INFLUENCE OF RAT CENTRAL THALAMIC NEURONS ON FORAGING BEHAVIOR IN A HAZARDOUS ENVIRONMENT.

Foraging entails a complex balance between approach and avoidance alongside sensorimotor and homeostatic processes under the control of multiple cortical and subcortical areas. Recently, it has become clear that several thalamic nuclei located near the midline regulate motivated behaviors. However, one midline thalamic nucleus that project to key nodes in the foraging network, the central medial (CMT) nucleus, has received little attention so far. Therefore, the present study examined CMT contributions to foraging behavior using inactivation and unit recording techniques in male rats. Inactivation of CMT or the basolateral amygdala (BLA) with muscimol abolished the rats' normally cautious behavior in the foraging task. Moreover, CMT neurons showed large but heterogeneous activity changes during the foraging task, with many neurons decreasing or increasing their discharge rates, with a modest bias for the latter. A generalized linear model revealed that the nature (inhibitory vs. excitatory) and relative magnitude of the activity modulations seen in CMT neurons differed markedly from those of principal BLA cells but were very similar to those of fast-spiking BLA interneurons. Together, these findings suggest that CMT is an important regulator of foraging behavior. In the Discussion, we consider how CMT is integrated in the network of structures that regulate foraging.SIGNIFICANCE STATEMENTForaging entails a complex balance between approach and avoidance alongside sensorimotor and homeostatic processes under the control of multiple cortical and subcortical areas. Although the central medial thalamic (CMT) nucleus is connected to many nodes in this network, its role in the regulation of foraging behavior has not been investigated so far. Here, we examined CMT contributions to foraging behavior using inactivation and unit recording techniques. We found that CMT inactivation abolishes the rats' normally cautious foraging behavior and that CMT neurons show large but heterogeneous changes in firing rates during the foraging task. Together, these results suggest that CMT is an important regulator of foraging behavior.

Basolateral amygdala neurons are activated during threat expectation.

Fear conditioning studies have led to the view that the amygdala contains neurons that signal threat and in turn elicit defensive behaviors through their brain stem and hypothalamic targets. In agreement with this model, a prior unit-recording study in rats performing a seminaturalistic foraging task revealed that many lateral amygdala (LA) neurons are predator responsive. In contrast, our previous study emphasized that most basolateral (BL) amygdala neurons are inhibited at proximity of the predator. However, the two studies used different methods to analyze unit activity, complicating comparisons between them. By applying the same method to the sample of BL neurons we recorded previously, the present study revealed that most principal cells are inhibited by the predator and only 4.5% are activated. Moreover, two-thirds of these cells were also activated by nonthreatening stimuli. In fact, fitting unit activity with a generalized linear model revealed that the only task variables associated with a prevalent positive modulation of BL activity were expectation of the predator's presence and whether the prior trial had been a failure or success. At odds with the threat-coding model of the amygdala, actual confrontation with the predator was usually associated with a widespread inhibition of principal BL neurons. NEW & NOTEWORTHY The basolateral amygdala (BL) is thought to contain neurons that signal threat, in turn eliciting defensive behaviors. In contrast, the present study reports that very few principal BL cells are responsive to threats and that most of them are also activated by nonthreatening stimuli. Yet, expectation of the threat's presence was associated with a prevalent positive modulation of BL activity; actual confrontation with the threat was associated with a widespread inhibition.

Gamma Oscillations in the Basolateral Amygdala: Biophysical Mechanisms and Computational Consequences.

The basolateral nucleus of the amygdala (BL) is thought to support numerous emotional behaviors through specific microcircuits. These are often thought to be comprised of feedforward networks of principal cells (PNs) and interneurons. Neither well-understood nor often considered are recurrent and feedback connections, which likely engender oscillatory dynamics within BL. Indeed, oscillations in the gamma frequency range (40 - 100 Hz) are known to occur in the BL, and yet their origin and effect on local circuits remains unknown. To address this, we constructed a biophysically and anatomically detailed model of the rat BL and its local field potential (LFP) based on the physiological and anatomical literature, along with in vivo and in vitro data we collected on the activities of neurons within the rat BL. Remarkably, the model produced intermittent gamma oscillations (∼50 - 70 Hz) whose properties matched those recorded in vivo, including their entrainment of spiking. BL gamma-band oscillations were generated by the intrinsic circuitry, depending upon reciprocal interactions between PNs and fast-spiking interneurons (FSIs), while connections within these cell types affected the rhythm's frequency. The model allowed us to conduct experimentally impossible tests to characterize the synaptic and spatial properties of gamma. The entrainment of individual neurons to gamma depended on the number of afferent connections they received, and gamma bursts were spatially restricted in the BL. Importantly, the gamma rhythm synchronized PNs and mediated competition between ensembles. Together, these results indicate that the recurrent connectivity of BL expands its computational and communication repertoire.

