Introduction:
Early life adversity consisting of trauma, poverty, or tumultuous environment impacts the lives of over 30% of children in the United States (
1American Psychiatric Association (2018): Stress in America Survey: Stress and Generation Z. Washington, DC: American Psychiatric Publishing.
). In humans, ELA is associated with poor cognitive and emotional health and increased risk for mood disorders such as depression as well as increased risk for substance use disorders (
2Psychoneuroimmunology of early-life stress: the hidden wounds of childhood trauma?.
,
3Adverse early-life experiences and neurological disease: Age-old questions and novel answers.
,
4- Silvers J.A.
- Goff B.
- Gabard-Durnam L.J.
- Gee D.G.
- Fareri D.S.
- Caldera C.
- et al.
Vigilance, the Amygdala, and Anxiety in Youths With a History of Institutional Care.
,
5- Green J.G.
- Mclaughlin K.A.
- Berglund P.A.
- Gruber M.J.
- Sampson N.A.
- Zaslavsky A.M.
- et al.
Childhood Adversities and Adult Psychiatric Disorders in the National Comorbidity Survey Replication I: Associations With First Onset of DSM-IV Disorders.
,
6Socioeconomic status and the developing brain.
). Human imaging studies suggest altered development of specific brain circuits following ELA, including reward circuits (
7- McLaughlin K.A.
- Weissman D.
- Bitrán D.
Childhood Adversity and Neural Development: A Systematic Review.
,
8- Callaghan B.L.
- Sullivan R.M.
- Howell B.
- Tottenham N.
The international society for developmental psychobiology Sackler symposium: Early adversity and the maturation of emotion circuits-A cross-species analysis.
). It is crucial to understand the nature of these associations because ELA and its consequences, unlike genetic contributors to vulnerability to psychopathologies, may be amenable to prevention. In human studies, it is difficult to demonstrate causality between ELA and adverse adult outcomes and to establish the underlying mechanisms, necessitating use of experimental animal models. Using an animal model in which ELA is induced by an impoverished environment that provokes aberrant maternal care, we have identified later life disruptions of reward-related behaviors (
9Toward Understanding How Early-Life Stress Reprograms Cognitive and Emotional Brain Networks.
,
10- Bolton J.L.
- Molet J.
- Regev L.
- Chen Y.
- Rismanchi N.
- Haddad E.
- et al.
Anhedonia Following Early-Life Adversity Involves Aberrant Interaction of Reward and Anxiety Circuits and Is Reversed by Partial Silencing of Amygdala Corticotropin-Releasing Hormone Gene.
,
11- Bolton J.L.
- Ruiz C.M.
- Rismanchi N.
- Sanchez G.A.
- Castillo E.
- Huang J.
- et al.
Early-life adversity facilitates acquisition of cocaine self-administration and induces persistent anhedonia.
,
12- Levis S.C.
- Bentzley B.S.
- Molet J.
- Bolton J.L.
- Perrone C.R.
- Baram T.Z.
- et al.
On the early-life origins of vulnerability to opioid addiction.
,
13- Molet J.
- Heins K.
- Zhuo X.
- Mei Y.T.
- Regev L.
- Baram T.Z.
- et al.
Fragmentation and high entropy of neonatal experience predict adolescent emotional outcome.
). These disruptions indicate dysfunction of the reward circuit, as seen in several human mood disorders that commonly follow ELA (
5- Green J.G.
- Mclaughlin K.A.
- Berglund P.A.
- Gruber M.J.
- Sampson N.A.
- Zaslavsky A.M.
- et al.
Childhood Adversities and Adult Psychiatric Disorders in the National Comorbidity Survey Replication I: Associations With First Onset of DSM-IV Disorders.
,
14Oltean LE, Șoflău R, Miu A, Szentágotai-Tătar A (2022): Childhood adversity and impaired reward processing: A meta-analysis. Child Abuse Negl 105596.
). These findings provide an impetus to employ this animal model for advancing our understanding of how transient ELA enduringly disrupts the operations of reward circuits.
