To Approach or Avoid: An Introductory Overview of the Study of Anxiety Using Rodent Assays

Anxiety is a widely studied phenomenon in behavioral neuroscience, but the recent literature lacks an overview of the major conceptual framework underlying anxiety research to introduce young researchers to the field. In this mini-review article, which is aimed toward new undergraduate and graduate students, we discuss how researchers exploit the approach-avoidance conflict, an internal conflict rodents face between exploration of novel environments and avoidance of danger, to inform rodent assays that allow for the measurement of anxiety-related behavior in the laboratory. We review five widely-used rodent anxiety assays, consider the pharmacological validity of these assays, and discuss neural circuits that have recently been shown to modulate anxiety using the assays described. Finally, we offer related lines of inquiry and comment on potential future directions

A shared neural ensemble links distinct contextual memories encoded close in time.

Recent studies suggest that a shared neural ensemble may link distinct memories encoded close in time. According to the memory allocation hypothesis, learning triggers a temporary increase in neuronal excitability that biases the representation of a subsequent memory to the neuronal ensemble encoding the first memory, such that recall of one memory increases the likelihood of recalling the other memory. Here we show in mice that the overlap between the hippocampal CA1 ensembles activated by two distinct contexts acquired within a day is higher than when they are separated by a week. Several findings indicate that this overlap of neuronal ensembles links two contextual memories. First, fear paired with one context is transferred to a neutral context when the two contexts are acquired within a day but not across a week. Second, the first memory strengthens the second memory within a day but not across a week. Older mice, known to have lower CA1 excitability, do not show the overlap between ensembles, the transfer of fear between contexts, or the strengthening of the second memory. Finally, in aged mice, increasing cellular excitability and activating a common ensemble of CA1 neurons during two distinct context exposures rescued the deficit in linking memories. Taken together, these findings demonstrate that contextual memories encoded close in time are linked by directing storage into overlapping ensembles. Alteration of these processes by ageing could affect the temporal structure of memories, thus impairing efficient recall of related information

Dorsal Periaqueductal gray ensembles represent approach and avoidance states

Animals must balance needs to approach threats for risk assessment and to avoid danger. The dorsal periaqueductal gray (dPAG) controls defensive behaviors, but it is unknown how it represents states associated with threat approach and avoidance. We identified a dPAG threatavoidance ensemble in mice that showed higher activity farther from threats such as the open arms of the elevated plus maze and a predator. These cells were also more active during threat avoidance behaviors such as escape and freezing, even though these behaviors have antagonistic motor output. Conversely, the threat approach ensemble was more active during risk assessment behaviors and near threats. Furthermore, unsupervised methods showed that avoidance/approach states were encoded with shared activity patterns across threats. Lastly, the relative number of cells in each ensemble predicted threat avoidance across mice. Thus, dPAG ensembles dynamically encode threat approach and avoidance states, providing a flexible mechanism to balance risk assessment and danger avoidance

Dorsal premammillary projection to periaqueductal gray controls escape vigor from innate and conditioned threats

Escape from threats has paramount importance for survival. However, it is unknown if a single circuit controls escape vigor from innate and conditioned threats. Cholecystokinin (cck)-expressing cells in the hypothalamic dorsal premammillary nucleus (PMd) are necessary for initiating escape from innate threats via a projection to the dorsolateral periaqueductal gray (dlPAG). We now show that in mice PMd-cck cells are activated during escape, but not other defensive behaviors. PMd-cck ensemble activity can also predict future escape. Furthermore, PMd inhibition decreases escape speed from both innate and conditioned threats. Inhibition of the PMd-cck projection to the dlPAG also decreased escape speed. Intriguingly, PMd-cck and dlPAG activity in mice showed higher mutual information during exposure to innate and conditioned threats. In parallel, human functional magnetic resonance imaging data show that a posterior hypothalamic-to-dlPAG pathway increased activity during exposure to aversive images, indicating that a similar pathway may possibly have a related role in humans. Our data identify the PMd-dlPAG circuit as a central node, controlling escape vigor elicited by both innate and conditioned threats. &nbsp;</p

Dorsal premammillary projection to periaqueductal gray controls escape vigor from innate and conditioned threats

Escape from threats has paramount importance for survival. However, it is unknown if a single circuit controls escape vigor from innate and conditioned threats. Cholecystokinin (cck)-expressing cells in the hypothalamic dorsal premammillary nucleus (PMd) are necessary for initiating escape from innate threats via a projection to the dorsolateral periaqueductal gray (dlPAG). We now show that in mice PMd-cck cells are activated during escape, but not other defensive behaviors. PMd-cck ensemble activity can also predict future escape. Furthermore, PMd inhibition decreases escape speed from both innate and conditioned threats. Inhibition of the PMd-cck projection to the dlPAG also decreased escape speed. Intriguingly, PMd-cck and dlPAG activity in mice showed higher mutual information during exposure to innate and conditioned threats. In parallel, human functional magnetic resonance imaging data show that a posterior hypothalamic-to-dlPAG pathway increased activity during exposure to aversive images, indicating that a similar pathway may possibly have a related role in humans. Our data identify the PMd-dlPAG circuit as a central node, controlling escape vigor elicited by both innate and conditioned threats

Sparse genetically defined neurons refine the canonical role of periaqueductal gray columnar organization.