Midline thalamic inputs to the amygdala: Ultrastructure and synaptic targets.

One of the main subcortical inputs to the basolateral nucleus of the amygdala (BL) originates from a group of dorsal thalamic nuclei located at or near the midline, mainly from the central medial (CMT), and paraventricular (PVT) nuclei. Although similarities among the responsiveness of BL, CMT, and PVT neurons to emotionally arousing stimuli suggest that these thalamic inputs exert a significant influence over BL activity, little is known about the synaptic relationships that mediate these effects. Thus, the present study used Phaseolus vulgaris-leucoagglutinin (PHAL) anterograde tracing and electron microscopy to shed light on the ultrastructural properties and synaptic targets of CMT and PVT axon terminals in the rat BL. Virtually all PHAL-positive CMT and PVT axon terminals formed asymmetric synapses. Although CMT and PVT axon terminals generally contacted dendritic spines, a substantial number ended on dendritic shafts. To determine whether these dendritic shafts belonged to principal or local-circuit cells, calcium/calmodulin-dependent protein kinase II (CAMKIIα) immunoreactivity was used as a selective marker of principal BL neurons. In most cases, dendritic shafts postsynaptic to PHAL-labeled CMT and PVT terminals were immunopositive for CaMKIIα. Overall, these results suggest that CMT and PVT inputs mostly target principal BL neurons such that when CMT or PVT neurons fire, little feed-forward inhibition counters their excitatory influence over principal cells. These results are consistent with the possibility that CMT and PVT inputs constitute major determinants of BL activity.

Vigilance-Associated Gamma Oscillations Coordinate the Ensemble Activity of Basolateral Amygdala Neurons.

Principal basolateral amygdala (BL) neurons profoundly influence motivated behaviors, yet few of them are activated by emotionally valenced stimuli. Here, we show that a likely explanation for this paradox is the synchronizing influence of the high-gamma rhythm. High-gamma (75-95 Hz) entrained BL firing more strongly than all other rhythms. It was most pronounced during states of increased vigilance, when rats were apprehensive. Relative to behavioral states, high-gamma produced minor changes in firing rates yet dramatic increases in synchrony. Moreover, connected pairs of cells showed similarly high levels of entrainment and synchronization. Unexpectedly, prefrontal- and accumbens-projecting cells, respectively, showed high and low entrainment by high-gamma, indicating that this rhythm differentially synchronizes the activity of BL neurons projecting to specific sites. Overall, our findings suggest that individual BL neurons encode information not only by changing their firing rates, but also by synchronizing their collective activity, amplifying their impact on target structures.

Differential Recruitment of Competing Valence-Related Amygdala Networks during Anxiety.

The basolateral amygdala (BL) is involved in fear and anxiety, but it is currently unclear how the same network supports these two states. To address this question, we trained rats on appetitive and aversive conditioning in different contexts. Distinct groups of BL neurons displayed increased activity during appetitive (CS-R) versus aversive (CS-S) conditioned stimuli (R cells and S cells, respectively), and they were typically inhibited by the other CS. When the CS-S was presented in the safe context, rats entered a long-lasting, anxiety-like state characterized by increased inter-CS freezing and impaired reward seeking. During this state, a subset of BL cells ("state cells") showed sustained shifts in baseline activity whose time course matched that of the behavioral changes. Many state cells with increased firing rates were S cells, whereas R cells only included state cells with reduced firing rates. Thus, anxiety involves persistent activity changes that are differentially expressed by subsets of valence-specific BL neurons.

Basolateral amygdala nucleus responses to appetitive conditioned stimuli correlate with variations in conditioned behaviour.