Here we aimed to determine which regions and neuronal populations within the brain, and specifically within reward circuits, were activated by ELA and are thus candidates for mediating the behavioral consequences of ELA. To this end, we employed TRAP2 mice expressing iCre-ER
T2 recombinase at the locus of the immediate early gene, cFos, to genetically label neurons that are activated during ELA with the red fluorescent reporter tdTomato using Ai14, a knock-in allele of the Rosa26 locus (
15- DeNardo L.A.
- Liu C.D.
- Allen W.E.
- Adams E.L.
- Friedmann D.
- Fu L.
- et al.
Temporal evolution of cortical ensembles promoting remote memory retrieval.
). To date this technique had not been utilized during the first week of life. Using this approach, we established the paraventricular nucleus of the thalamus (PVT) as a key region engaged by these experiences.
The PVT is a dorsal midline thalamic nucleus that is a crucial component of the limbic system and emotional processing network and is engaged by emotionally salient stimuli of either valence (
16- Barson J.R.
- Mack N.R.
- Gao W.J.
The Paraventricular Nucleus of the Thalamus Is an Important Node in the Emotional Processing Network.
,
17- Hsu D.T.
- Kirouac G.J.
- Zubieta J.K.
- Bhatnagar S.
Contributions of the paraventricular thalamic nucleus in the regulation of stress, motivation, and mood.
). The PVT utilizes information derived from remote emotionally salient experiences to gate the expression of approach and avoidance behaviors and to influence responses to stress (
17- Hsu D.T.
- Kirouac G.J.
- Zubieta J.K.
- Bhatnagar S.
Contributions of the paraventricular thalamic nucleus in the regulation of stress, motivation, and mood.
,
18- Bhatnagar S.
- Viau V.
- Chu A.
- Soriano L.
- Meijer O.C.
- Dallman F.
A Cholecystokinin-Mediated Pathway to the Paraventricular Thalamus Is Recruited in Chronically Stressed Rats and Regulates Hypothalamic-Pituitary-Adrenal Function.
,
19- Otis J.M.
- Zhu M.
- Namboodiri V.M.K.
- Cook C.A.
- Kosyk O.
- Matan A.M.
- et al.
Paraventricular thalamus projection neurons integrate cortical and hypothalamic signals for cue-reward processing.
,
20Paraventricular thalamus balances danger and reward.
,
21- Choi E.A.
- Jean-Richard-dit-Bressel P.
- Clifford C.W.G.
- McNally G.P.
Paraventricular thalamus controls behavior during motivational conflict.
). The PVT projects to many brain regions with important roles in stress and reward (
22Placing the paraventricular nucleus of the thalamus within the brain circuits that control behavior.
,
23Projections from the Paraventricular Nucleus of the Thalamus to the Forebrain, With Special Emphasis on the Extended Amygdala.
,
24- Dong X.
- Li S.
- Kirouac G.J.
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.
) and is thus poised to regulate behaviors related to stress and reward following salient early life experiences. Indeed, our prior work using cFos expression had indicated that the PVT is activated by positive early life experience in the form of augmented maternal care (
25- Fenoglio K.A.
- Chen Y.
- Baram T.Z.
Neuroplasticity of the Hypothalamic-Pituitary-Adrenal Axis Early in Life Requires Recurrent Recruitment of Stress-Regulating Brain Regions.
), yet, whether this applies to experiences of the opposite valence and whether this activation may influence future behaviors remains unknown.
Methods and Materials:
Animals
Fos2A-iCreER (Jax #030323) and Ai14 (Jax #007914) mice were received from The Jackson Laboratory or bred in house. All mice were housed in a temperature-controlled, quiet and uncrowded facility on a 12-hr. light, 12 hr. dark schedule (lights on at 0630 hr., lights off at 1830 hr.), except Fos2A-iCreER+/+ litters. These litters were maintained on a 12-hr. reverse light cycle (lights on at 0000 hr., lights off at 1200 hr.) to enable perfusion during the mice’s early active period, and we have previously found no difference in any behavioral test between normally-housed and reverse-light-cycle-housed mice when tested at the same point of their circadian cycle. Mice were provided with ad libitum access to water and food (Envigo Teklad, 2020x, global soy protein-free extruded). Fos2A-iCreER mice bred with the Ai14 reporter mice were employed for TRAP2 studies; Fos2A-iCreER+/+ were employed for the endogenous cFos validation studies. All experiments were performed in accordance with National Institutes of Health guidelines and were approved by the University of California-Irvine Animal Care and Use Committee.
