Analysis of Monosynaptic Inputs to Thalamic Paraventricular Nucleus Neurons Innervating the Shell of the Nucleus Accumbens and Central Extended Amygdala.

The paraventricular nucleus of the thalamus (PVT) sends dense projections to the shell of the nucleus accumbens (NAcSh), dorsolateral region of the bed nucleus of the stria terminalis (BSTDL) and the lateral region of central nucleus of the amygdala (CeL). Projection specific modulation of these pathways has been shown to regulate appetitive and aversive behavioral responses. The present investigation applied an intersectional monosynaptic rabies tracing approach to quantify the brain-wide sources of afferent input to PVT neurons that primarily project to the NAcSh, BSTDL and CeL. The results demonstrate that these projection neurons receive monosynaptic input from similar brain regions. The prefrontal cortex and the ventral subiculum of the hippocampus were major sources of input to the PVT projection neurons. In addition, the lateral septal nucleus, thalamic reticular nucleus and the hypothalamic medial preoptic area, dorsomedial, ventromedial, and arcuate nuclei were sources of input. The subfornical organ, parasubthalamic nucleus, periaqueductal gray matter, lateral parabrachial nucleus, and nucleus of the solitary tract were consistent but lesser sources of input. This input-output relationship is consistent with recent observations that PVT neurons have axons that bifurcate extensively to divergently innervate the NAcSh, BSTDL and CeL.

Convergence of monosynaptic inputs from neurons in the brainstem and forebrain on parabrachial neurons that project to the paraventricular nucleus of the thalamus.

The paraventricular nucleus of the thalamus (PVT) projects to areas of the forebrain involved in regulating behavior. Homeostatic challenges and salient cues activate the PVT and evidence shows that the PVT regulates appetitive and aversive responses. The brainstem is a source of afferents to the PVT and the present study was done to determine if the lateral parabrachial nucleus (LPB) is a relay for inputs to the PVT. Retrograde tracing experiments with cholera toxin B (CTB) demonstrate that the LPB contains more PVT projecting neurons than other regions of the brainstem including the catecholamine cell groups. The hypothesis that the LPB is a relay for signals to the PVT was assessed using an intersectional monosynaptic rabies tracing approach. Sources of inputs to LPB included the reticular formation; periaqueductal gray (PAG); nucleus cuneiformis; and superior and inferior colliculi. Distinctive clusters of input cells to LPB-PVT projecting neurons were also found in the dorsolateral bed nucleus of the stria terminalis (BSTDL) and the lateral central nucleus of the amygdala (CeL). Anterograde viral tracing demonstrates that LPB-PVT neurons densely innervate all regions of the PVT in addition to providing collateral innervation to the preoptic area, lateral hypothalamus, zona incerta and PAG but not the BSTDL and CeL. The paper discusses the anatomical evidence that suggests that the PVT is part of a network of interconnected neurons involved in arousal, homeostasis, and the regulation of behavioral states with forebrain regions potentially providing descending modulation or gating of signals relayed from the LPB to the PVT.

Extensive divergence of projections to the forebrain from neurons in the paraventricular nucleus of the thalamus.

Neurons in the paraventricular nucleus of the thalamus (PVT) respond to emotionally salient events and project densely to subcortical regions known to mediate adaptive behavioral responses. The areas of the forebrain most densely innervated by the PVT include striatal-like subcortical regions that consist of the shell of the nucleus accumbens (NAcSh), the dorsolateral region of the bed nucleus of the stria terminalis (BSTDL) and the lateral-capsular division of the central nucleus of the amygdala (CeL). A recent tracing experiment demonstrated that the PVT is composed of two intermixed populations of neurons that primarily project to either the dorsomedial (dmNAcSh) or ventromedial region of the NAcSh (vmNAcSh) with many of the vmNAcSh projecting neurons providing collateral innervation of the BSTDL and CeL. The present study used triple injections of the retrograde tracer cholera toxin B to provide a detailed map of the location of PVT neurons that provide collaterals to the vmNAcSh, BSTDL and CeL. These neurons were intermixed throughout the PVT and did not form uniquely localized subpopulations. An intersectional viral anterograde tracing approach was used to demonstrate that regardless of its presumed target of innervation (dmNAcSh, vmNAcSh, BSTDL, or CeL), most neurons in the PVT provide collateral innervation to a common set of forebrain regions. The paper shows that PVT-dmNAcSh projecting neurons provide the most divergent projection system and that these neurons express the immediate early gene product cFos following an aversive incident. We propose that the PVT may regulate a broad range of responses to physiological and psychological challenges by simultaneously influencing functionally diverse regions of the forebrain that include the cortex, striatal-like regions in the basal forebrain and a number of hypothalamic nuclei.

