Fos Labeling Research Articles

Early perturbations to fluid homeostasis alter development of hypothalamic feeding circuits with context-specific changes in ingestive behavior.

Drinking and feeding are tightly coordinated homeostatic events and the paraventricular nucleus of the hypothalamus (PVH) represents a possible node of neural integration for signals related to energy and fluid homeostasis. We used TRAP2;Ai14 and Fos labeling to visualize neurons in the PVH and median preoptic nucleus (MEPO) responding to both water deprivation and hunger. Moreover, we determined that structural and functional development of dehydration-sensitive inputs to the PVH from the MEPO precedes those of agouti-related peptide (AgRP) neurons, which convey hunger signals and are known to be developmentally programmed by nutrition. We also determined that osmotic hyperstimulation of neonatal mice led to enhanced AgRP inputs to the PVH in adulthood, as well as disruptions to ingestive behaviors during high-fat diet feeding and dehydration-anorexia. Thus, development of feeding circuits is impacted not only by nutritional signals, but also by early perturbations to fluid homeostasis with context-specific consequences for coordination of ingestive behavior.

Distribution of c‐Fos Labeling Elicited by Intragastric Copper Sulfate: Comparison with Labeling Induced by Galvanic Vestibular Stimulation

The neural pathways that produce nausea and emesis are poorly defined. Although it is widely postulated that a “final common pathway” is activated during emetic responses elicited by any stimulus, this hypothesis has not been adequately tested. In a prior study (Balaban et al., PLoS ONE 9(1): e86730, 2014) we mapped the distribution of Fos labeling in the feline brainstem elicited by galvanic vestibular stimulation (GVS) that evoked retching and other indicators of nausea. As a comparison, in the present study we mapped Fos labeling patterns induced by intragastric administration of copper sulfate (CuSO4), which produces emesis by activating vagal afferent fibers. Emetic responses slowly build‐up during GVS, but occur rapidly following CuSO4 administration. In 10 felines (6 male, 4 female), doses of CuSO4 ranging from 83 to 322 mg were administered through an intragastric catheter or gavage, and ~2 hours later animals were perfused with paraformaldehyde. The brain was sectioned and processed for Fos labeling as in our prior study (Balaban et al., PLoS ONE 9(1): e86730, 2014). Three additional animals served as controls and did not receive intragastric CuSO4. Some brainstem areas exhibited Fos labeling following either GVS or CuSO4 treatments, as shown in Fig. 1: the commissural, medial and lateral regions of nucleus tractus solitarii, lateral reticular nucleus, medullary lateral tegmental field, subtrigeminal nucleus, medullary raphe nuclei, parabrachial nucleus, and locus coeruleus. However, the labeling patterns also diverged: CuSO4 administration produced virtually no labeling in the vestibular nuclei (which was heavy following GVS), more labeling in nucleus tractus solitarii and the medullary raphe nuclei, and less labeling in the pontine raphe nuclei. These findings support the hypothesis that some brainstem areas mediate nausea and emesis induced by any stimulus, independent of how quickly the responses develop, but also suggest that unique pathways may be activated by particular emetic stimuli.

SMART: An Open-Source Extension of WholeBrain for Intact Mouse Brain Registration and Segmentation.

Mapping immediate early gene (IEG) expression across intact mouse brains allows for unbiased identification of brain-wide activity patterns underlying complex behaviors. Accurate registration of sample brains to a common anatomic reference is critical for precise assignment of IEG-positive (“active”) neurons to known brain regions of interest (ROIs). While existing automated voxel-based registration methods provide a high-throughput solution, they require substantial computing power, can be difficult to implement and fail when brains are damaged or only partially imaged. Additionally, it is challenging to cross-validate these approaches or compare them to any preexisting literature based on serial coronal sectioning. Here, we present the open-source R package SMART (Semi-Manual Alignment to Reference Templates) that extends the WholeBrain R package framework to automated segmentation and semi-automated registration of intact mouse brain light-sheet fluorescence microscopy (LSFM) datasets. The SMART package was created for novice programmers and introduces a streamlined pipeline for aligning, registering, and segmenting LSFM volumetric datasets across the anterior-posterior (AP) axis, using a simple “choice game” and interactive menus. SMART provides the flexibility to register whole brains, partial brains or discrete user-chosen images, and is fully compatible with traditional sectioned coronal slice-based analyses. We demonstrate SMART’s core functions using example datasets and provide step-by-step video tutorials for installation and implementation of the package. We also present a modified iDISCO+ tissue clearing procedure for uniform immunohistochemical labeling of the activity marker Fos across intact mouse brains. The SMART pipeline, in conjunction with the modified iDISCO+ Fos procedure, is ideally suited for examination and orthogonal cross-validation of brain-wide neuronal activation datasets.