The Limited Bedding and Nesting (LBN) Model of Early-life Adversity (ELA)
Fos
2A-iCreER dams bred with Ai14 males were singly housed on embryonic day (E)17 and monitored for birth of pups every 12 hours. On the morning of postnatal day (P)2, Fos
2A-iCreER+/-::Ai14+/- litters and Fos
2A-iCreER+/+ litters were culled to a maximum of 8 pups, including both sexes, and the ELA paradigm was initiated as previously described (
26- Molet J.
- Maras P.M.
- Avishai-Eliner S.
- Baram T.Z.
Naturalistic rodent models of chronic early-life stress.
). Control dams and pups were placed in cages with a standard amount of corn cob bedding (400 ml) and cotton nestlet material (one 5x5 cm square). ELA dams and pups were provided with one-half cotton nestlet placed on a 2.5 cm tall, fine gauge plastic-coated aluminum mesh platform above sparse corn cob bedding on the cage floor. Accumulation of ammonia was avoided by placing cages in a room with robust ventilation. Both rearing groups were left completely undisturbed until the morning of P6, at which point pups were briefly removed from the cage, placed on a warming pad, and injected subcutaneously with 150 mg/kg tamoxifen (cat#T5648, Millipore Sigma) dissolved in corn oil (cat#C8267, Millipore Sigma; Fig. S1). Pups were then returned to their cage and left undisturbed until the morning of P10. Dams and pups were then transferred to standard cages where maternal behaviors rapidly normalize and the ELA pups’ stress dissipates. P6 was chosen as the timepoint for tamoxifen injections because it is roughly at the midpoint of the ELA period and should allow for optimal tagging of neurons activated by ELA rearing.
Brain processing and analyses
On the morning of P14 (for Fos2A-iCreER+/-::Ai14+/- litters), or at approximately 1400 hr. of P10 (for Fos2A-iCreER+/+ litters), the dam was removed from the home cage, and the cage was placed on a warming pad. Pups were euthanized with sodium pentobarbital and transcardially perfused with ice cold phosphate-buffered saline (PBS; pH=7.4) followed by 4% paraformaldehyde in 0.1M sodium phosphate buffer (pH=7.4). Perfused brains were post-fixed in 4% paraformaldehyde in 0.1M PBS (pH=7.4) for 4-6 hrs. before cryoprotection in 25% sucrose in PBS. Brains were then frozen and coronally sectioned at a thickness of 30 μm (1:5 series) using a Leica CM1900 cryostat (Leica Microsystems, Wetzlar, Germany). Fos2A-iCreER+/-::Ai14+/- sections were mounted on gelatin-coated slides and coverslipped with Vectashield containing DAPI (cat. #H-1200, Vector Laboratories, Burlingame, CA, USA). P14 was chosen as the sacrifice timepoint for Fos2A-iCreER+/-::Ai14+/- litters because optimal expression/accumulation of the tdTomato reporter is not achieved until at least a week following the tamoxifen injection.
Immunohistochemistry
Avidin-biotin complex (ABC)-amplified, diaminobenzidine (DAB) reactions were used to visualize cFos and CRFR1 on free-floating sections. Sections were first washed in PBS containing 0.3% triton (PBST; 3 X 5 min.) followed by quenching of endogenous peroxidase activity by incubation in 0.3% H2O2 for 20 min. Sections were blocked in 5% normal donkey serum (NDS) or normal goat serum (NGS) in PBST for one hr. Sections were incubated with 1:40,000 rabbit anti-cFos (cat# ABE457, Millipore Sigma, Temecula, CA) for 3 days at 4°C or 1:2,000 goat anti-CRFR1 (cat# EB08035, Everest Biotech, Ramona, CA) for 16 hrs. at 4°C. Following 3 x 5 min. washes in PBST, sections were incubated with 1:500 biotinylated goat anti-rabbit antibody (cat# BA-1000-1.5, Vector Laboratories, Burlingame, CA) or 1:500 biotinylated donkey anti-goat antibody (cat# 705-065-147, Jackson ImmunoResearch, West Grove, PA) in 2% NDS or NGS for 2 hrs. Sections were washed in PBST (3 x 5 min.) and then incubated in 1% ABC solution (Vectastain, Vector Laboratories, Burlingame, CA) and washed again in PBST (3 x 5 min.). The immunoreaction product was visualized using solution containing 0.005% H2O2 and 0.05% DAB. Sections were mounted onto gelatin-coated slides or co-labeled for tdTomato expression.