Schizophrenia-associated LRRTM1 regulates cognitive behavior through controlling synaptic function in the mediodorsal thalamus.

Reduced activity of the mediodorsal thalamus (MD) and abnormal functional connectivity of the MD with the prefrontal cortex (PFC) cause cognitive deficits in schizophrenia. However, the molecular basis of MD hypofunction in schizophrenia is not known. Here, we identified leucine-rich-repeat transmembrane neuronal protein 1 (LRRTM1), a postsynaptic cell-adhesion molecule, as a key regulator of excitatory synaptic function and excitation-inhibition balance in the MD. LRRTM1 is strongly associated with schizophrenia and is highly expressed in the thalamus. Conditional deletion of Lrrtm1 in the MD in adult mice reduced excitatory synaptic function and caused a parallel reduction in the afferent synaptic activity of the PFC, which was reversed by the reintroduction of LRRTM1 in the MD. Our results indicate that chronic reduction of synaptic strength in the MD by targeted deletion of Lrrtm1 functionally disengages the MD from the PFC and may account for cognitive, social, and sensorimotor gating deficits, reminiscent of schizophrenia.

The Paraventricular Nucleus of the Thalamus as an Integrating and Relay Node in the Brain Anxiety Network.

The brain anxiety network is composed of a number of interconnected cortical regions that detect threats and execute appropriate defensive responses via projections to the shell of the nucleus accumbens (NAcSh), dorsolateral region of the bed nucleus of the stria terminalis (BSTDL) and lateral region of the central nucleus of the amygdala (CeL). The paraventricular nucleus of the thalamus (PVT) is anatomically positioned to integrate threat- and arousal-related signals from cortex and hypothalamus and then relay these signals to neural circuits in the NAcSh, BSTDL, and CeL that mediate defensive responses. This review describes the anatomical connections of the PVT that support the view that the PVT may be a critical node in the brain anxiety network. Experimental findings are reviewed showing that the arousal peptides orexins (hypocretins) act at the PVT to promote avoidance of potential threats especially following exposure of rats to a single episode of footshocks. Recent anatomical and experimental findings are discussed which show that neurons in the PVT provide divergent projections to subcortical regions that mediate defensive behaviors and that the projection to the NAcSh is critical for the enhanced social avoidance displayed in rats exposed to footshocks. A theoretical model is proposed for how the PVT integrates cortical and hypothalamic signals to modulate the behavioral responses associated with anxiety and other challenging situations.

A projection from the paraventricular nucleus of the thalamus to the shell of the nucleus accumbens contributes to footshock stress-induced social avoidance.

The paraventricular nucleus of the thalamus (PVT) is an area of the dorsal midline thalamus that contributes to footshock induced anxiety. The PVT sends a dense projection to the shell of the nucleus accumbens (NAcSh) and the present study explored if this projection is involved in the behavioral changes produced by a single exposure of rats to inescapable footshocks. The inhibitory Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) hM4Di was transduced in PVT neurons that project to the NAcSh. Rats were exposed to an episode of moderately intense footshock (1.5 mA × 2 s × 5) and assigned to either high-responder (HR) or low-responder groups (LR) according to their level of fear generalization 24 h later. The effect of chemogenetic inhibition of the PVT-NAcSh projection on anxiety- and fear-like behaviors was assessed at approximately 2 weeks post-footshock. HR showed a higher level of social avoidance compared to non-shocked animals and LR. The elevated level of social avoidance was attenuated in the HR treated with the hM4Di agonist clozapine (0.01 mg/kg, i.p.) or clozapine N-oxide (CNO) administrations in the NAcSh while avoidance of open spaces and contextual fear expression were not affected. Analysis of protein product of the early to immediate gene cfos indicated that these effects were mediated by dynorphin neurons in the NAcSh. This study provides evidence for a role of a projection from the PVT to the NAcSh in stress-induced social avoidance independent of anxiety to non-social stimuli and contextual fear mechanisms.

