Probing Neuro-Endocrine Interactions Through Remote Magnetothermal Adrenal Stimulation.

Exposure to stressful or traumatic stimuli may alter hypothalamic-pituitary-adrenal (HPA) axis and sympathoadrenal-medullary (SAM) reactivity. This altered reactivity may be a component or cause of mental illnesses. Dissecting these mechanisms requires tools to reliably probe HPA and SAM function, particularly the adrenal component, with temporal precision. We previously demonstrated magnetic nanoparticle (MNP) technology to remotely trigger adrenal hormone release by activating thermally sensitive ion channels. Here, we applied adrenal magnetothermal stimulation to probe stress-induced HPA axis and SAM changes. MNP and control nanoparticles were injected into the adrenal glands of outbred rats subjected to a tone-shock conditioning/extinction/recall paradigm. We measured MNP-triggered adrenal release before and after conditioning through physiologic (heart rate) and serum (epinephrine, corticosterone) markers. Aversive conditioning altered adrenal function, reducing corticosterone and blunting heart rate increases post-conditioning. MNP-based organ stimulation provides a novel approach to probing the function of SAM, HPA, and other neuro-endocrine axes and could help elucidate changes across stress and disease models.

Effects of Immediate Aversive Stimulation on Haloperidol-Induced Catalepsy in Rats.

In animal models, the administration of the dopaminergic D2 antagonist haloperidol affects the nigrostriatal pathway, inducing catalepsy, a state of immobility similar to Parkinson's disease (PD) bradykinesia and akinesia. In PD, the motor impairments are due to difficulties in selecting and executing motor actions, associated with dopamine loss in basal ganglia and cortical targets. Motor and affective limbic networks seem to be integrated via a striato-nigro-striatal network, therefore, it is not surprising that the motor impairments in PD can be influenced by the patient's emotional state. Indeed, when exposed to aversive stimuli or life-threatening events, immobile patients are capable of performing sudden movements, a phenomenon known as paradoxical kinesia. Thus, the present study investigated the effects of unconditioned and conditioned aversive stimulation on haloperidol-induced catalepsy in rats. First, male Wistar rats received intraperitoneal administration of saline or haloperidol (1 or 2 mg/kg) and were evaluated in the catalepsy bar test to assess the cataleptic state induced by the different doses of haloperidol over time. Next, we evaluated the effects of two types of unconditioned aversive stimuli-100 lux light (1 and 20 s) or 0.6 mA footshock (1 s)-on the catalepsy. Finally, we evaluated the effects of light conditioned stimuli (Light-CS), previously paired with footshocks, on the cataleptic state. Catalepsy was observed following haloperidol 1 and 2 mg/kg administration. Exposure to footshocks, but not to light, significantly reduced step-down latency during the catalepsy test. Although unconditioned light did not affect catalepsy, paired Light-CS did reduce step-down latency. Here, we have provided evidence of face validity for the study of paradoxical kinesia. In addition to demonstrating that immediate exposure to an aversive stimulus is capable of disrupting the cataleptic state, our findings show that haloperidol-induced catalepsy seems to be differently influenced depending on the modality of aversive stimulation. Our data suggest that the selective recruitment of threat response systems may bypass the dysfunctional motor circuit leading to the activation of alternative routes to drive movement.

Lost in translation: no effect of repeated optogenetic cortico-striatal stimulation on compulsivity in rats.

The orbitofrontal cortex-ventromedial striatum (OFC-VMS) circuitry is widely believed to drive compulsive behavior. Hyperactivating this pathway in inbred mice produces excessive and persistent self-grooming, which has been considered a model for human compulsivity. We aimed to replicate these findings in outbred rats, where there are few reliable compulsivity models. Male Long-Evans rats implanted with optical fibers into VMS and with opsins delivered into OFC received optical stimulation at parameters that produce OFC-VMS plasticity and compulsive grooming in mice. We then evaluated rats for compulsive self-grooming at six timepoints: before, during, immediately after, and 1 h after each stimulation, 1 and 2 weeks after the ending of a 6-day stimulation protocol. To further test for effects of OFC-VMS hyperstimulation, we ran animals in three standard compulsivity assays: marble burying, nestlet shredding, and operant attentional set-shifting. OFC-VMS stimulation did not increase self-grooming or induce significant changes in nestlet shredding, marble burying, or set-shifting in rats. Follow-on evoked potential studies verified that the stimulation protocol altered OFC-VMS synaptic weighting. In sum, although we induced physiological changes in the OFC-VMS circuitry, we could not reproduce in a strongly powered study in rats a model of compulsive behavior previously reported in mice. This suggests possible limitations to translation of mouse findings to species higher on the phylogenetic chain.