Activation of Brainstem Neurons During Mesencephalic Locomotor Region-Evoked Locomotion in the Cat

The distribution of locomotor-activated neurons in the brainstem of the cat was studied by c-Fos immunohistochemistry in combination with antibody-based cellular phenotyping following electrical stimulation of the mesencephalic locomotor region (MLR) – the anatomical constituents of which remain debated today, primarily between the cuneiform (CnF) and the pedunculopontine tegmental nuclei (PPT). Effective MLR sites were co-extensive with the CnF nucleus. Animals subject to the locomotor task showed abundant Fos labeling in the CnF, parabrachial nuclei of the subcuneiform region, periaqueductal gray, locus ceruleus (LC)/subceruleus (SubC), Kölliker–Fuse, magnocellular and lateral tegmental fields, raphe, and the parapyramidal region. Labeled neurons were more abundant on the side of stimulation. In some animals, Fos-labeled cells were also observed in the ventral tegmental area, medial and intermediate vestibular nuclei, dorsal motor nucleus of the vagus, n. tractus solitarii, and retrofacial nucleus in the ventrolateral medulla. Many neurons in the reticular formation were innervated by serotonergic fibers. Numerous locomotor-activated neurons in the parabrachial nuclei and LC/SubC/Kölliker–Fuse were noradrenergic. Few cholinergic neurons within the PPT stained for Fos. In the medulla, serotonergic neurons within the parapyramidal region and the nucleus raphe magnus were positive for Fos. Control animals, not subject to locomotion, showed few Fos-labeled neurons in these areas. The current study provides positive evidence for a role for the CnF in the initiation of locomotion while providing little evidence for the participation of the PPT. The results also show that MLR-evoked locomotion involves the parallel activation of reticular and monoaminergic neurons in the pons/medulla, and provides the anatomical and functional basis for spinal monoamine release during evoked locomotion. Lastly, the results indicate that vestibular, cardiovascular, and respiratory centers are centrally activated during MLR-evoked locomotion. Altogether, the results show a complex pattern of neuromodulatory influences of brainstem neurons by electrical activation of the MLR.

Recruitment of central angiotensin II type 1 receptor associated neurocircuits in carbon dioxide associated fear

Individuals with fear-associated conditions such as panic disorder (PD) and posttraumatic stress disorder (PTSD) display increased emotional responses to interoceptive triggers, such as CO2 inhalation, that signal a threat to physiological homeostasis. Currently, effector systems and mechanisms underlying homeostatic modulation of fear memory are not well understood. In this regard, the renin angiotensin system (RAS), particularly the angiotensin receptor type 1 (AT1R), a primary homeostatic regulatory target, has gained attention. RAS polymorphisms have been reported in PD and PTSD, and recent studies report AT1R-mediated modulation of fear extinction. However, contribution of AT1Rs in fear evoked by the interoceptive threat of CO2 has not been investigated. Using pharmacological, behavioral, and AT1R/ACE gene transcription analyses, we assessed central AT1R recruitment in CO2-associated fear. CO2 inhalation led to significant AT1R and ACE mRNA upregulation in homeostatic regulatory regions, subfornical organ (SFO) and paraventricular nucleus (PVN), in a temporal manner. Intracerebroventricular infusion of selective AT1R antagonist, losartan, significantly attenuated freezing during CO2 inhalation, and during re-exposure to CO2 context, suggestive of AT1R modulation of contextual fear. Regional Fos mapping in losartan-treated mice post-behavior revealed significantly attenuated labeling in areas regulating defensive behavior, contextual fear, and threat responding; such as, the bed nucleus of stria terminalis, dorsal periaqueductal gray, hypothalamic nuclei, hippocampus, and prefrontal areas such as the prelimbic, infralimbic, and anterior cingulate cortices. Sub-regions of the amygdala did not show CO2-associated AT1R regulation or altered Fos labeling. Collectively, our data suggests central AT1R recruitment in modulation of fear behaviors associated with CO2 inhalation via engagement of neurocircuits regulating homeostasis and defensive behaviors. Our data provides mechanistic insights into the interoceptive regulation of fear, relevant to fear related disorders such as PD and PTSD.

Aging affects isoproterenol-induced water drinking, astrocyte density, and central neuronal activation in female Brown Norway rats.

