Effects of Repetitive Mild Traumatic Brain Injury on Corticotropin-Releasing Factor Modulation of Lateral Habenula Excitability and Motivated Behavior.

Mild traumatic brain injury (mTBI) is a significant health burden due to mTBI-related chronic debilitating cognitive and psychiatric morbidities. Recent evidence from our laboratory suggests a possible dysregulation within reward/motivational circuit function at the level of a subcortical structure, the lateral habenula (LHb), where we demonstrated a causal role for hyperactive LHb in mTBI-induced motivational deficits in self-care grooming behavior in young adult male mice when exposed to mTBI injury during late adolescence (at ~8 weeks old). Here we extended this observation by further characterizing neurobehavioral effects of this repetitive closed head injury model of mTBI in both young adult male and female mice on LHb excitability, corticotropin releasing factor (CRF) modulation of LHb activity, and behavioral responses of motivation to self-care behavior, and approach versus avoidance behavior in the presence of a social- or threat-related stimulus. We show that mTBI increases LHb spontaneous tonic activity in female mice similar to what we previously observed in male mice as well as promoting LHb neuronal hyperexcitability and hyperpolarization-induced LHb bursting in both male and female mice. Interestingly, mTBI only increases LHb intrinsic excitability in male mice coincident with higher levels of the hyperpolarization-activated cation currents (HCN/Ih) and reduces levels of the M-type potassium currents while potentiating M-currents without altering intrinsic excitability in LHb neurons of female mice. Since persistent dysregulation of brain CRF systems is suggested to contribute to chronic psychiatric morbidities and that LHb neurons are highly responsive to CRF, we then tested whether LHb CRF subsystem becomes engaged following mTBI. We found that in vitro inhibition of CRF receptor type 1 (CRFR1) within the LHb reverses mTBI-induced enhancement of LHb tonic activity and hyperexcitability in both sexes, suggesting that an augmented intra-LHb CRF-CRFR1-mediated signaling contributes to the overall LHb hyperactivity following mTBI. Behaviorally, mTBI diminishes motivation for self-care grooming in female mice as in male mice. mTBI also alters defensive behaviors in the looming shadow task by shifting the innate defensive behaviors towards more passive action-locking rather than escape behaviors in response to an aerial threat in both male and female mice as well as prolonging the latency to escape responses in female mice. While, this model of mTBI reduces social preference in male mice, it induces higher social novelty seeking during the novel social encounters in both male and female mice. Overall, our study provides further translational validity for the use of this preclinical model of mTBI for investigation of mTBI-related reward circuit dysfunction and mood/motivation-related behavioral deficits in both sexes while uncovering a few sexually dimorphic neurobehavioral effects of this model that may differentially affect young males and females when exposed to this type of mTBI injury during late adolescence.

Effects of Repetitive Mild Traumatic Brain Injury on Corticotropin-Releasing Factor Modulation of Lateral Habenula Excitability and Motivated Behavior.