Dopamine D2 receptors in the expression and extinction of contextual and cued conditioned fear in rats.

Dopamine seems to mediate fear conditioning through its action on D2 receptors in the mesolimbic pathway. Systemic and local injections of dopaminergic agents showed that D2 receptors are preferentially involved in the expression, rather than in the acquisition, of conditioned fear. To further examine this issue, we evaluated the effects of systemic administration of the dopamine D2-like receptor antagonists sulpiride and haloperidol on the expression and extinction of contextual and cued conditioned fear in rats. Rats were trained to a context-CS or a light-CS using footshocks as unconditioned stimuli. After 24 h, rats received injections of sulpiride or haloperidol and were exposed to the context-CS or light-CS for evaluation of freezing expression (test session). After another 24 h, rats were re-exposed to the context-CS or light-CS, to evaluate the extinction recall (retest session). Motor performance was assessed with the open-field and catalepsy tests. Sulpiride, but not haloperidol, significantly reduced the expression of contextual and cued conditioned fear without affecting extinction recall. In contrast, haloperidol, but not sulpiride, had cataleptic and motor-impairing effects. The results reinforce the importance of D2 receptors in fear conditioning and suggest that dopaminergic mechanisms mediated by D2 receptors are mainly involved in the expression rather than in the extinction of conditioned freezing.

Influence of aversive stimulation on haloperidol-induced catalepsy in rats.

Catalepsy - an immobile state in which individuals fail to change imposed postures - can be induced by haloperidol. In rats, the pattern of haloperidol-induced catalepsy is very similar to that observed in Parkinson's disease (PD). As some PD symptoms seem to depend on the patient's emotional state, and as anxiety disorders are common in PD, it is possible that the central mechanisms regulating emotional and cataleptic states interplay. Previously, we showed that haloperidol impaired contextual-induced alarm calls in rats, without affecting footshock-evoked calls. Here, we evaluated the influence of distinct aversive stimulations on the haloperidol-induced catalepsy. First, male Wistar rats were subjected to catalepsy tests to establish a baseline state after haloperidol or saline administration. Next, distinct cohorts were exposed to open-field; elevated plus-maze; open-arm confinement; inescapable footshocks; contextual conditioned fear; or corticosterone administration. Subsequently, catalepsy tests were performed again. Haloperidol-induced catalepsy was verified in all drug-treated animals. Exposure to open-field, elevated plus-maze, open-arm confinement, footshocks, or administration of corticosterone had no significant effect on haloperidol-induced catalepsy. Contextual conditioned fear, which is supposed to promote a more intense fear, increased catalepsy over time. Our findings suggest that only specific defensive circuitries modulate the nigrostriatal system mediating the haloperidol-induced cataleptic state.

Dopamine D2-like receptors modulate freezing response, but not the activation of HPA axis, during the expression of conditioned fear.

Considering the complexity of aversive information processing and defensive response expression, a combined action of stress modulators may be required for an optimal performance during threatening situations. Dopamine is now recognized as one of the most active modulators underlying states of fear and anxiety. On the other hand, activation of hypothalamic-pituitary-adrenocortical (HPA) axis, which leads to the release of corticosterone in rodents, has been considered a key part of the stress response. The current study is an extension of prior work investigating modulatory effects of dopamine and corticosterone on conditioned fear expression. We have showed that corticosterone, acting through mineralocorticoid receptors in the ventral tegmental area (VTA), upregulates dopaminergic system in the basolateral amygdala (BLA), enabling the expression of conditioned freezing response. The novel question addressed here is whether VTA-BLA dopaminergic signaling is necessary for increases in corticosterone during conditioned fear expression. Using site-specific treatment with D2-like agonist quinpirole (VTA) and D2-like antagonist sulpiride (BLA), we evaluated freezing and plasma corticosterone in rats exposed to a light used as aversive conditioned stimulus (CS). Intra-VTA quinpirole and intra-BLA sulpiride significantly decreased freezing expression in the conditioned fear test, but this anxiolytic-like effect of the dopaminergic drugs was not associated with changes in plasma corticosterone concentrations. Altogether, data suggest that interferences with the ability of the CS to activate the dopaminergic VTA-BLA pathway reduce the expression of freezing, but activation of the HPA axis seems to occur upstream of the recruitment of dopaminergic mechanisms in conditioned fear states.

Mineralocorticoid receptors in the ventral tegmental area regulate dopamine efflux in the basolateral amygdala during the expression of conditioned fear.

