A Photoactivated Protein Degrader for Optical Control of Synaptic Function.

Hundreds of proteins determine the function of synapses, and synapses define the neuronal circuits that subserve myriad brain, cognitive, and behavioral functions. It is thus necessary to precisely manipulate specific proteins at specific sub-cellular locations and times to elucidate the roles of particular proteins and synapses in brain function. We developed PHOtochemically TArgeting Chimeras (PHOTACs) as a strategy to optically degrade specific proteins with high spatial and temporal precision. PHOTACs are small molecules that, upon wavelength-selective illumination, catalyze ubiquitylation and degradation of target proteins through endogenous proteasomes. Here we describe the design and chemical properties of a PHOTAC that targets Ca 2+ /calmodulin-dependent protein kinase II alpha (CaMKIIα), which is abundant and crucial for baseline synaptic function of excitatory neurons. We validate the PHOTAC strategy, showing that the CaMKIIα-PHOTAC is effective in mouse brain tissue. Light activation of CaMKIIα-PHOTAC removed CaMKIIα from regions of the mouse hippocampus only within 25 µm of the illuminated brain surface. The optically-controlled degradation decreases synaptic function within minutes of light activation, measured by the light-initiated attenuation of evoked field excitatory postsynaptic potential (fEPSP) responses to physiological stimulation. The PHOTACs methodology should be broadly applicable to other key proteins implicated in synaptic function, especially for evaluating their precise roles in the maintenance of long-term potentiation and memory within subcellular dendritic domains.

Dorsal hippocampal damage disrupts the auditory context-dependent attenuation of taste neophobia in mice.

Rodents exhibit neophobia for novel tastes, demonstrated by an initial reluctance to drink novel-tasting, potentially-aversive solutions. Taste neophobia attenuates across days if the solution is not aversive, demonstrated by increased consumption as the solution becomes familiar. This attenuation of taste neophobia is context dependent, which has been demonstrated by maintained reluctance to drink the novel tasting solution if the subject has to drink it after being brought to a novel environment. This spatial context-dependent attenuation of taste neophobia has been described and likely depends on the integrity of the dorsal hippocampus because this brain area is crucial for representing space and spatial context associations, but is unnecessary for processing taste memories per se. Whether changing the non-spatial auditory context causes a similar effect on attenuation of taste neophobia and the potential role of the dorsal hippocampus in processing this decidedly non-spatial information has not been determined. Here we demonstrate that changing the non-spatial auditory context affects the attenuation of taste neophobia in mice, and investigate the consequence of hippocampal lesion. The results demonstrate that the non-spatial auditory context-dependent attenuation of taste neophobia in mice is lost following NMDA excitotoxic lesions of the CA1 region of the dorsal hippocampus. These findings demonstrate that the dorsal hippocampus is crucial for the modulation non-associative taste learning by auditory context, neither of which provide information about space.

Intravenous antiarrhythmic doses of lidocaine increase the survival rate of CA1 neurons and improve cognitive outcome after transient global cerebral ischemia in rats.

Brain ischemia is often a consequence of cardiac or neurologic surgery. Prophylactic pharmacological neuroprotection would be beneficial for patients undergoing surgery to reduce brain damage due to ischemia. We examined the effects of two antiarrhythmic doses of lidocaine (2 or 4 mg/kg) on rats in a model of transient global cerebral ischemia. The occlusion of both common carotid arteries combined with hypotension for 10 min induced neuronal loss in the CA1 region of the hippocampus (18±12 vs. 31±4 neurons/200 µm linear distance of the cell body layer, X±SD; P<0.01). Lidocaine (4 mg/kg) 30 min before, during and 60 min after ischemia increased dorsal hippocampal CA1 neuronal survival 4 weeks after global cerebral ischemia (30±9 vs. 18±12 neurons/200 µm; P<0.01). There was no significant cell loss after 10 min of ischemia in the CA3 region, the dentate region or the amygdalae; these regions were less sensitive than the CA1 region to ischemic damage. Lidocaine not only increased hippocampal CA1 neuronal survival, but also preserved cognitive function associated with the CA1 region. Using an active place avoidance task, there were fewer entrances into an avoidance zone, defined by relevant distal room-bound cues, in the lidocaine groups. The untreated ischemic group had an average, over the nine sessions, of 21±12 (X±SD) entrances into the avoidance zone per session; the 4 mg/kg lidocaine group had 7±8 entrances (P<0.05 vs. untreated ischemic) and the non-ischemic control group 7±5 entrances (P<0.01 vs. untreated ischemic). Thus, a clinical antiarrhythmic dose of lidocaine increased the number of surviving CA1 pyramidal neurons and preserved cognitive function; this indicates that lidocaine is a good candidate for clinical brain protection.

Evidence for hippocampal role in place avoidance other than merely memory storage.