Mild traumatic brain injury (mTBI) is a significant health burden due to mTBI-related chronic debilitating cognitive and psychiatric morbidities. Recent evidence from our laboratory suggests a possible dysregulation within reward/motivational circuit function at the level of a subcortical structure, the lateral habenula (LHb), where we demonstrated a causal role for hyperactive LHb in mTBI-induced motivational deficits in self-care grooming behavior in young adult male mice when exposed to mTBI injury during late adolescence (at ~8 weeks old). Here we extended this observation by further characterizing neurobehavioral effects of this repetitive closed head injury model of mTBI in both young adult male and female mice on LHb excitability, corticotropin releasing factor (CRF) modulation of LHb activity, and behavioral responses of motivation to self-care behavior, and approach versus avoidance behavior in the presence of a social- or threat-related stimulus. We show that mTBI increases LHb spontaneous tonic activity in female mice similar to what we previously observed in male mice as well as promoting LHb neuronal hyperexcitability and hyperpolarization-induced LHb bursting in both male and female mice. Interestingly, mTBI only increases LHb intrinsic excitability in male mice coincident with higher levels of the hyperpolarization-activated cation currents (HCN/Ih) and reduces levels of the M-type potassium currents while potentiating M-currents without altering intrinsic excitability in LHb neurons of female mice. Since persistent dysregulation of brain CRF systems is suggested to contribute to chronic psychiatric morbidities and that LHb neurons are highly responsive to CRF, we then tested whether LHb CRF subsystem becomes engaged following mTBI. We found that in vitro inhibition of CRF receptor type 1 (CRFR1) within the LHb normalizes mTBI-induced enhancement of LHb tonic activity and hyperexcitability in both sexes, suggesting that an augmented intra-LHb CRF-CRFR1-mediated signaling contributes to the overall LHb hyperactivity following mTBI. Behaviorally, mTBI diminishes motivation for self-care grooming in female mice as in male mice. mTBI also alters defensive behaviors in the looming shadow task by shifting the innate defensive behaviors towards more passive action-locking rather than escape behaviors in response to an aerial threat in both male and female mice as well as prolonging the latency to escape responses in female mice. While, this model of mTBI reduces social preference in male mice, it induces higher social novelty seeking during the novel social encounters in both male and female mice. Overall, our study provides further translational validity for the use of this preclinical model of mTBI for investigation of mTBI-related reward circuit dysfunction and mood/motivation-related behavioral deficits in both sexes while uncovering a few sexually dimorphic neurobehavioral effects of this model that may differentially affect young males and females when exposed to this type of mTBI injury during late adolescence.

Dysregulation of Kappa Opioid Receptor Neuromodulation of Lateral Habenula Synaptic Function following a Repetitive Mild Traumatic Brain Injury.

Mild traumatic brain injury (mTBI) increases the risk of cognitive deficits, affective disorders, anxiety and substance use disorder in affected individuals. Substantial evidence suggests a critical role for the lateral habenula (LHb) in pathophysiology of psychiatric disorders. Recently, we demonstrated a causal link between persistent mTBI-induced LHb hyperactivity due to synaptic excitation/inhibition (E/I) imbalance and motivational deficits in self-care grooming behavior in young adult male mice using a repetitive closed head injury mTBI model. One of the major neuromodulatory systems that is responsive to traumatic brain and spinal cord injuries, influences affective states and also modulates LHb activity is the dynorphin/kappa opioid receptor (Dyn/KOR) system. However, the effects of mTBI on KOR neuromodulation of LHb function is unknown. To address this, we first used retrograde tracing to anatomically verify that the mouse LHb indeed receives Dyn/KOR expressing projections. We identified several major KOR-expressing and Dyn-expressing synaptic inputs projecting to the mouse LHb. We then functionally evaluated the effects of in vitro KOR modulation of spontaneous synaptic activity within the LHb of male and female sham and mTBI mice at 4week post-injury using the repetitive closed head injury mTBI model. Similar to what we previously reported in the LHb of male mTBI mice, mTBI presynaptically diminished spontaneous synaptic activity onto LHb neurons, while shifting synaptic E/I toward excitation in female mouse LHb. Furthermore, KOR activation in either mouse male/female LHb generally suppressed spontaneous glutamatergic transmission without altering GABAergic transmission, resulting in a significant reduction in E/I ratios and decreased excitatory synaptic drive to LHb neurons of male and female sham mice. Interestingly following mTBI, while responses to KOR activation at LHb glutamatergic synapses were observed comparable to those of sham, LHb GABAergic synapses acquired an additional sensitivity to KOR-mediated inhibition. Thus, in contrast to sham LHb, we observed a reduction in GABA release probability in response to KOR stimulation in mTBI LHb, resulting in a chronic loss of KOR-mediated net synaptic inhibition within the LHb. Overall, our findings uncovered the previously unknown sources of major Dyn/KOR-expressing synaptic inputs projecting to the mouse LHb. Further, we demonstrate that an engagement of intra-LHb Dyn/KOR signaling provides a global suppression of excitatory synaptic drive to the mouse LHb which could act as an inhibitory braking mechanism to prevent LHb hyperexcitability. The additional engagement of KOR-mediated modulatory action on LHb GABAergic transmission by mTBI could contribute to the E/I imbalance after mTBI, with Dyn/KOR signaling serving as a disinhibitory mechanism for LHb neurons in male and female mTBI mice.