Pheromone-Induced Odor Associative Fear Learning in Rats.

Alarm pheromones alert conspecifics to the presence of danger. Can pheromone communication aid in learning specific cues? Such facilitation has an evident evolutionary advantage. We use two associative learning paradigms to test this hypothesis. The first is stressed cage mate-induced conditioning. One pair-housed adult rat received 4 pairings of terpinene + shock over 30 min. Ten minutes after return to the home cage, its companion rat was removed and exposed to terpinene. Single-housed controls were exposed to either terpinene or shock only. Companion rats showed terpinene-specific freezing, which was prevented by ß-adrenoceptor blockade. Using Arc to index neuronal activation in response to terpinene re-exposure, stressed cage-mate induced associative learning was measured. Companion rats showed increased neuronal activity in the accessory olfactory bulb, while terpinene + shock-conditioned rats showed increased activity in the main olfactory bulb. Both groups had enhanced activity in the anterior basolateral amygdala and central amygdala. To test involvement of pheromone mediation, in the 2nd paradigm, we paired terpinene with soiled bedding from odor + shock rats or a rat alarm pheromone. Both conditioning increased rats' freezing to terpinene. Blocking NMDA receptors in the basolateral amygdala prevented odor-specific learning suggesting shock and pheromone-paired pathways converge in the amygdala. An alarm pheromone thus enables cue-specific learning as well as signalling danger.

Boosting of Thalamic D2 Dopaminergic Transmission: A Potential Strategy for Drug-Seeking Attenuation.

This commentary focuses on novel findings by Clark et al. (2017) published in eNeuro, which show that dopamine D2 receptors (D2Rs) in the paraventricular nucleus of the thalamus (PVT) are involved in cocaine sensitization. We extend the discussion on how their findings contribute to our understanding of the role of the PVT in drug seeking by providing new insight on the role of the PVT in the regulation of food-seeking and fear responses. We also consider the significance of the neuroanatomical findings reported by Clark et al., that the PVT is reciprocally connected with areas of the brain involved in addiction and discuss the implications associated with the source and type of dopaminergic fibers innervating this area of the thalamus.

Collateralization of projections from the paraventricular nucleus of the thalamus to the nucleus accumbens, bed nucleus of the stria terminalis, and central nucleus of the amygdala.

The paraventricular nucleus of the thalamus (PVT) is a midline thalamic nucleus with dense projections to the nucleus accumbens (NAc), dorsolateral region of the bed nucleus of the stria terminalis (BSTDL) and the lateral/capsular region of the central nucleus of the amygdala (CeL/CeC). Recent experimental evidence indicates that the PVT is involved in both appetitive and aversive behaviors. However, it is unknown if subgroups of neurons in the PVT innervate different subcortical targets or if the same neurons issue collaterals to multiple areas. To address this issue, we injected two different fluorescent retrograde tracers, cholera toxin subunit B conjugated to Alexa Fluor-488 or Alexa Fluor-594, into different pairs of the subcortical targets including different parts of the NAc (shell, core, dorsomedial shell, and ventromedial shell), BSTDL, and amygdala (basolateral amygdala and CeL/CeC). The results indicate a moderate to high level of collateralization of projections from neurons in the PVT to NAc, BSTDL, and CeL/CeC suggesting a potential importance of the PVT in simultaneously coordinating the activity of key regions of the brain involved in mediating emotional and motivational behaviors. We also observed a difference in the subcortical targets innervated by the anterior PVT (aPVT) and posterior PVT (pPVT) showing that more neurons in the aPVT innervate the dorsomedial part of the NAc shell, while more neurons in the pPVT innervate the ventromedial NAc shell, BSTDL, and CeL/CeC. This observation is suggestive of a potential functional difference between the aPVT and pPVT.

Role of the orexin (hypocretin) system in contextual fear conditioning in rats.

