Edited by: Agnes Gruart, Pablo de Olavide University, Spain
Reviewed by: Alexander C. Jackson, University of Connecticut, USA; Cristina Marquez, Instituto de Neurociencias de Alicante (CSIC), Spain
*Correspondence: Joel D. Hahn
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Our understanding of the extrinsic connections of the lateral hypothalamic area (LHA) has deepened in recent years. In particular, a series of studies using neural pathway-tracing methods to investigate the macroconnections of histologically differentiated LHA regions, have revealed that the neural connections of these regions are substantially distinct, and have robust connections with neural circuits controlling survival behaviors. To begin testing functional associations suggested by the distinct LHA region neural connections, the present study has investigated the role of the LHA juxtadorsomedial region (LHAjd) in the control of social defeat (a socially-relevant defensive behavior). Male rats received bilateral cytotoxic lesions targeted to the LHAjd. A resident-intruder paradigm was then employed to investigate the effect of these lesions on defensive behavioral responses. Behavioral data were collected during three phases of testing: (1) pre-encounter habituation to testing context; (2) encounter with a dominant conspecific in the testing context; and (3) post-encounter context. Statistical analysis of behavioral measures revealed a significant decrease in risk assessment behaviors during post-encounter context testing in lesioned intruders compared to sham-lesioned and intact rats. However, changes in defensive behavioral measures during the habituation, or during resident-intruder encounters, did not reach significance. We discuss these data in relation to LHAjd (and neighboring LHA region) neural connections, and in relation to current advances in understanding of the neural control of defensive behaviors. A refined model for the neural circuits that are central to the control of socially-relevant defensive behaviors is outlined. We also consider possible broader implications of these data for disorders of behavioral control.
In rats, as in other animals, social defeat can occur as a consequence of social conflict with a dominant aggressor (Blanchard et al.,
The hypothalamus plays a critical role in the control of survival behaviors (for reviews see Risold et al.,
These correlated data coalesced in two recent articles: the first of these reported that socially-defeated rats had a robust increase in the expression of the immediate early gene product cFos in the LHAjd after exposure to a social defeat-associated context (Faturi et al.,
Animals were maintained in accordance with the guidelines of the Brazilian Association for Laboratory Animal Science (Sociedade Brasileira de Ciência em Animais de Laboratório; COBEA), and the Guide for the Care and Use of Laboratory Animals (National Research Council, USA,
Male Wistar rats (
Methods for the resident-intruder behavioral experiments followed those described previously (Ribeiro-Barbosa et al.,
For 10 days, each Wistar rat (NMDA lesion, sham lesion, or intact) was isolated in its home cage. At the beginning of the light phase the rat was transferred in its home cage from a housing room to an adjacent procedure room. The home cage access panel was then raised for 10 min, allowing egress and free exploration of an enclosed Plexiglas corridor (100 cm length × 30 cm height × 12.5 cm width) and (at the other end of the corridor) a second cage of identical construction to the home cage, into which were placed food pellets the rat could obtain. A small amount of fresh bedding was placed in the testing apparatus (corridor and second cage) prior to habituation. After the 10-min habituation period the rat was returned in its home cage to the housing room. The corridor and second cage of the apparatus were cleaned between each habituation session.
After 10 days of habituation to context, on the next day, the second cage (food compartment) was replaced with the Long Evans pair resident home cage (with the female removed for the duration of the encounter). The Wistar male intruder was allowed access to the resident home cage following the habituation protocol of the prior 10 days, and once inside the resident’s cage, the access panel was lowered to prevent egress. Only experienced resident males were used for resident-intruder encounters. If a clear attack (bite) occurred within the first 10 min of an encounter, the resident and intruder were allowed to remain together for a further 10 min after the first attack; if an attack did not occur in the first 10 min, the pair were separated (and these intruders were excluded from subsequent testing and analysis,
On the day after an encounter and social defeat, socially-defeated intruders were allowed to explore the testing context for 5 min. That is, a shortened version of the habituation protocol was followed, with the resident’s home cage placed at the other end of the connecting corridor, and with the resident removed from its home cage for the duration of the experiment.