For co-labeling to visualize the cFos reporter, tdTomato, benzidine dihydrochloride (BDHC) reactions were used following DAB staining. Sections were quenched in a solution of 50% methanol and 0.2% H2O2 in PBST for 5 min. followed by 100% methanol containing 0.2% H2O2 for 20 min. Sections were washed in PBST (3 x 5 min.) then blocked in 2% NGS for 30 min. Sections were then incubated with 1:10,000 rabbit anti-RFP (cat# 600-401-379, Rockland Immunochemicals, Pottstown, PA) for 3 days at 4°C. Following primary antibody incubation, sections were washed in PBST (3 x 5 min.) and incubated in 1:500 biotinylated goat anti-rabbit antibody in 2% NGS for 2 hrs. Sections were then washed in PBST (3 x 5 min.) and incubated in 1% ABC solution, followed by additional washes in PBST (3 x 5 min.). Sections were washed in 1X acidic buffer (3 x 5 min.; cat# 003850, Bioenno, Santa Ana, CA) then incubated in a buffer containing 0.025% sodium nitroprusside and 0.01–0.02% benzidine dihydrochloride (BDHC) for 5–10 min. The granular blue deposits were visualized by immersing the sections in fresh incubation solution containing 0.003% H2O2 for 3 min. Sections were washed in 1X acidic buffer (3 x 5 min.) and mounted onto gelatin-coated slides. All mounted sections were dehydrated and coverslipped with Permount mounting medium (cat# SP15-500, Fisher Scientific, Hampton, NH).
For fluorescent labeling of CRH, the tyramide signal amplification technique was used (
27- Chen Y.
- Molet J.
- Gunn B.G.
- Ressler K.
- Baram T.Z.
Diversity of reporter expression patterns in transgenic mouse lines targeting corticotropin- releasing hormone-expressing neurons.
). Free-floating sections were blocked in 5% NGS in PBST for one hour and then incubated in rabbit anti-CRH antiserum (1:20,000; gifted by Dr. W. Vale, Salk Institute, La Jolla, CA) for 14 days at 4°C. Following washing in PBST (3 x 5 min.), sections were incubated in horseradish peroxidase-conjugated anti-rabbit IgG (1:1000; cat# NEF812001EA, Perkin Elmer, Boston, MA) for 1.5 hours. Fluorescein tyramide conjugate was diluted in in amplification buffer (1:150; cat# NEL701A001KT, Perkin Elmer, Boston, MA) and applied to sections in the dark for 5-6 min., followed by washing and mounting on gelatin-coated slides. Sections were coverslipped with Vectashield containing DAPI.
Image acquisition
Images of sections processed with DAB and BDHC were collected using a Nikon Eclipse E400 light microscope with 10x and 20x objective lenses. Confocal images were collected using an LSM-510 confocal microscope (Zeiss, Dublin, CA, USA) with an Apochromat 10x, 20x, or 63x objective. Virtual z-sections of 1 μm were taken at 0.2–0.5 μm intervals. Image frame was digitized at 12-bit using a 1024 X 1024-pixel frame size.
Analyses and statistical considerations
tdTomato
+, CRFR1
+, CRH
+, and cFos
+ neuron numbers were counted manually in FIJI (
28- Schindelin J.
- Arganda-Carreras I.
- Frise E.
- Kaynig V.
- Longair M.
- Pietzsch T.
- et al.
Fiji: An open-source platform for biological-image analysis.
). All quantifications and analyses were performed using stereological principles including systematic unbiased sampling and without knowledge of group assignment. Statistical analyses were carried out using GraphPad Prism software (GraphPad, San Diego, CA, USA). Differences between CTL and ELA groups of both sexes were assessed using two-way ANOVA. To examine significance of cell number differences throughout the anteroposterior axis of the PVT, we used two-way mixed ANOVA with the Sidak multiple comparisons
post hoc test.