AKAP150-anchored PKA regulates synaptic transmission and plasticity, neuronal excitability and CRF neuromodulation in the mouse lateral habenula.

The scaffolding A-kinase anchoring protein 150 (AKAP150) is critically involved in kinase and phosphatase regulation of synaptic transmission/plasticity, and neuronal excitability. Emerging evidence also suggests that AKAP150 signaling may play a key role in brain's processing of rewarding/aversive experiences, however its role in the lateral habenula (LHb, as an important brain reward circuitry) is completely unknown. Using whole cell patch clamp recordings in LHb of male wildtype and ΔPKA knockin mice (with deficiency in AKAP-anchoring of PKA), here we show that the genetic disruption of PKA anchoring to AKAP150 significantly reduces AMPA receptor-mediated glutamatergic transmission and prevents the induction of presynaptic endocannabinoid-mediated long-term depression in LHb neurons. Moreover, ΔPKA mutation potentiates GABAA receptor-mediated inhibitory transmission while increasing LHb intrinsic excitability through suppression of medium afterhyperpolarizations. ΔPKA mutation-induced suppression of medium afterhyperpolarizations also blunts the synaptic and neuroexcitatory actions of the stress neuromodulator, corticotropin releasing factor (CRF), in mouse LHb. Altogether, our data suggest that AKAP150 complex signaling plays a critical role in regulation of AMPA and GABAA receptor synaptic strength, glutamatergic plasticity and CRF neuromodulation possibly through AMPA receptor and potassium channel trafficking and endocannabinoid signaling within the LHb.

Effects of prenatal opioid exposure on synaptic adaptations and behaviors across development.

In this review, we focus on prenatal opioid exposure (POE) given the significant concern for the mental health outcomes of children with parents affected by opioid use disorder (OUD) in the view of the current opioid crisis. We highlight some of the less explored interactions between developmental age and sex on synaptic plasticity and associated behavioral outcomes in preclinical POE research. We begin with an overview of the rich literature on hippocampal related behaviors and plasticity across POE exposure paradigms. We then discuss recent work on reward circuit dysregulation following POE. Additional risk factors such as early life stress (ELS) could further influence synaptic and behavioral outcomes of POE. Therefore, we include an overview on the use of preclinical ELS models where ELS exposure during key critical developmental periods confers considerable vulnerability to addiction and stress psychopathology. Here, we hope to highlight the similarity between POE and ELS on development and maintenance of opioid-induced plasticity and altered opioid-related behaviors where similar enduring plasticity in reward circuits may occur. We conclude the review with some of the limitations that should be considered in future investigations. This article is part of the Special Issue on 'Opioid-induced addiction'.

Involvement of Lateral Habenula Dysfunction in Repetitive Mild Traumatic Brain Injury-Induced Motivational Deficits.

Affective disorders including depression (characterized by reduced motivation, social withdrawal, and anhedonia), anxiety, and irritability are frequently reported as long-term consequences of mild traumatic brain injury (mTBI) in addition to cognitive deficits, suggesting a possible dysregulation within mood/motivational neural circuits. One of the important brain regions that control motivation and mood is the lateral habenula (LHb), whose hyperactivity is associated with depression. Here, we used a repetitive closed-head injury mTBI model that is associated with social deficits in adult male mice and explored the possible long-term alterations in LHb activity and motivated behavior 10-18 days post-injury. We found that mTBI increased the proportion of spontaneous tonically active LHb neurons yet decreased the proportion of LHb neurons displaying bursting activity. Additionally, mTBI diminished spontaneous glutamatergic and GABAergic synaptic activity onto LHb neurons, while synaptic excitation and inhibition (E/I) balance was shifted toward excitation through a greater suppression of GABAergic transmission. Behaviorally, mTBI increased the latency in grooming behavior in the sucrose splash test suggesting reduced self-care motivated behavior following mTBI. To show whether limiting LHb hyperactivity could restore motivational deficits in grooming behavior, we then tested the effects of Gi (hM4Di)-DREADD-mediated inhibition of LHb activity in the sucrose splash test. We found that chemogenetic inhibition of LHb glutamatergic neurons was sufficient to reverse mTBI-induced delays in grooming behavior. Overall, our study provides the first evidence for persistent LHb neuronal dysfunction due to an altered synaptic integration as causal neural correlates of dysregulated motivational states by mTBI.