Ninety (90) minutes after the start of the post-encounter context testing, rats were deeply anesthetized (sodium pentobarbital 40 mg/kg, IP), and then perfused transcardially with ice-cooled 0.9% saline, followed by ice-cooled 4% paraformaldehyde in 0.1 M phosphate buffer pH 7.4. The perfusion-fixed brains were removed and placed overnight in a solution of 20% sucrose in 0.1M phosphate buffer pH 7.4 at 4°C. They were then frozen on dry-ice and sectioned on a sliding microtome in the transverse (coronal) plane into four stepwise collated series (40 μM thickness). One of the series was processed for detection of Nissl substance (thionine stain) to confirm cannulae placement and cytotoxic lesion extent. For additional analysis of the lesions a second series of sections was processed for immunohistochemical (IHC) detection of NeuN (Anti-NeuN, MAB377, clone A60, Millipore, USA); the remaining series of sections were transferred to an anti-freeze solution and stored at −20°C for future use. For NeuN IHC, (in brief) the sections were incubated overnight in primary antibody (1:1000 dilution), then for 90 min at room temperature in a solution of biotinylated goat anti-mouse IgG (Vector Laboratories, Burlingame, CA, USA; 1:200 dilution). The sections were then exposed to an avidin–biotin horseradish peroxidase (HRP) reagent (ABC Elite Kit; Vector Laboratories) for 90 min. To visualize the location of the bound NeuN antibodies, the sections were exposed for 10-min to a solution containing 0.02% of a chromogen (3,3-diaminobenzidine tetrahydrochloride—DAB; Sigma, St Louis, MO, USA) and 0.3% nickel–ammonium sulfate in 0.05 M Tris–buffer (pH 7.6), followed by the addition of hydrogen peroxide (1:3000 dilution) and a further 10 min incubation, resulting in a dark blue-black product. The reaction was stopped by extensive washing in potassium phosphate-buffered saline pH 7.4 (KPBS). Sections were mounted on gelatin-coated slides, air-dried, dehydrated through an ascending series of alcohols, cleared with xylene, and coverslipped with DePeX (Sigma). During antibody incubation steps sections were refrigerated; antibodies were diluted in KPBS, that was also used for multiple washes between the incubation steps.
Data were quantified cumulatively for the intruder for a period sufficient for quantification for the three phases of the experiment (habituation, encounter and context re-exposure). Measurements for the first 5 min of habituation to context, and 5-min post-encounter context testing, included: (1) spatiotemporal measurements: time spent in home cage, corridor and second cage; and (2) duration of the following behaviors: risk assessment, exploration, rearing and grooming. Measurements for 10-min of resident-intruder agonistic encounters included the duration of following behaviors: passive defense, active defense, locomotion, grooming and social investigation. Data quantification (behavioral scoring) from video recordings was done by a trained observer using dedicated analysis software (The Observer, version XT; Noldus, Netherlands). Only intruders that had suffered a clear social defeat were used in the present analysis. The criteria used for scoring encoded behavioral measures are as follows:
Risk assessment: (1) crouch-sniff (animal immobile with its back arched but actively sniffing and scanning the environment); and (2) stretch postures (animal’s body stretched forward, either motionless or moving slowly toward the second/resident cage).
Exploration: (1) fearless locomotion (locomotion with arched back); and (2) upright position (animal actively exploring the environment, standing over its rear paws and leaning on the wall with the fore paws).
Rearing: animal standing over its rear paws without wall contact.
Grooming: self-cleaning behavior.
Social investigation: intruder animal sniffing and making light exploratory paw contact with the body of the resident animal.
Locomotion: forward movement.
Passive defense: animal motionless and supine (on-the-back submissive posture).
Active defense: including (1) intruder animal pushing away the resident animal; (2) assuming an upright position with sparse boxing; and (3) attempts to flee from the resident.