Discussion
The principal findings in this set of experiments are (
1American Psychiatric Association (2018): Stress in America Survey: Stress and Generation Z. Washington, DC: American Psychiatric Publishing.
) genetic tagging of neurons activated during the neonatal period in mice is feasible, with high sensitivity and fidelity; (
2Psychoneuroimmunology of early-life stress: the hidden wounds of childhood trauma?.
) the PVT is the major brain region activated by early-life adversity; (
3Adverse early-life experiences and neurological disease: Age-old questions and novel answers.
) sex is an important determinant of neuronal activation by early-life experiences, and (
4- Silvers J.A.
- Goff B.
- Gabard-Durnam L.J.
- Gee D.G.
- Fareri D.S.
- Caldera C.
- et al.
Vigilance, the Amygdala, and Anxiety in Youths With a History of Institutional Care.
) neurons expressing the CRH receptor, CRFR1, likely a target of CRH signaling, are preferentially activated by ELA in a sex-dependent manner and are poised to contribute to the mechanisms by which it contributes to alterations in adult behaviors.
Using the TRAP2 transgenic mouse, we identify here region-specific neuronal activation during the early postnatal period in the mouse. Reporter expression is highly congruent with native cFos. Unlike native cFos expression, the TRAP2 system allows for labeling of neuronal activity over a much longer time period (up to approximately 36 hours) following tamoxifen administration (
31- Guenthner C.J.
- Miyamichi K.
- Yang H.H.
- Heller H.C.
- Luo L.
Permanent genetic access to transiently active neurons via TRAP: Targeted recombination in active populations.
). This characteristic is advantageous when visualizing neuronal activity during a chronic stimulus, such as early-life adversity. The finding that activity-dependent genetic labeling of neurons in P6 mice is possible is novel and demonstrates that cFos can be robustly expressed in the brains of neonatal mice. This transgenic system can therefore be an effective and advantageous tool for the investigation of early life neuronal activation within the brain and its consequences later in life.
An important consideration when pursuing activity-dependent labeling is the particular gene locus governing CRE expression. Immediate early genes (IEGs) represent a well-described connection between neuronal activity and subsequent gene expression changes (
39Pinaud R, Tremere LA, De Weerd P (2005): Critical Calcium-Regulated Biochemical and Gene Expression Programs Involved in Experience-Dependent Plasticity. Plasticity in the Visual System: From Genes to Circuits. Boston: Springer, pp 153-180.
), and thus provide a useful strategy for targeting active cell populations for genetic access. While several IEGs are expressed in the brain, cFos is known to be expressed in the neonatal rodent brain, and this expression in the PVT is dependent upon an ongoing stimulus (
25- Fenoglio K.A.
- Chen Y.
- Baram T.Z.
Neuroplasticity of the Hypothalamic-Pituitary-Adrenal Axis Early in Life Requires Recurrent Recruitment of Stress-Regulating Brain Regions.
,
40- Gallo F.T.
- Katche C.
- Morici J.F.
- Medina J.H.
- Weisstaub N.V.
Immediate early genes, memory and psychiatric disorders: Focus on c-Fos, Egr1 and Arc.
). cFos has been shown to be directly involved in the long-term consequences of neuronal activation on transcriptional and circuit-level changes (
41- West A.E.
- Griffith E.
- Greenberg M.E.
Regulation of transcription factors by neuronal activity.
). Unlike other IEGs that act rapidly via direct influences on synapses and cellular function, cFos functions through more protracted pathways via regulation of downstream target genes (
25- Fenoglio K.A.
- Chen Y.
- Baram T.Z.
Neuroplasticity of the Hypothalamic-Pituitary-Adrenal Axis Early in Life Requires Recurrent Recruitment of Stress-Regulating Brain Regions.
,
40- Gallo F.T.
- Katche C.
- Morici J.F.
- Medina J.H.
- Weisstaub N.V.
Immediate early genes, memory and psychiatric disorders: Focus on c-Fos, Egr1 and Arc.