Despite the recognized involvement of corticosteroids in the modulation of emotional behavior, the specific role of mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs) in the expression of conditioned fear responses is still open to investigation. The present study sought to clarify the involvement of both types of corticosteroid receptors in two different brain regions--the ventral tegmental area (VTA) and the basolateral amygdala complex (BLA)--on the expression of conditioned fear. The first experiment assessed the effects of intra-VTA or intra-BLA administration of spironolactone (MR antagonist) or mifepristone (GR antagonist) on the expression of conditioned freezing to a light-CS and on motor performance in the open-field test. Intra-VTA spironolactone, but not mifepristone, attenuated the expression of the conditioned freezing response whereas intra-BLA spironolactone or mifepristone had no significant effects. These treatments did not affect motor performance in the open-field test. Since dopamine is released in the BLA from the VTA during the expression of conditioned fear, the anxiolytic-like effect of decreased corticosteroid activity in the first experiment could be associated with changes in dopaminergic neurotransmission. The second experiment, using in vivo microdialysis, investigated the role of MRs in the VTA on dopamine levels in the BLA during the expression of conditioned fear. Blocking MRs locally in the VTA with spironolactone reduced dopamine efflux in the BLA and decreased the expression of conditioned freezing in response to the CS. Taken together, the data indicate that corticosterone, acting locally on MRs in the VTA, stimulates dopamine efflux in the BLA, which facilitates the expression of conditioned freezing to a light-CS.

Conditioned fear response is modulated by a combined action of the hypothalamic-pituitary-adrenal axis and dopamine activity in the basolateral amygdala.

The present study sought to determine the extent to which the combined activity of the hypothalamic-pituitary-adrenal (HPA) axis and dopaminergic systems is important for the expression of conditioned fear responses. The first experiment examined changes in plasma corticosterone concentration and the conditioned freezing response in rats treated with the dopamine D2 receptor agonist quinpirole (0.25 mg/kg), the dopamine D2 receptor antagonist sulpiride (40 mg/kg), corticosterone (3 or 6 mg/kg), or the corticosterone synthesis blocker metyrapone (30 mg/kg) and subjected to a conditioned fear test. A second experiment assessed the effects of corticosterone (3 or 6 mg/kg) and metyrapone (30 or 60 mg/kg) on fear-potentiated startle. A third experiment assessed the HPA axis modulation of conditioned fear using in vivo microdialysis targeted at dopaminergic neurotransmission in the basolateral amygdala (BLA). Quinpirole and sulpiride decreased conditioned freezing but did not affect plasma corticosterone concentration. Corticosterone and metyrapone did not affect fear-potentiated startle, but metyrapone attenuated conditioned freezing, suggesting that the expression of conditioned freezing requires HPA axis activation. Metyrapone inhibited the increase in dopamine levels in the BLA in response to the conditioned stimulus, whereas corticosterone had no significant effect. These results suggest that HPA axis activation is an initial step in an integrated neuroendocrine-neurochemical-behavioral response when the organism evaluates a threat associated with an environmental stimulus and triggers defense reactions to cope with this situation.

Selective involvement of GABAergic mechanisms of the dorsal periaqueductal gray and inferior colliculus on the memory of the contextual fear as assessed by the fear potentiated startle test.

The inferior colliculus (IC) together with the dorsal periaqueductal gray (dPAG), the amygdala and the medial hypothalamus make part of the brain aversion system, which has mainly been related to the organization of unconditioned fear. However, the involvement of the IC and dPAG in the conditioned fear is still unclear. It is certain that GABA has a regulatory role on the aversive states generated and elaborated in these midbrain structures. In this study, we evaluated the effects of injections of the GABA-A receptor agonist muscimol (1.0 and 2.0 nmol/0.2 microL) into the IC or dPAG on the freezing and fear-potentiated startle (FPS) responses of rats submitted to a context fear conditioning. Intra-IC injections of muscimol did not cause any significant effect on the FPS or conditioned freezing but enhanced the startle reflex in non-conditioned animals. In contrast, intra-dPAG injections of muscimol caused significant reduction in FPS and conditioned freezing without changing the startle reflex in non-conditioned animals. Thus, intra-dPAG injections of muscimol produced the expected inhibitory effects on the anxiety-related responses, the FPS and the freezing whereas these injections into the IC produced quite opposite effects suggesting that descending inhibitory pathways from the IC, probably mediated by GABA-A mechanisms, exert a regulatory role on the lower brainstem circuits responsible for the startle reflex.