by bacteria growing in liquid culture. We have applied pattern discovery/recognition algorithms (ProteomeQuest) to analyze headspace gas spectra generated by microDMx to reliably discern multiple species of bacteria in vitro: Escherichia coli, Bacillus subtilis, Bacillus thuringiensis, and Mycobacterium smegmatis. The overall accuracy for identifying volatile profiles of a species within the 95% confidence interval for the two highest accuracy models evolved was between 70.4 and 89.3% based upon the coordinated expression of between 5 and 11 features. These encouraging in vitro results suggest that the microDMx technology, coupled with bioinformatics data analysis, has potential for diagnosis of bacterial infections.