During threat exposure, survival depends on defensive reactions. Prior works linked large glutamatergic populations in the midbrain periaqueductal gray (PAG) to defensive freezing and flight, and established that the overarching functional organization axis of the PAG is along anatomically-defined columns. Accordingly, broad activation of the dorsolateral column induces flight, while activation of the lateral or ventrolateral (l and vl) columns induces freezing. However, the PAG contains diverse cell types that vary in neurochemistry. How these cell types contribute to defense remains unknown, indicating that targeting sparse, genetically-defined populations may reveal how the PAG generates diverse behaviors. Though prior works showed that broad excitation of the lPAG or vlPAG causes freezing, we found in mice that activation of lateral and ventrolateral PAG (l/vlPAG) cholecystokinin-expressing (CCK) cells selectively caused flight to safer regions within an environment. Furthermore, inhibition of l/vlPAG-CCK cells reduced predator avoidance without altering other defensive behaviors like freezing. Lastly, l/vlPAG-CCK activity decreased when approaching threat and increased during movement to safer locations. These results suggest CCK cells drive threat avoidance states, which are epochs during which mice increase distance from threat and perform evasive escape. Conversely, l/vlPAG pan-neuronal activation promoted freezing, and these cells were activated near threat. Thus, CCK l/vlPAG cells have opposing function and neural activation motifs compared to the broader local ensemble defined solely by columnar boundaries. In addition to the anatomical columnar architecture of the PAG, the molecular identity of PAG cells may confer an additional axis of functional organization, revealing unexplored functional heterogeneity

A shared neural ensemble links distinct contextual memories encoded close in time.

Recent studies suggest that a shared neural ensemble may link distinct memories encoded close in time. According to the memory allocation hypothesis, learning triggers a temporary increase in neuronal excitability that biases the representation of a subsequent memory to the neuronal ensemble encoding the first memory, such that recall of one memory increases the likelihood of recalling the other memory. Here we show in mice that the overlap between the hippocampal CA1 ensembles activated by two distinct contexts acquired within a day is higher than when they are separated by a week. Several findings indicate that this overlap of neuronal ensembles links two contextual memories. First, fear paired with one context is transferred to a neutral context when the two contexts are acquired within a day but not across a week. Second, the first memory strengthens the second memory within a day but not across a week. Older mice, known to have lower CA1 excitability, do not show the overlap between ensembles, the transfer of fear between contexts, or the strengthening of the second memory. Finally, in aged mice, increasing cellular excitability and activating a common ensemble of CA1 neurons during two distinct context exposures rescued the deficit in linking memories. Taken together, these findings demonstrate that contextual memories encoded close in time are linked by directing storage into overlapping ensembles. Alteration of these processes by ageing could affect the temporal structure of memories, thus impairing efficient recall of related information

Coordination of escape and spatial navigation circuits orchestrates versatile flight from threats

Naturalistic escape requires versatile context-specific flight with rapid evaluation of local geometry to identify and use efficient escape routes. It is unknown how spatial navigation and escape circuits are recruited to produce context-specific flight. Using mice, we show that activity in cholecystokinin-expressing hypothalamic dorsal premammillary nucleus (PMd-cck) cells is sufficient and necessary for context-specific escape that adapts to each environment's layout. In contrast, numerous other nuclei implicated in flight only induced stereotyped panic-related escape. We reasoned the dorsal premammillary nucleus (PMd) can induce context-specific escape because it projects to escape and spatial navigation nuclei. Indeed, activity in PMd-cck projections to thalamic spatial navigation circuits is necessary for context-specific escape induced by moderate threats but not panic-related stereotyped escape caused by perceived asphyxiation. Conversely, the PMd projection to the escape-inducing dorsal periaqueductal gray projection is necessary for all tested escapes. Thus, PMd-cck cells control versatile flight, engaging spatial navigation and escape circuits

Breakdown of spatial coding and interneuron synchronization in epileptic mice

Temporal lobe epilepsy causes severe cognitive deficits, but the circuit mechanisms remain unknown. Interneuron death and reorganization during epileptogenesis may disrupt the synchrony of hippocampal inhibition. To test this, we simultaneously recorded from the CA1 and dentate gyrus in pilocarpine-treated epileptic mice with silicon probes during head-fixed virtual navigation. We found desynchronized interneuron firing between the CA1 and dentate gyrus in epileptic mice. Since hippocampal interneurons control information processing, we tested whether CA1 spatial coding was altered in this desynchronized circuit, using a novel wire-free miniscope. We found that CA1 place cells in epileptic mice were unstable and completely remapped across a week. This spatial instability emerged around 6 weeks after status epilepticus, well after the onset of chronic seizures and interneuron death. Finally, CA1 network modeling showed that desynchronized inputs can impair the precision and stability of CA1 place cells. Together, these results demonstrate that temporally precise intrahippocampal communication is critical for spatial processing