Spatial navigation is used as a popular animal model of higher cognitive functions in people. The data suggest that the hippocampus is important for both storing spatial memories and for performing spatial computations necessary for navigation. Animals use multiple behavioral strategies to solve spatial tasks often using multiple memory systems. We investigated how inactivation of the rat hippocampus affects performance in a place avoidance task to determine if the role of the hippocampus in this task could be attributed to memory storage/retrieval or to the computations needed for navigation. Injecting tetrodotoxin (TTX) into both hippocampi impaired conditioned place avoidance, but after injecting only one hippocampus, the rats learned the place avoidance as well as without any injections. Retention of the place avoidance learned with one hippocampus was not impaired when the injection was switched to the hippocampus that had not been injected during learning. The result suggests that during learning, the hippocampus did not store the place avoidance memory.

Quantifying location-specific information in the discharge of rat hippocampal place cells.

The informational content in the location-specific discharge of rat hippocampal cells is usually quantified by an average for the entire behaviorally accessible space. In contrast to such "global" information measures, we consider here information that can be obtained from "local" spike counts at each position. The properties of these local information measures are first illustrated using simulated data with predetermined distributions of location-specific spike counts. Next, place cell recordings from rats foraging in a cylindrical arena with two cue cards on its walls are analyzed; time windows as short as 100 ms were used to accumulate spike counts in locations. We show that information at the centers of firing fields is higher for fields nearer to the cues. Neither firing rates or "global" information measures detected differences between fields near and far from the cues. Thus, analyses of the location-specific information provides a new valuable tool for studying the location-specific activity of rat hippocampal cells. Generalizations of location-specific information can be used to investigate place cell responses to other factors such as running speed or the state of the hippocampal EEG in addition to current position.

Hippocampus-dependent retrieval and hippocampus-independent extinction of place avoidance navigation, and stress-induced out-of-context activation of a memory revealed by reversible lesion experiments in rats.

The use of reversible lesion techniques in memory research was pioneered in the laboratory of Jan Bures and Olga Buresova. We use the occasion of Jan's 75th birthday to briefly review the experimental utility of this approach. Two experiments from our current research are reported in which reversible lesioning methods are used to ask otherwise experimentally untenable questions about memory retrieval. The first experiment used intra-hippocampal injections of tetrodotoxin to temporarily inactivate the hippocampus during retrieval of a well-learned place avoidance navigation memory. This revealed that the hippocampus is necessary for place avoidance retrieval but that the extinction of place avoidance can occur independently of retrieving the memory and intact hippocampal function. The second experiment used KCl-induced cortical spreading depression in an interhippocampal transfer paradigm to demonstrate that a Y-maze memory that is learned by only one cortical hemisphere can be made to transfer to the other hemisphere by forcing the rat to swim, a unique stressful experience that occurred in a different apparatus, different behavioral context, and involved different behaviors than the Y-maze training. This demonstrates, we believe for the first time behaviorally, that memories can be activated outside of the behavioral context of their acquisition and expression in rats.

On the location-specific positional and extra-positional information in the discharge of rat hippocampal cells.

The informational content in the location-specific discharge of rat hippocampal cells is usually quantified by an average for the entire experimental space. In contrast, in the present work the information that can be obtained from spike counts at each position is considered. Along with the local positional information, measures for the total and extra-positional information are introduced. Their properties are studied and illustrated on simulated and experimentally obtained data. It is demonstrated that these information measures provide a new valuable tool for studying the location-specific activity of rat hippocampal cells even on time scales as short as 100 ms. The measures can be used to investigate place cell responses to arbitrary signals in addition to current position.

Properties of the extra-positional signal in hippocampal place cell discharge derived from the overdispersion in location-specific firing.

There is a good deal of evidence that in the rodent, an internal model of the external world is encoded by hippocampal pyramidal cells, called 'place cells'. During free exploration, the activity of place cells is higher within a small part of the space, called the firing field, and virtually silent elsewhere. We have previously shown that the spiking activity during passes through the firing field is characterized not only by the high firing rate, but also by its very high variability ('overdispersion'). This overdispersion indicates that place cells carry information in addition to position. Here we demonstrate by simulations of an integrate-and-fire neuronal model that while a rat is foraging in an open space this additional information may arise from a process that alternatingly modulates the inputs to place cells by about 10% with a mean period of about 1 s. We propose that the overdispersion reflects switches of the rats attention between different spatial reference frames of the environment. This predicts that the overdispersion will not be observed in rats that use only room-based cues for navigation. We show that while place cell firing is overdispersed in rats during foraging in an open arena, the firing is less overdispersed during the same behavior in the same environment, when the rats have been trained to use only room-based and not arena-based cues to navigate.

Transient sex differences in the between-sessions but not in the within-session memory underlying an active place avoidance task in weanling rats.