). Additionally, the role of cFos in memory is well-established, including a role in mediating responses following acquisition of contextual memories (
40- Gallo F.T.
- Katche C.
- Morici J.F.
- Medina J.H.
- Weisstaub N.V.
Immediate early genes, memory and psychiatric disorders: Focus on c-Fos, Egr1 and Arc.
). While cFos is expressed following activation in both excitatory and inhibitory neurons and therefore cannot distinguish between the two, the vast majority of the PVT neurons are excitatory glutamatergic neurons (
42- Frassoni C.
- Spreafico R.
- Bentivoglio M.
Glutamate, aspartate and co-localization with calbindin in the medial thalamus. An immunohistochemical study in the rat.
,
43Gupta A, Gargiulo AT, Curtis GR, Badve PS, Pandey S, Barson JR (2018): Pituitary adenylate cyclase-activating polypeptide-27 (PACAP-27) in the thalamic paraventricular nucleus is stimulated by ethanol drinking. Alcohol. Clin. Exp. Res 42:1650–1660.
). Therefore, the lack of distinction between excitatory and inhibitory neurons is not of consequence in this context. These characteristics highlight cFos as an excellent tool for understanding brain activity in early life.
Using TRAP2, we find that the PVT is prominently activated during exposure to ELA as compared to normal rearing in male mice. By contrast, in females, there is little additional apparent neuronal activation in ELA- versus control-reared groups. In both sexes, the selectivity of PVT engagement by early-life experiences is striking, as other brain regions related to stress and reward contain few experience-engaged neurons, and this activation does not differ between control and ELA mice.
In adult rodents, the PVT is activated by stimuli of both positive valence (e.g. drugs of abuse; 44) and negative valence (e.g. footshock; 45), but activation of the PVT by emotionally salient stimuli early in life is not well characterized. We have previously identified PVT activation in 9-day old rat pups by sensory input from their mothers, a positive emotionally salient experience (
46- Bhatnagar S.
- Huber R.
- Nowak N.
- Trotter P.
Lesions of the posterior paraventricular thalamus block habituation of hypothalamic-pituitary-adrenal responses to repeated restraint.
). Here we show that the PVT is activated by ELA taking place during postnatal days 2-9 in mice. This is important, because adult studies demonstrate that activation of the PVT by stressful events influences responses to an additional stress later in life (
46- Bhatnagar S.
- Huber R.
- Nowak N.
- Trotter P.
Lesions of the posterior paraventricular thalamus block habituation of hypothalamic-pituitary-adrenal responses to repeated restraint.
,
47- Do-Monte F.H.
- Quinõnes-Laracuente K.
- Quirk G.J.
A temporal shift in the circuits mediating retrieval of fear memory.
). Our demonstration that the PVT is selectively engaged by ELA renders this brain region a candidate for mediating the long-lasting consequences of ELA on future responses to stress throughout life.
In addition to altered stress responses (
32- Bolton J.L.
- Short A.K.
- Simeone K.
- Daglian J.
- Baram T.Z.
Programming of Stress-Sensitive Neurons and Circuits by Early-Life Experiences.
,
48- Bolton J.L.
- Short A.K.
- Othy S.
- Kooiker C.L.
- Shao M.
- Gunn B.G.
- et al.
Early stress-induced impaired microglial pruning of excitatory synapses on immature CRH-expressing neurons provokes aberrant adult stress responses.
,
49- Brunton P.J.
- Russell J.A.
Prenatal social stress in the rat programmes neuroendocrine and behavioural responses to stress in the adult offspring: Sex-specific effects.
), ELA leads to deficits in motivated reward behaviors (
11- Bolton J.L.
- Ruiz C.M.
- Rismanchi N.
- Sanchez G.A.
- Castillo E.
- Huang J.
- et al.
Early-life adversity facilitates acquisition of cocaine self-administration and induces persistent anhedonia.
,
50- Kangas B.D.
- Short A.K.
- Luc O.T.
- Stern H.S.
- Baram T.Z.
- Pizzagalli D.A.
A cross-species assay demonstrates that reward responsiveness is enduringly impacted by adverse, unpredictable early-life experiences.