Repetitive mild traumatic brain injury induces persistent alterations in spontaneous synaptic activity of hippocampal CA1 pyramidal neurons.

Mild traumatic brain injury (mTBI) or concussion is the most common form of TBI which frequently results in persistent cognitive impairments and memory deficits in affected individuals [1]. Although most studies have investigated the role of hippocampal synaptic dysfunction in earlier time points following a single injury, the long-lasting effects of mTBI on hippocampal synaptic transmission following multiple brain concussions have not been well-elucidated. Using a repetitive closed head injury (3XCHI) mouse model of mTBI, we examined the alteration of spontaneous synaptic transmission onto hippocampal CA1 pyramidal neurons by recording spontaneous excitatory AMPA receptor (AMPAR)- and inhibitory GABAAR-mediated postsynaptic currents (sEPSCs and sIPSCs, respectively) in adult male mice 2-weeks following the injury. We found that mTBI potentiated postsynaptic excitatory AMPAR synaptic function while depressed postsynaptic inhibitory GABAAR synaptic function in CA1 pyramidal neurons. Additionally, mTBI slowed the decay time of AMPAR currents while shortened the decay time of GABAAR currents suggesting changes in AMPAR and GABAAR subunit composition by mTBI. On the other hand, mTBI reduced the frequency of sEPSCs while enhanced the frequency of sIPSCs resulting in a lower ratio of sEPSC/sIPSC frequency in CA1 pyramidal neurons of mTBI animals compared to sham animals. Altogether, our results suggest that mTBI induces persistent postsynaptic modifications in AMPAR and GABAAR function and their synaptic composition in CA1 neurons while triggering a compensatory shift in excitation/inhibition (E/I) balance of presynaptic drives towards more inhibitory synaptic drive to hippocampal CA1 cells. The persistent mTBI-induced CA1 synaptic dysfunction and E/I imbalance could contribute to deficits in hippocampal plasticity that underlies long-term hippocampal-dependent learning and memory deficits in mTBI patients long after the initial injury.

Lateral Habenula Beyond Avoidance: Roles in Stress, Memory, and Decision-Making With Implications for Psychiatric Disorders.

In this Perspective review, we highlight some of the less explored aspects of lateral habenula (LHb) function in contextual memory, sleep, and behavioral flexibility. We provide evidence that LHb is well-situated to integrate different internal state and multimodal sensory information from memory-, stress-, motivational-, and reward-related circuits essential for both survival and decision making. We further discuss the impact of early life stress (ELS) on LHb function as an example of stress-induced hyperactivity and dysregulation of neuromodulatory systems within the LHb that promote anhedonia and motivational deficits following ELS. We acknowledge that recent technological advancements in manipulation and recording of neural circuits in simplified and well-controlled behavioral paradigms have been invaluable in our understanding of the critical role of LHb in motivation and emotional regulation as well as the involvement of LHb dysfunction in stress-induced psychopathology. However, we also argue that the use of ethologically-relevant behaviors with consideration of complex aspects of decision-making is warranted for future studies of LHb contributions in a wide range of psychiatric illnesses. We conclude this Perspective with some of the outstanding issues for the field to consider where a multi-systems approach is needed to investigate the complex nature of LHb circuitry interactions with environmental stimuli that predisposes psychiatric disorders.

Potentiation of glutamatergic synaptic transmission onto lateral habenula neurons following early life stress and intravenous morphine self-administration in rats.