In the lateral amygdala (LA), training-induced increases in neuronal responsiveness to conditioned stimuli (CSs) reflect potentiated sensory responses that drive conditioned behaviours (CRs) via LA's targets. The basolateral nucleus of the amygdala (BL) receives LA inputs and projects to various subcortical sites that can drive aversive and appetitive CRs. Consistent with this, BL neurons also develop increased responses to CSs that predict rewarding or aversive outcomes. This increased BL activity is thought to reflect the potentiated sensory responses of LA neurons. Here we contrast the CS-related activity of BL neurons when rats produced the expected CR or not, to show that cells activated by appetitive CSs mainly encode behavioural output, not CS identity. The strong dependence of BL activity on behaviour irrespective of CS identity suggests that feedforward connectivity from LA to BL can be overridden by other BL inputs.

Amygdala Signaling during Foraging in a Hazardous Environment.

We recorded basolateral amygdala (BL) neurons in a seminaturalistic foraging task. Rats had to leave their nest to retrieve food in an elongated arena inhabited by a mechanical predator. There were marked trial-to-trial variations in behavior. After poking their head into the foraging arena and waiting there for a while, rats either retreated to their nest or initiated foraging. Before initiating foraging, rats waited longer on trials that followed failed than successful trials indicating that prior experience influenced behavior. Upon foraging initiation, most principal cells (Type-1) reduced their firing rate, while in a minority (Type-2) it increased. When rats aborted foraging, Type-1 cells increased their firing rates, whereas in Type-2 cells it did not change. Surprisingly, the opposite activity profiles of Type-1 and Type-2 units were also seen in control tasks devoid of explicit threats or rewards. The common correlate of BL activity across these tasks was movement velocity, although an influence of position was also observed. Thus depending on whether rats initiated movement or not, the activity of BL neurons decreased or increased, regardless of whether threat or rewards were present. Therefore, BL activity not only encodes threats or rewards, but is closely related to behavioral output. We propose that higher order cortical areas determine task-related changes in BL activity as a function of reward/threat expectations and internal states. Because Type-1 and Type-2 cells likely form differential connections with the central amygdala (controlling freezing), this process would determine whether movement aimed at attaining food or exploration is suppressed or facilitated. Significance statement: For decades, amygdala research has been dominated by pavlovian and operant conditioning paradigms. This work has led to the view that amygdala neurons signal threats or rewards, in turn causing defensive or approach behaviors. However, the artificial circumstances of conditioning studies bear little resemblance to normal life. In natural conditions, subjects are simultaneously presented with potential threats and rewards, forcing them to engage in a form of risk assessment. We examined this process using a seminaturalistic foraging task. In constant conditions of threats and rewards, amygdala activity could be high or low, depending on the rats' decisions on a given trial. Therefore, amygdala activity does not only encode threats or rewards but is also closely related to behavioral output.

Morphology, PKCδ expression, and synaptic responsiveness of different types of rat central lateral amygdala neurons.

Recent findings implicate the central lateral amygdala (CeL) in conditioned fear. Indeed, CeL contains neurons exhibiting positive (CeL-On) or negative (CeL-Off) responses to fear-inducing conditioned stimuli (CSs). In mice, these cells differ in their expression of protein kinase Cδ (PKCδ) and physiological properties. CeL-Off cells are PKCδ(+) and late firing (LF), whereas CeL-On cells are PKCδ(-) and express a regular-spiking (RS) or low-threshold bursting (LTB) phenotype. However, the scarcity of LF cells in rats raises questions about the correspondence between the organization of CeL in mice and rats. Therefore, we studied the PKCδ expression, morphological properties, synaptic responsiveness, and fear conditioning-induced plasticity of rat CeL neurons. No PKCδ(+) LF cells were encountered, but ≈20-25% of RS and LTB neurons were PKCδ(+). Compared with RS neurons, a higher proportion of LTB cells projected to central medial amygdala (CeM) and they had fewer primary dendritic branches, yet the amplitude of excitatory postsynaptic potentials (EPSPs) evoked by lateral amygdala (LA) stimulation was similar in RS and LTB cells. In contrast, LA-evoked inhibitory postsynaptic potentials (IPSPs) had a higher amplitude in LTB than RS neurons. Finally, fear conditioning did not induce plasticity at LA inputs to RS or LTB neurons. These findings point to major species differences in the organization of CeL. Since rat LTB cells are subjected to stronger feedforward inhibition, they are more likely to exhibit inhibitory CS responses than RS cells. This is expected to cause a disinhibition of CeM fear output neurons and therefore an increase in fear expression.