Spatial abilities were tested in male and female rats by training them to avoid an area in which there was a mild footshock while the arena rotated at 1 revolution/minute. The to-be-avoided area was stable in the coordinates of the room, so extramaze landmarks had to be used for accurate navigation, as the rotation made intramaze cues and substrate-based path integration useless for the avoidance. From Postnatal Day (PD) 19, rats were trained for 22 consecutive days. When the shock area was the same across sessions male rats reached optimal performance on PDs 23-24, 10 days before female rats, but when the location of the shock changed daily there were no sex differences. The results indicate that there are separate memory components underlying spatial competence: a within-session component that develops similarly in male and female rats and a between-sessions component that lasts at least 24 hr and appears earlier in male than in female rats.

New spatial cognition tests for mice: passive place avoidance on stable and active place avoidance on rotating arenas.

Dry arenas are a convenient tool for assessing the spatial navigation abilities of rodents. In this paper, mice must avoid a punished sector of a dry arena from which they are expelled by a puff of compressed air. The position of the punished sector is defined relative to the coordinate system of the room. In a stable environment the mice can use both extramaze and intramaze landmarks to orient themselves accurately. However, when the shock area is defined by extramaze landmarks, continuous rotation of the arena at 1 rpm makes it impossible to solve the avoidance task using arena-based cues or idiothesis. The avoidance can only be solved by paying attention to extramaze cues. Our protocol tested spatial abilities on stable and rotating arenas. The acquisition of the task was manifested under both conditions by a significant improvement of performance within the first session (short-term memory component) and at the beginning of the 24-h delayed second session (long-term memory component).

Inactivating one hippocampus impairs avoidance of a stable room-defined place during dissociation of arena cues from room cues by rotation of the arena.

Unilateral intrahippocampal injections of tetrodotoxin were used to temporarily inactivate one hippocampus during specific phases of training in an active allothetic place avoidance task. The rat was required to use landmarks in the room to avoid a room-defined sector of a slowly rotating circular arena. The continuous rotation dissociated room cues from arena cues and moved the arena surface through a part of the room in which foot-shock was delivered. The rat had to move away from the shock zone to prevent being transported there by the rotation. Unilateral hippocampal inactivations profoundly impaired acquisition and retrieval of the allothetic place avoidance. Posttraining unilateral hippocampal inactivation also impaired performance in subsequent sessions. This allothetic place avoidance task seems more sensitive to hippocampal disruption than the standard water maze task because the same unilateral hippocampal inactivation does not impair performance of the variable-start, fixed hidden goal task after procedural training. The results suggest that the hippocampus not only encodes allothetic relationships amongst landmarks, it also organizes perceived allothetic stimuli into systems of mutually stable coordinates. The latter function apparently requires greater hippocampal integrity.

Substratal idiothetic navigation of rats is impaired by removal or devaluation of extramaze and intramaze cues.

The spatial orientation of vertebrates is implemented by two complementary mechanisms: allothesis, processing the information about spatial relationships between the animal and perceptible landmarks, and idiothesis, processing the substratal and inertial information produced by the animal's active or passive movement through the environment. Both systems allow the animal to compute its position with respect to perceptible landmarks and to the already traversed portion of the path. In the present study, we examined the properties of substratal idiothesis deprived of relevant exteroceptive information. Rats searching for food pellets in an arena formed by a movable inner disk and a peripheral immobile belt were trained in darkness to avoid a 60 degrees sector; rats that entered this sector received a mild foot shock. The punished sector was defined in the substratal idiothetic frame, and the rats had to determine the location of the shock sector with the use of substratal idiothesis only, because all putative intramaze cues were made irrelevant by angular displacements of the disk relative to the belt. Striking impairment of place avoidance by this "shuffling procedure" indicates that effective substratal idiothesis must be updated by exteroceptive intramaze cues.

Conjoint control of hippocampal place cell firing by two visual stimuli. I. The effects of moving the stimuli on firing field positions.

To better understand how hippocampal place cell activity is controlled by sensory stimuli, and to further elucidate the nature of the environmental representation provided by place cells, we have made recordings in the presence of two distinct visual stimuli under standard conditions and after several manipulations of these stimuli. In line with a great deal of earlier work, we find that place cell activity is constant when repeated recordings are made in the standard conditions in which the centers of the two stimuli, a black card and a white card, are separated by 135 degrees on the wall of a cylindrical recording chamber. Rotating the two stimuli by 45 degrees causes equal rotations of place cell firing fields. Removing either card and rotating the other card also causes fields to rotate equally, showing that the two stimuli are individually salient. Increasing or decreasing the card separation (card reconfiguration) causes a topological distortion of the representation of the cylinder floor such that field centers move relative to each other. We also found that either kind of reconfiguration induces a position-independent decrease in the intensity of place cell firing. We argue that these results are not compatible with either of two previously stated views of the place cell representation; namely, a nonspatial theory in which each place cell is tuned to an arbitrarily selected subset of available stimuli or a rigid map theory. We propose that our results imply that the representation is map-like but not rigid; it is capable of undergoing stretches without altering the local arrangement of firing fields.