After testing for homogeneity of variance (Levene’s test), the behavioral data were analyzed using a parametric a univariate analysis of variance (ANOVA) for each dependent variable followed by a
For the rats that received NMDA lesions targeted to the LHAjd, only those with bilateral lesions substantially restricted to the LHAjd were included in the present analysis (
Experimental groups | ||||
---|---|---|---|---|
Intact ( |
LHAjd lesion ( |
Sham lesion ( |
Statistics ( |
|
Home cage | 61.4 ± 13.7 | 23.4 ± 10.6 | 55, 4 ± 10.5 | 2.82, 0.093 |
Corridor | 86.1 ± 9.5 | 107 ± 12.5 | 112.8 ± 14.7 | 1.39, 0.280 |
Resident cage | 152.7 ± 12.6 | 169.8 ± 20 | 132 ± 7.7 | 1.5, 0.255 |
Risk assessment | 3.3 ± 1.6 | 12.4 ± 6.5 | 11.2 ± 5 | 2.83, 0.092 |
Exploration | 274.2 ± 9.9 | 271.8 ± 5.4 | 281.2 ± 6.3 | 0.3, 0.743 |
Rearing | 3 ± 1.1 | 3.4 ± 1.4 | 3 ± 3 | 0.34, 0.712 |
Grooming | 20 ± 10.4 | 12.4 ± 5.4 | 4.4 ± 3.4 | 0.92, 0.422 |
Behavioral interactions during the resident-intruder encounter were comparable to those described previously: a typically short (<30 s) latency to first attack by the dominant aggressor (resident), and predominantly passive subordinate (intruder) defensive responses (remaining mainly motionless; Faturi et al.,
Experimental groups | ||||
---|---|---|---|---|
Intact ( |
LHAjd lesion ( |
Sham lesion ( |
Statistics ( |
|
Passive defense | 525.5 ± 20.9 | 360.6 ± 82.4 | 438.6 ± 53.4 | 2.73, 0.099 |
Active defense | 58.5 ± 19.5 | 182.0 ± 50.8 | 120.8 ± 39.5 | 3.31, 0.066 |
Locomotion | 11.9 ± 4.37 | 17.8 ± 13.8 | 21.1 ± 11.1 | 0.23, 0.791 |
Grooming | 0.2 ± 0.2 | 4.0 ± 2.8 | 8.7 ± 4.2 | 1.7, 0.217 |
Social investigation | 3.8 ± 3.8 | 35.4 ± 23.8 | 10.4 ± 4.8 | 1.41, 0.275 |
Experimental groups | ||||
---|---|---|---|---|
Intact ( |
LHAjd lesion ( |
Sham lesion ( |
Statistics ( |
|
Home cage | 98.1 ± 29.7 | 101.0 ± 19.9 | 114.8 ± 47.8 | 0.06, 0.93 |
Corridor | 154.9 ± 29.1 | 94.4 ± 15.6 | 107.8 ± 32.8 | 1.08, 0.364 |
Resident cage | 47.0 ± 25.7 | 104.8 ± 29.9 | 77.6 ± 20.7 | 1.6, 0.234 |
Risk assessment | 180.9 ± 7.7 | 81.0 ± 8.9* | 182.0 ± 38.3 | 10.16, 0.0018 |
Exploration | 107.3 ± 10.4 | 207.4 ± 7.0 | 111.2 ± 36.2 | 3.915, 0.044 |
Rearing | 0.6 ± 0.3 | 1.8 ± 0.8 | 1.2 ± 0.4 | 1.02, 0.382 |
Grooming | 9.9 ± 4.4 | 9.6 ± 5.1 | 4.6 ± 2.0 | 0.40, 0.677 |
The experimental paradigm used in the present study was established in a recent study that showed rats exposed to a single social defeat event, and then re-exposed to the defeat-associated context, displayed robust and reproducible defensive behavioral responses (Faturi et al.,
The PMd and PAGd are both extensively characterized key nodes for the control of defensive behavioral responses to different types of threat stimuli (Cezario et al.,
In addition to the LHAjd, PMd and PAGd, several other interconnected gray matter regions (within and outside of hypothalamus) show increased cFos expression in response to social defeat, or in response to re-exposure to the context in which a social defeat occurred (Motta et al.,
Furthermore, the MPO, MPN, VMHvl, TU and PMv, in addition to being implicated by cFos analysis in socially-relevant defensive responses (Motta et al.,
Taking the present results together with the indicated similarities in responses to social defeat, in terms of neuroactivational responses of the LHAjd and the conspecific/social-responsive hypothalamic circuit (Motta et al.,
In support of a possible general role for the LHAjd in defensive behavioral control, in addition to direct connections with the conspecific/social-responsive hypothalamic circuit, the LHAjd also connects directly with a conceptual predator-responsive hypothalamic circuit involving the anterior hypothalamic nucleus (AHN) and the VMH dorsomedial part (VMHdm; see Figure 11 in Motta et al.,