Early life stress presents an important risk factor for drug addiction and comorbid depression and anxiety through persistent effects on the mesolimbic dopamine pathways. Using an early life stress model for child neglect (a single 24 h episode of maternal deprivation, MD) in rats, recent published works from our lab show that MD induces dysfunction in the ventral tegmental area and its negative controller, the lateral habenula (LHb). MD-induced potentiation of glutamatergic synaptic transmission onto LHb neurons shifts the coordination of excitation/inhibition (E/I) balance towards excitation, resulting in an increase in the overall spontaneous neuronal activity with elevation in bursting and tonic firing, and in the intrinsic excitability of LHb neurons in early adolescent male rats. Here, we explored how MD affects intravenous morphine self-administration (MSA) acquisition and sucrose preference as well as glutamatergic synaptic function in LHb neurons of adult male rats self-administering morphine. We found that MD-induced increases in LHb neuronal and glutamatergic synaptic activity and E/I ratio persisted into adulthood. Moreover, MD significantly reduced morphine intake, triggered anhedonia-like behaviour in the sucrose preference test and was associated with persistent glutamatergic potentiation 24 h after the last MSA session. MSA also altered the decay time kinetics of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (AMPAR) currents in LHb neurons of control rats during this time period. Our data highlight that early life stress-induced glutamatergic plasticity in LHb may dampen the positive reinforcing and motivational properties of both natural rewards and opioids, and may contribute to the development of anhedonia and dysphoric states associated with opioids.

Characterization of proinflammatory markers in the ventral tegmental area across mouse models of chronic stress.

Despite the high prevalence of major depressive disorder (MDD), understanding of the biological underpinnings remains limited. Rodent models suggest that changes in activity and output of dopamine (DA) neurons in the ventral tegmental area (VTA) are important for depressive-like phenotypes. Additionally, brain inflammatory processes are thought to contribute to MDD pathology and inflammation in the VTA has been linked to changes in VTA DA neuronal activity. Thus, we sought to determine whether there is increased inflammatory signaling in the VTA following forms of chronic stress that induce depressive-like symptoms. First, we subjected male mice to either physical or vicarious chronic social defeat stress (CSDS), paradigms known to induce long-term depressive-like behavior and changes in VTA signaling. Second, we subjected male and female mice to subchronic variable stress (SCVS), a paradigm that induces depressive-like behavior only in female mice. We then isolated mRNA from the VTA and assessed proinflammatory gene regulation via RT-PCR. Our results show that physical, but not vicarious, CSDS increases interleukin 1ß (IL-1ß) mRNA expression and this inversely correlates with social interaction score. In contrast, IL-1ß expression was unchanged in male or female mice following SCVS. No significant increases in VTA ionized calcium binding adapter molecule 1 (Iba1) and glial fibrillary acidic protein (GFAP) immunochemistry were detected following CSDS that would be indicative of a robust inflammatory response. In conclusion, we show that chronic stressors distinctively alter expression of proinflammatory genes in the VTA and changes may depend on the severity and time-course of the stress exposure.

Early life stress dysregulates kappa opioid receptor signaling within the lateral habenula.

The lateral habenula (LHb) is an epithalamic brain region associated with value-based decision making and stress evasion through its modulation of dopamine (DA)-mediated reward circuitry. Specifically, increased activity of the LHb is associated with drug addiction, schizophrenia and stress-related disorders such as depression, anxiety and posttraumatic stress disorder. Dynorphin (Dyn)/Kappa opioid receptor (KOR) signaling is a mediator of stress response in reward circuitry. Previously, we have shown that maternal deprivation (MD), a severe early life stress, increases LHb spontaneous neuronal activity and intrinsic excitability while blunting the response of LHb neurons to extrahypothalamic corticotropin-releasing factor (CRF) signaling, another stress mediator. CRF pathways also interact with Dyn/KOR signaling. Surprisingly, there has been little study of direct KOR regulation of the LHb despite its distinct role in stress, reward and aversion processing. To test the functional role of Dyn/KOR signaling in the LHb, we utilized ex-vivo electrophysiology combined with pharmacological tools in rat LHb slices. We show that activation of KORs by a KOR agonist (U50,488) exerted differential effects on the excitability of two distinct sub-populations of LHb neurons that differed in their expression of hyperpolarization-activated cation currents (HCN, Ih). Specifically, KOR stimulation increased neuronal excitability in LHb neurons with large Ih currents (Ih+) while decreasing neuronal excitability in small/negative Ih (Ih-) neurons. We found that an intact fast-synaptic transmission was required for the effects of U50,488 on the excitability of both Ih- and Ih+ LHb neuronal subpopulations. While AMPAR-, GABAAR-, or NMDAR-mediated synaptic transmission alone was sufficient to mediate the effects of U50,488 on excitability of Ih- neurons, either GABAAR- or NMDAR-mediated synaptic transmission could mediate these effects in Ih+ neurons. Consistently, KOR activation also altered both glutamatergic and GABAergic synaptic transmission where stimulation of presynaptic KORs uniformly suppressed glutamate release onto LHb neurons while primarily decreased or in some cases increased GABA release. We also found that MD significantly increased immunolabeled Dyn (the endogenous KOR agonist) labeling in neuronal fibers in LHb while significantly decreasing mRNA levels of KORs in LHb tissues compared to those from non-maternally deprived (non-MD) control rats. Moreover, the U50,488-mediated increase in LHb neuronal firing observed in non-MD rats was absent following MD. Altogether, this is the first demonstration of the existence of functional Dyn/KOR signaling in the LHb that can be modulated in response to severe early life stressors such as MD.