Conjoint control of hippocampal place cell firing by two visual stimuli. Ii. A vector-field theory that predicts modifications of the representation of the environment.

Changing the angular separation between two visual stimuli attached to the wall of a recording cylinder causes the firing fields of place cells to move relative to each other, as though the representation of the floor undergoes a topological distortion. The displacement of the firing field center of each cell is a vector whose length is equal to the linear displacement and whose angle indicates the direction that the field center moves in the environment. Based on the observation that neighboring fields move in similar ways, whereas widely separated fields tend to move relative to each other, we develop an empirical vector-field model that accounts for the stated effects of changing the card separation. We then go on to show that the same vector-field equation predicts additional aspects of the experimental results. In one example, we demonstrate that place cell firing fields undergo distortions of shape after the card separation is changed, as though different parts of the same field are affected by the stimulus constellation in the same fashion as fields at different locations. We conclude that the vector-field formalism reflects the organization of the place-cell representation of the environment for the current case, and through suitable modification may be very useful for describing motions of firing patterns induced by a wide variety of stimulus manipulations.

Functional inactivation of dorsal hippocampus impairs active place avoidance in rats.

This paper assesses the contribution of hippocampus to the spatial orientation of Long-Evans rats in a new place avoidance task. The animals learn to avoid a mild footshock in a segment of a rotating arena. Since the punished region is defined in the coordinate system of the stationary room the subject is forced to move away from the prohibited segment even if it is immobile. After bilateral injection of tetrodotoxin (5 ng in 1 microl of saline) into the dorsal hippocampus rats were not able to avoid the punished place while a similar injection of saline did not affect performance. The results suggest the task is suitable for assessing the hippocampus-dependent spatial abilities of laboratory rodents.

Understanding hippocampal activity by using purposeful behavior: place navigation induces place cell discharge in both task-relevant and task-irrelevant spatial reference frames.

Continuous rotation of an arena in a cue-rich room dissociates the stationary room-bound information from the rotating arena-bound information. This disrupted spatial discharge in the majority of place cells from rats trained to collect randomly scattered food. In contrast, most place cell firing patterns recorded from rats trained to solve a navigation task on the rotating arena were preserved during the rotation. Spatial discharge was preserved in both the task-relevant stationary and the task-irrelevant rotating reference frames, but firing was more organized in the task-relevant frame. It is concluded that, (i) the effects of environmental manipulations can be understood with confidence only when the rat's purposeful behavior is used to formulate interpretations of the data, and (ii) hippocampal place cell activity is organized in multiple overlapping spatial reference frames.

Continuous place avoidance task reveals differences in spatial navigation in male and female rats.

A new place navigation test was used to estimate the spatial orientation abilities of male and female rats. Animals had to avoid a room frame defined area on a rotating arena, entering of which was punished by mild footshock, i.e. rats had to avoid the same place in the room but different parts of the floor, which was rotated through the punished zone. Because of the rotation of the arena (one revolution per min), animals could not rely on intramaze cues and only extramaze landmarks could be used for accurate navigation. During 8 consecutive days rats were exposed to daily 40-min sessions, consisting of 20-min acquisition and 20-min extinction (shock discontinued). The position of the punished sector centered around one of the four mutually perpendicular azimuths was daily changed in a predetermined sequence. The results showed no male female differences during acquisition and better performance of males during extinction. The performance of females was not affected by estral cycle-related hormonal changes. The findings are discussed in the light of controversial results of research into sex differences in spatial abilities.

Is the hippocampus of the rat part of a specialized navigational system?

The spatial mapping theory of hippocampal function proposes that the rat hippocampus is specialized for navigational computations, computations that allow the animal to solve difficult spatial problems. In this paper, we review evidence obtained by recording place cells and other "spatially tuned" cells from freely moving rats. Our main conclusion is that the nature of the signals carried by these cells and the ways in which the signals transform after changing the environment imply that the hippocampus and associated structures are able to represent aspects of the geometry of the environment.

Both here and there: simultaneous expression of autonomous spatial memories in rats.

Foraging rats learned to avoid footshock that was present in a part of a circular arena that was either stable or rotating slowly in a lighted room. The rotation dissociated spatial information in the separate reference frames of the room and arena. After learning to avoid the shocked region in either condition, in the absence of shock, memory for this place was expressed by simultaneous avoidance of an area defined in the reference frame of the room as well as of an area defined in the reference frame of the rotating arena. Spatial memories in these distinct reference frames were acquired, retrieved, and extinguished autonomously.