In the present study an additional and intriguing finding was an increase in active- and decrease in passive defensive responses in LHAjd lesioned intruders during resident-intruder encounters. Although these behavioral changes did not quite reach significance (Table
Continuing a consideration of a potentially broad role for the LHAjd in control of defensive behavioral responses, in addition to LHAjd connections with the PMd, and to the previously mentioned PAGd, the LHAjd also has robust downstream connections with several other PAG divisions, notably including the PAG lateral (PAGl) and precommissural (PRC) divisions (Hahn and Swanson,
Turning from LHAjd downstream connections to those upstream, three sites are prominent: within the striatum the rostral part of the lateral septal nucleus (LSr), and medial nucleus of the amygdala (MEA), and within the cerebral cortex the subiculum (SUB; dorsal and ventral, but mostly its intermediate part—SUBi; see Figure 11 in Hahn and Swanson,
More broadly, it is worth noting that significantly increased MEAad and MEApd cFos associated with agonistic encounters was previously reported in Syrian hamsters in both dominant and subordinate males, and also following copulation (Kollack-Walker and Newman,
In addition to the MEA, the other part of striatum with robust LHAjd connections is the LSr. The LSr in turn receives a major input from the Ammon’s horn and SUB (Risold and Swanson,
Given the close connectional associations between the LSr and LHAjd, and the results of the present study, it is salient to note that socially-defeated rats re-exposed to the defeat context show a significant increase in LSr cFos expression (Faturi et al.,
A consideration of hippocampal neural connections in defensive behavioral responses was revisited in a recent article that compared and contrasted the pattern of cFos expression in male rats associated with the stressful threats posed by either entrapped immobilization, or an encounter with an aggressive conspecific (Motta and Canteras,
As a final point of discussion, and by way of considering possible broader implications of the present results, one might ask how convergent hippocampal-septal and amygdala input to the LHAjd may contribute to the control of defensive behavioral responses? As noted previously, in socially-defeated rats re-exposed to the defeat context, visuospatial context cues alone do not appear sufficient to elicit conditioned defensive responses, which also require the presence of (at least) olfactory cues previously associated with the dominant aggressor (Faturi et al.,
One possible example is post-traumatic stress disorder (PTSD) that is typified by context and cue disassociated (“inappropriate”) defensive (or aggressive) behavior following a traumatic (highly stressful and threatening) event or episode (American Psychiatric Association,
The present results provide the first direct evidence of a functional role for the LHAjd in the control of socially-relevant defensive behavioral responses, and conditioned context-dependent responses in particular. Additionally, the results are consistent with a previous report indicating increased activation of the LHAjd in the same behavioral model (Faturi et al.,
JDH and NSC designed the experiments. JDH and MJR carried out the experiments. MJR did most of the analysis (with additional input from NSC and JDH). MVCB did the statistical analysis. JDH wrote the article (with editorial input from NSC and MJR).
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
This work was supported by a grant to JDH from the University of Southern California (USC) and the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, Br), and by a grant to NSC from FAPESP (2014/05432-9). Additional support for NSC, and MVCB was provided by the National Council for Scientific and Technological Development (Brazil). MJR was supported by a FAPESP fellowship (2012/13804-8).