). Activation of subsets of PVT neurons may mechanistically contribute to these consequences: The PVT is required for the retrieval of remote emotionally salient experiences (i.e. those that have occurred >24 hrs. ago) and their influence on the regulation of motivated behaviors in adult rodents (
47- Do-Monte F.H.
- Quinõnes-Laracuente K.
- Quirk G.J.
A temporal shift in the circuits mediating retrieval of fear memory.
,
51- Keyes P.C.
- Adams E.L.
- Chen Z.
- Bi L.
- Nachtrab G.
- Wang V.J.
- et al.
Orchestrating Opiate-Associated Memories in Thalamic Circuits.
,
52- Padilla-Coreano N.
- Do-Monte F.H.
- Quirk G.J.
A time-dependent role of midline thalamic nuclei in the retrieval of fear memory.
). The PVT and its specific projections contribute to a variety of reward-related behaviors, including motivation for feeding (
53Oxytocin Receptor-Expressing Neurons in the Paraventricular Thalamus Regulate Feeding Motivation through Excitatory Projections to the Nucleus Accumbens Core.
), binge ethanol drinking (
54- Levine O.B.
- Skelly M.J.
- Miller J.D.
- Rivera-Irizarry J.K.
- Rowson S.A.
- DiBerto J.F.
- et al.
The paraventricular thalamus provides a polysynaptic brake on limbic CRF neurons to sex-dependently blunt binge alcohol drinking and avoidance behavior in mice.
) and heroin relapse (
55- Giannotti G.
- Gong S.
- Fayette N.
- Herson P.S.
- Ford C.P.
- Peters J.
- et al.
Extinction blunts paraventricular thalamic contributions to heroin relapse.
). Disruptions in similar reward-related behaviors characterize numerous mood disorders, including substance use disorders and depression, for which ELA is a risk factor (
2Psychoneuroimmunology of early-life stress: the hidden wounds of childhood trauma?.
,
5- Green J.G.
- Mclaughlin K.A.
- Berglund P.A.
- Gruber M.J.
- Sampson N.A.
- Zaslavsky A.M.
- et al.
Childhood Adversities and Adult Psychiatric Disorders in the National Comorbidity Survey Replication I: Associations With First Onset of DSM-IV Disorders.
,
56- Levis S.C.
- Baram T.Z.
- Mahler S.V.
Neurodevelopmental origins of substance use disorders: Evidence from animal models of early-life adversity and addiction.
). Thus, the robust and differential engagement of the PVT during ELA reported here suggests that the PVT may encode these early-life experiences and utilize this information later in life to impact reward-related behaviors. These hypotheses will be subjects of future studies.
We find that ELA exerts the largest increase in activation in specific subregions of the PVT. The specific topology of this differential activation sheds light on the potential connectivity and functions of ELA-engaged PVT neurons: The posterior (p)PVT sends strong projections to regions including the ventromedial nucleus accumbens shell, central amygdala (CeA), basolateral amygdala (BLA) and bed nucleus of the stria terminalis, whereas the anterior (a)PVT projects in a more diffuse manner to regions including the dorsomedial nucleus accumbens shell, suprachiasmatic nucleus, and ventral subiculum (
23Projections from the Paraventricular Nucleus of the Thalamus to the Forebrain, With Special Emphasis on the Extended Amygdala.
,
57- Moga M.M.
- Weis R.P.
- Moore R.Y.
Efferent projections of the paraventricular thalamic nucleus in the rat.
,
58Projections of the paraventricular and paratenial nuclei of the dorsal midline thalamus in the rat.
). In addition to this heterogeneity in projection patterns, different contributions to motivated behaviors are attributed to the aPVT versus pPVT. For example, inactivation of the aPVT, but not pPVT, decreases sucrose seeking when an expected sucrose reward is omitted (
59- Do-Monte F.H.
- Minier-Toribio A.
- Quiñones-Laracuente K.
- Medina-Colón E.M.
- Quirk G.J.
Thalamic Regulation of Sucrose Seeking during Unexpected Reward Omission.
). Similarly, injection of neurotensin into the pPVT, but not aPVT, is sufficient to curb excessive ethanol consumption in rats (
60- Pandey S.