Physiological identification and infralimbic responsiveness of rat intercalated amygdala neurons.

Intercalated (ITC) amygdala neurons are thought to play a critical role in the extinction of conditioned fear. However, several factors hinder progress in studying ITC contributions to extinction. First, although extinction is usually studied in rats and mice, most ITC investigations were performed in guinea pigs or cats. Thus it is unclear whether their connectivity is similar across species. Second, we lack criteria to identify ITC cells on the basis of their discharge pattern. As a result, key predictions of ITC extinction models remain untested. Among these, ITC cells were predicted to be strongly excited by infralimbic inputs, explaining why infralimbic inhibition interferes with extinction. To study the connectivity of ITC cells, we labeled them with neurobiotin during patch recordings in slices of the rat amygdala. This revealed that medially located ITC cells project topographically to the central nucleus and to other ITC clusters located more ventrally. To study the infralimbic responsiveness of ITC cells, we performed juxtacellular recording and labeling of amygdala cells with neurobiotin in anesthetized rats. All ITC cells were orthodromically responsive to infralimbic stimuli, and their responses usually consisted of high-frequency (~350 Hz) trains of four to six spikes, a response pattern never seen in neighboring amygdala nuclei. Overall, our results suggest that the connectivity of ITC cells is conserved across species and that ITC cells are strongly responsive to infralimbic stimuli, as predicted by extinction models. The unique response pattern of ITC cells to infralimbic stimuli can now be used to identify them in fear conditioning experiments.

Dissociation between rewarding and psychomotor effects of opiates: differential roles for glutamate receptors within anterior and posterior portions of the ventral tegmental area.

The rewarding effects of drugs of abuse are thought to be dependent on the mesocorticolimbic dopamine system, which originates in the ventral tegmental area (VTA) and projects into the nucleus accumbens (NAC) and other forebrain regions. Heroin, by inhibiting GABAergic interneurons in the VTA, induces local dopaminergic activation and release in the NAC terminals. The role of other basic neurotransmitter systems, such as glutamate in the VTA, in mediating the rewarding effect of addictive drugs, is less established. We explored whether blockade of glutamate receptors in subregions of the VTA modulate the rewarding properties and/or the development of psychomotor changes induced by opiates. Administration of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; an AMPA/kainate receptor antagonist) into the anterior VTA blocked the rewarding effects of opiates in both the conditioned place preference and the self-administration paradigms without affecting the gradual increase of the psychomotor response to opiates. In contrast, administration of CNQX into the posterior VTA did not affect the rewarding properties of opiates, but blocked the initial sedative effect of opiates and the gradual increase of the psychomotor response to the drug. These findings suggest a critical role for glutamate receptors in the VTA in opiate reward, as well as behavioral and anatomical dissociation between the rewarding and psychomotor effects of opiates.

Three-dimensional distribution of the electric field induced in the brain by transcranial magnetic stimulation using figure-8 and deep H-coils.

The H-coils are a novel development in transcranial magnetic stimulation (TMS), designed to achieve effective stimulation of deep neuronal regions without inducing unbearable fields cortically, thus broadly expanding the potential feasibility of TMS for research and for treating various neurologic disorders. This study compared the field distribution of two H-coil versions, termed H1 and H2, and of a standard figure-of-eight coil. Three-dimensional electrical field distributions of the H1 and H2-coils, designed for effective stimulation of prefrontal regions, and of a standard figure-8 coil, were measured in a head model filled with physiologic saline solution. With stimulator output at 120% of the hand motor threshold, suprathreshold field is induced by the H1-coil at lateral and medial frontal regions at depths of up to 4 to 5 cm, and by the H2-coil at medial prefrontal regions up to 2 to 3 cm, and at lateral frontal regions up to 5 to 6 cm. The figure-8 coil induced suprathreshold field focally under the coil's central segment, at depths of up to 1.5 cm. The ability of the H-coils to stimulate effectively deeper neuronal structures is obtained at the cost of a wider electrical field distribution in the brain. However, the H-coils enable simultaneous stimulation of several brain regions, whereas the depth penetration in each region can be controlled either by adjusting the stimulator output, and/or by varying the distance between various coil elements and the skull.