Orexin (hypocretin) neurons located in the posterior hypothalamus send projections to multiple areas of the brain involved in arousal and experimental evidence indicates that these neurons play a role in the physiological and behavioral responses to stress. This study was done to determine if the orexin system was involved in mediating the fear associated with shock context (5×2s of 1.5mA). First, real-time RT-PCR was used to examine changes in the mRNA levels for prepro-orexin (ppOX), the orexin-1 receptor (OX1R) and the orexin-2 receptor (OX2R) at two weeks post-shock. We found that the mRNA levels for ppOX and OX1R were increased in the posterior hypothalamus of shocked rats. In contrast, no significant difference was found in the midline thalamus or the locus coeruleus/parabrachial region. Second, the study examined if systemic injections of antagonists for orexin receptors attenuated the freezing related to contextual fear. The OX1R antagonist SB334867 (20 or 30mg/kg; i.p.) decreased freezing while the same doses of the OX2R antagonist TCSOX229 had no effect. The dual orexin antagonist TCS1102 (20mg/kg; i.p.) also decreased the freezing to the shock context. The results of the present study show upregulation of orexin activity and of the OX1R in the hypothalamus following exposure of rats to footshocks and highlight a specific role of OX1R in contextual fear.

Effects of footshocks on anxiety-like behavior and mRNA levels of precursor peptides for corticotropin releasing factor and opioids in the forebrain of the rat.

Corticotropin releasing factor (CRF) and dynorphin are neuropeptides that are associated with the negative emotional states. Experimental evidence indicates that dynorphin neurons located in the nucleus accumbens and CRF neurons in the bed nucleus of the stria terminalis (BST) and the central nucleus of the amygdala (CeA) mediate anxiety-like behaviors immediately after the stressful experience (24-48h). The present study was done to evaluate if changes in the levels of the mRNA for these peptides in the striatum, BST, and CeA were associated with the long-lasting avoidance of novelty, a measure of an anxiety-like state, in a subset of rats exposed to unpredictable and moderately intense footshocks (5×2s of 1.5mA). Shocked rats with enhanced fear to a novel tone 24h after the footshocks (high responders; HR) displayed long-lasting avoidance in the elevated T-maze whereas shocked rats with low levels of acute fear (low responders; LR) had low levels of avoidance similar to nonshocked rats. An increase in the level of proCRF mRNA was detected in the CeA of the HR compared to LR and nonshocked rats but not in other areas of the brain sampled. In contrast, prodynorphin and proenkephalin mRNA levels in the striatum, BST and CeA were not different between HR, LR and nonshocked rats. This study provides evidence that CRF neurons in the CeA may play a role in the anxiety-like state produced in a subset of rats exposed to footshocks.

Placing the paraventricular nucleus of the thalamus within the brain circuits that control behavior.

This article reviews the anatomical connections of the paraventricular nucleus of the thalamus (PVT) and discusses some of the connections by which the PVT could influence behavior. The PVT receives neurochemically diverse projections from the brainstem and hypothalamus with an especially strong innervation from peptide producing neurons. Anatomical evidence is also presented which suggests that the PVT relays information from neurons involved in visceral or homeostatic functions. In turn, the PVT is a major source of projections to the nucleus accumbens, the bed nucleus of the stria terminalis and the central nucleus of the amygdala as well as the cortical areas associated with these subcortical regions. The PVT is activated by conditions and cues that produce states of arousal including those with appetitive or aversive emotional valences. The paper focuses on the potential contribution of the PVT to circadian rhythms, fear, anxiety, food intake and drug-seeking. The information in this paper highlights the potential importance of the PVT as being a component of the brain circuits that regulate reward and defensive behavior with the hope of generating more research in this relatively understudied region of the brain.

Blocking of orexin receptors in the paraventricular nucleus of the thalamus has no effect on the expression of conditioned fear in rats.

The paraventricular nucleus of the thalamus (PVT) projects to the central nucleus of the amygdala and recent experimental evidence indicates a role for the PVT in conditioned fear. Furthermore, the PVT contains a high density of orexin receptors and fibers and acute injections of orexin antagonist into the PVT produce anxiolytic effects. The present study was done to determine if administration of a dual orexin receptor antagonist (DORA) in the region of the PVT interferes with the expression of conditioned fear in rats exposed to cued and contextual conditioning paradigms. Infusion of 0.5 µl of the DORA N-biphenyl-2-yl-1-[(1-methyl-1H-benzimidazol-2yl) sulfanyl] acetyl-L-prolinamide at a concentration of 0.1, 1.0, and 10 nmol had no effect on the freezing produced by exposing rats to an auditory cue or the context associated with foot shock. In contrast, the 1.0 and 10 nmol doses were anxiolytic in the social interaction test. The results of the present study do not support a role for orexin receptors in the PVT in the expression of learned fear. The finding that the 1.0 and 10 nmol doses of DORA in the PVT region were anxiolytic in the social interaction test is consistent with other studies indicating a role for orexins in the PVT in anxiety-like behaviors.