Pubertal Testosterone Programs Adult Behavioral Adaptations to Sexual Experience through Infralimbic Cortex ΔFosB.

Acquisition of social proficiency entails behavioral adaptations to social experience, including both behavioral flexibility and inhibition of behaviors inappropriate in specific social contexts. Here, we investigated the contributions of testosterone and ΔFosB, a transcription factor linked to experience-dependent neural plasticity, to the adolescent maturation of social proficiency in male-female social interactions. To determine whether pubertal testosterone organizes circuits underlying social proficiency, we first compared behavioral adaptations to sexual experience in male Syrian hamsters that were deprived of testosterone during puberty (prepubertal castration; NoT@P) to those of males deprived of testosterone for an equivalent period of time in adulthood (postpubertal castration; T@P). All males were given testosterone replacement in adulthood for two weeks before sexual behavior testing, where males were allowed to interact with a receptive female once per week for five consecutive weeks. T@P males showed the expected decrease in ectopic (mis-directed) mounts with sexual experience, whereas NoT@P males did not. In addition, sexual experience induced FosB gene products expression in the infralimbic cortex (IL) in T@P, but not NoT@P, males. Overexpression of ΔFosB via an adeno-associated viral (AAV) vector in the IL of NoT@P males prior to sexual behavior testing was sufficient to produce a behavioral phenotype similar to that of experienced T@P males. Finally, overexpression of ΔFosB in IL increased the density of immature spines on IL dendrites. Our findings provide evidence that social proficiency acquired through sexual experience is organized by pubertal testosterone through the regulation of ΔFosB in the IL, possibly through increasing synaptic lability.

Determination of circuit-specific morphological adaptations in ventral tegmental area dopamine neurons by chronic morphine.

Chronic opiate exposure induces neuroadaptations in the mesocorticolimbic system including ventral tegmental area (VTA) dopamine (DA) neurons, whose soma size is decreased following opiate exposure. Yet it is now well documented that VTA DA neurons are heterogeneous, with notable differences between VTA DA neurons based on their projection target. Therefore, we sought to determine whether chronic morphine induced similar changes in the morphology of VTA DA neurons that project to the nucleus accumbens (NAc) and prefrontal cortex (PFC). We utilized Cre-dependent retrograde viral vectors in DA Cre driver lines to label VTA DA neurons that projected to NAc and PFC and assessed neuronal soma size. Consistent with previous data, the soma size of VTA DA neurons that projected to the NAc medial shell was decreased following morphine exposure. However, soma size of VTA DA neurons that projected to the NAc core was unaltered by morphine. Interestingly, morphology of PFC-projecting VTA DA neurons was also altered by morphine, but in this case soma size was increased compared to sham controls. Differences in basal soma size were also noted, suggesting stable differences in projection-specific morphology in addition to drug-induced changes. Together, these data suggest morphine-induced changes in VTA DA morphology occur within distinct VTA DA populations and that study of opiate-induced structural plasticity of individual VTA DA subcircuits may be critical for understanding addiction-related behavior.