- Badve P.S.
- Curtis G.R.
- Leibowitz S.F.
- Barson J.R.
Neurotensin in the posterior thalamic paraventricular nucleus: inhibitor of pharmacologically relevant ethanol drinking.
). A role in regulation of responses to chronic or repeated stress has also been identified exclusively for the pPVT (
18- Bhatnagar S.
- Viau V.
- Chu A.
- Soriano L.
- Meijer O.C.
- Dallman F.
A Cholecystokinin-Mediated Pathway to the Paraventricular Thalamus Is Recruited in Chronically Stressed Rats and Regulates Hypothalamic-Pituitary-Adrenal Function.
,
61- Heydendael W.
- Sharma K.
- Iyer V.
- Luz S.
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Orexins/hypocretins act in the posterior paraventricular thalamic nucleus during repeated stress to regulate facilitation to novel stress.
). Therefore, the distinct topological distribution of neuronal activation by ELA may suggest specific roles and projection targets that warrant further investigation.
Our finding that CRFR1-expressing PVT neurons are preferentially activated by ELA is intriguing. The receptor is well expressed during the first week of life in the rodent (
62- Avishai-Eliner S.
- Gilles E.E.
- Eghbal-Ahmadi M.
- Bar-El Y.
- Baram T.Z.
Altered regulation of gene and protein expression of hypothalamic-pituitary-adrenal axis components in an immature rat model of chronic stress.
) and was abundantly expressed in both the male and female PVT in our findings. Why and how might ELA augment activation of CRFR1 neurons? As shown in other brain regions, ELA might increase CRH expression and release in the PVT, activating receptor-expressing neurons. CRH release regulates CRFR1 expression in a biphasic manner (
63- Brunson K.L.
- Grigoriadis D.E.
- Lorang M.T.
- Baram T.Z.
Corticotropin-releasing hormone (CRH) downregulates the function of its receptor (CRF1) and induces CRF1 expression in hippocampal and cortical regions of the immature rat brain.
), such that it is conceivable that more cells in the PVT express CRFR1 above detection threshold in ELA versus control animals. The augmented activation of CRFR1-neurons by ELA is notable, because CRFR1 plays important roles in responses to stress, including mediating changes in reward-related behaviors following stress (
64- Kreibich A.S.
- Briand L.
- Cleck J.N.
- Ecke L.
- Rice K.C.
- Blendy J.A.
Stress-induced potentiation of cocaine reward: a role for CRFR1 and CREB.
,
65- Chen N.A.
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Knockdown of CRF1 receptors in the ventral tegmental area attenuates cue- and acute food deprivation stress-induced cocaine seeking in mice.
,
66- Vranjkovic O.
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Enhanced CRFR1-dependent regulation of a ventral tegmental area to prelimbic cortex projection establishes susceptibility to stress-induced cocaine seeking.
,
67- Lemos J.C.
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Severe stress switches CRF action in the nucleus accumbens from appetitive to aversive.
). Further analysis revealed a significant interaction between rearing condition and sex in the aPVT, whereby the effect of ELA on proportion of TRAPed aPVT neurons that express CRFR1 was significantly more pronounced in females as compared to males (Fig. S2). This, combined with the finding that overall PVT activation is greater in control-reared females as compared to control-reared males, suggests sex-dependent mechanisms of PVT activation in early life. Future studies will determine whether these CRFR1 neurons mediate the influence of ELA on adult reward behaviors.
In conclusion, the present studies describe a novel use of activity-dependent genetic tagging to demonstrate robust and selective activation of the PVT during ELA. Compared to typical rearing, PVT neurons activated during ELA are significantly more likely to express the receptor to CRH, CRFR1. Establishing the functional role of these ELA-engaged PVT neurons will be a crucial next step toward determining their role in disruptions of reward-related behaviors induced by ELA and will provide important information toward understanding the mechanisms underlying the consequences of ELA on mental health.
Article info
Publication history
Accepted:
January 13,
2023
Received in revised form:
January 9,
2023
Received:
November 28,
2022
Publication stage
In Press Accepted ManuscriptCopyright
© 2023 Published by Elsevier Inc. on behalf of Society of Biological Psychiatry.