The hypothalamus and periaqueductal gray are the sources of dopamine fibers in the paraventricular nucleus of the thalamus in the rat.

The paraventricular nucleus of the thalamus (PVT) sends a very dense projection to the nucleus accumbens. This area of the striatum plays a key role in motivation and recent experimental evidence indicates that the PVT may have a similar function. It is well known that a dopaminergic projection from the ventral tegmental area (VTA) to the nucleus accumbens is a key regulator of motivation and reward-related behavior. Dopamine (DA) fibers have also been localized in the PVT but the source of these fibers in the rat has not been unequivocally identified. The present study was done to re-examine this question. Small iontophoretic injections of cholera toxin B (CTb) were made in the PVT to retrogradely label tyrosine hydroxylase (TH) neurons. Neurons that were double-labeled for TH/CTb were found scattered in DA cell groups of the hypothalamus (ventrorostral A10, A11, A13, A15 DA cell groups) and the midbrain (dorsocaudal A10 embedded in the periaqueductal gray). In contrast, double-labeled neurons were absent in the retrorubral field (A8), substantia nigra (A9) and VTA (A10) of the midbrain. We conclude that DA fibers in the PVT do not originate from VTA but from a heterogeneous population of DA neurons located in the hypothalamus and periaqueductal gray.

Lesions of the posterior paraventricular nucleus of the thalamus attenuate fear expression.

The paraventricular nucleus of the thalamus (PVT) has generated interest because of its strong projections to areas of the brain associated with the regulation of emotional behaviors. The posterior aspect of the PVT (pPVT) is notable for its projection to the central nucleus of the amygdala which is essential for the expression of a conditioned fear response. The present study was done to determine if the pPVT is involved in the expression of fear by examining the effect of post-conditioning lesions of the pPVT. Male rats were trained to bar press for food pellets on a variable ratio schedule. Fear conditioning was done using auditory tones (30 s) that co-terminate with footschocks (0.65 mA, 1.0 s). Rats were anesthetized 24 h later and small bilateral electrolytic lesions of the pPVT were made. Fear expression to the tone was assessed using suppression of bar-pressing and freezing after one week of recovery from the surgical procedure. Small bilateral lesions of the pPVT increased bar-pressing for food and decreased freezing during the presentation of the conditioned tone. Lesions of the pPVT had no effect on fear extinction, fear conditioning to a novel tone, or the motivation for food as assessed using a progressive ratio (PR) schedule. The results of the experiment support a role for the pPVT in fear expression. In contrast, the pPVT does not appear to be involved in fear learning or extinction nor does it appear to play a role in the motivation of rats to bar press for food.

Contributions of the paraventricular thalamic nucleus in the regulation of stress, motivation, and mood.

The purpose of this review is to describe how the function and connections of the paraventricular thalamic nucleus (Pa) may play a role in the regulation of stress and negative emotional behavior. Located in the dorsal midline thalamus, the Pa is heavily innervated by serotonin, norepinephrine, dopamine (DA), corticotropin-releasing hormone, and orexins (ORX), and is the only thalamic nucleus connected to the group of structures comprising the amygdala, bed nucleus of the stria terminalis (BNST), nucleus accumbens (NAcc), and infralimbic/subgenual anterior cingulate cortex (sgACC). These neurotransmitter systems and structures are involved in regulating motivation and mood, and display abnormal functioning in several psychiatric disorders including anxiety, substance use, and major depressive disorders (MDD). Furthermore, rodent studies show that the Pa is consistently and potently activated following a variety of stressors and has a unique role in regulating responses to chronic stressors. These observations provide a compelling rationale for investigating the Pa in the link between stress and negative emotional behavior, and for including the Pa in the neural pathways of stress-related psychiatric disorders.

Blocking of corticotrophin releasing factor receptor-1 during footshock attenuates context fear but not the upregulation of prepro-orexin mRNA in rats.

Hypothalamic neuropeptides called orexins (hypocretins) are well known for their roles in promoting arousal. Orexins have also been shown to play a role in fear and anxiety produced by the exposure of rats to an acute episode of moderately intense footshocks. Recent evidence indicates that stress activates orexin neurons through a corticotropin releasing factor (CRF) mechanism. In this study, we examined the effect of a CRF receptor-1 (CRF-R1) antagonist antalarmin (20mg/kg, i.p.) given before shock exposure on subsequent expression of contextual fear and the levels of prepro-orexin (ppOX) mRNA in the hypothalamus. Antalarmin decreased fear and ultrasonic vocalization expression to the shock context at 2 and 10 days after shock exposure. However, antalarmin did not prevent the increases in ppOX mRNA produced by the shock experience. This study provides evidence that blocking of CRF-R1 at the time of footshocks attenuates contextual fear. While an increase in the activity of the orexin system may contribute to fear, this activation does not appear to be sufficient for fear expression.

Orexins (hypocretins) contribute to fear and avoidance in rats exposed to a single episode of footshocks.

Orexins (hypocretins) are peptides that have been shown to regulate behavioral arousal and wakefulness. Recent evidence indicates that orexin neurons are activated by stress and that orexins play a role in anxiety. The present paper describes a series of experiments that examined whether orexins are involved in the anxiety that resulted from exposing rats to an acute episode of footshocks (5 × 2 s of 1.5 mA shocks). We found that prepro-orexin (ppOX) mRNA was elevated in rats at 6 and 14 days after exposure to footshock and that ppOX mRNA levels were correlated with fear at 14 days post-shock. Systemic injections of the non-selective dual orexin receptor antagonist TCS-1102 (10 and 20 mg/kg, i.p.) were found to decrease fear and anxiety in rats 14 days after exposure to footshock. We also found that rats that exhibited a high level of immobility to a novel tone the day after the footshock episode (high responders, HR) showed significantly elevated levels of ppOX mRNA at 14 days post-shock compared to control rats. Furthermore, TCS-1102 (10 mg/kg, i.p.) was found to have anxiolytic effects that were specific for HR when tested in the elevated T-maze. This study provides evidence linking the orexin system to the anxiety produced by exposure of rats to a single episode of footshocks. It also provides preclinical evidence in support of the use of orexin antagonists for the treatment of anxiety in response to an acute episode of stress.

Effects of a post-shock injection of the kappa opioid receptor antagonist norbinaltorphimine (norBNI) on fear and anxiety in rats.

Exposure of rats to footshocks leads to an enduring behavioral state involving generalized fear responses and avoidance. Recent evidence suggests that the expression of negative emotional behaviors produced by a stressor is in part mediated by dynorphin and its main receptor, the kappa opioid receptor (KOR). The purpose of this study was to determine if a subcutaneous injection of the long-acting KOR antagonist norbinaltorphimine (norBNI; 15.0 and 30.0 mg/kg) given 2 days after an acute exposure of rats to footshooks (5×2 s episodes of 1.5 mA delivered over 5 min) attenuates the expression of lasting fear and anxiety. We report that exposure of rats to acute footshock produced long-lasting (>4 weeks) fear (freezing) and anxiety (avoidance of an open area in the defensive withdrawal test). The 30 mg dose of norBNI attenuated the fear expressed when shock rats were placed in the shock context at Day 9 but not Day 27 post-shock. The same dose of norBNI had no effect on the expression of generalized fear produced when shock rats were placed in a novel chamber at Days 8 and 24. In contrast, the 30 mg dose of norBNI produced consistent anxiolytic effects in shock and nonshock rats. First, the 30 mg dose was found to decrease the latency to enter the open field in the defensive withdrawal test done 30 days after the shock exposure. Second, the same high dose also had anxiolytic effects in both nonshock and shock rats as evidence by a decrease in the mean time spent in the withdrawal box. The present study shows that systemic injection of the KOR antagonist norBNI had mixed effect on fear. In contrast, norBNI had an anxiolytic effect which included the attenuation of the enhanced avoidance of a novel area produced by a prior shock experience.