Age-dependent impairments in the central control of compensatory responses to body fluid challenges have received scant experimental attention, especially in females. In the present study, we found that water drinking in response to β-adrenergic activation with isoproterenol (30 μg/kg, s.c.) was reduced by more than half in aged (25 mo) vs. young (5 mo) ovariectomized female Brown Norway rats. To determine whether this age-related decrease in water intake was accompanied by changes in central nervous system areas associated with fluid balance, we assessed astrocyte density and neuronal activation in the SFO, OVLT, SON, AP and NTS of these rats using immunohistochemical labeling for GFAP and c-fos, respectively. GFAP labeling intensity was increased in the SFO, AP, and NTS of aged females independent of treatment, and was increased in the OVLT of isoproterenol-treated rats independent of age. Fos immunolabeling in response to isoproterenol was reduced in both the SFO and the OVLT of aged females compared to young females, but was increased in the SON of female rats of both ages. Finally, fos labeling in the AP and caudal NTS of aged rats was elevated after vehicle control treatment and did not increase in response to isoproterenol as it did in young females. Thus, age-related declines in water drinking are accompanied by site-specific, age-related changes in astrocyte density and neuronal activation. We suggest that astrocyte density may alter the detection and/or processing of signals related to isoproterenol treatment, and thereby alter neuronal activation in areas associated with fluid balance.

Electrophysiological Evidence That the Retrosplenial Cortex Displays a Strong and Specific Activation Phased with Hippocampal Theta during Paradoxical (REM) Sleep.

It is widely accepted that cortical neurons are similarly more activated during waking and paradoxical sleep (PS; aka REM) than during slow-wave sleep (SWS). However, we recently reported using Fos labeling that only a few limbic cortical structures including the retrosplenial cortex (RSC) and anterior cingulate cortex (ACA) contain a large number of neurons activated during PS hypersomnia. Our aim in the present study was to record local field potentials and unit activity from these two structures across all vigilance states in freely moving male rats to determine whether the RSC and the ACA are electrophysiologically specifically active during basal PS episodes. We found that theta power was significantly higher during PS than during active waking (aWK) similarly in the RSC and hippocampus (HPC) but not in ACA. Phase-amplitude coupling between HPC theta and gamma oscillations strongly and specifically increased in RSC during PS compared with aWK. It did not occur in ACA. Further, 68% and 43% of the units recorded in the RSC and ACA were significantly more active during PS than during aWK and SWS, respectively. In addition, neuronal discharge of RSC but not of ACA neurons increased just after the peak of hippocampal theta wave. Our results show for the first time that RSC neurons display enhanced spiking in synchrony with theta specifically during PS. We propose that activation of RSC neurons specifically during PS may play a role in the offline consolidation of spatial memories, and in the generation of vivid perceptual scenery during dreaming.SIGNIFICANCE STATEMENT Fifty years ago, Michel Jouvet used the term paradoxical to define REM sleep because of the simultaneous occurrence of a cortical activation similar to waking accompanied by muscle atonia. However, we recently demonstrated using functional neuroanatomy that only a few limbic structures including the retrosplenial cortex (RSC) and anterior cingulate cortex (ACA) are activated during PS. In the present study, we show for the first time that the RSC and ACA contain neurons firing more during PS than in any other state. Further, RSC neurons are firing in phase with the hippocampal theta rhythm. These data indicate that the RSC is very active during PS and could play a key role in memory consolidation taking place during this state.

Do Trigeminal Nerves that Innervate the Nose and External Nares Project to Medullary Dorsal Horn Locations Activated during Voluntary Diving in Rats?

Underwater diving initiates the diving response, an autonomic cardiorespiratory reflex that includes parasympathetically‐mediated bradycardia, a sympathetically‐mediated increase in total peripheral resistance, and apnea. The initial afferent node of the reflex circuitry includes the ventral tip of the medullary dorsal horn (MDH), between the pyramidal decussation and obex. This region is also the location of the central terminations of trigeminal nerves that innervate the nose and external nares. Accordingly, the objective of this research was to compare the pattern of neuronal activation of secondary neurons activated by voluntary diving, as determined by neuronal Fos production, with the central terminations of specific trigeminal nerves that innervate the nose and external nares, as determined by the transganglionic tracer Wheat Germ Agglutinin (WGA). Sprague‐Dawley rats (N=12) were trained to voluntarily dive through a 5 m underwater maze. Six rats then received bilateral AEN sectioning, while 6 rats received sham surgery. Nine days post‐surgery all rats had 24 dive trials over 2 hours to activate brainstem neurons to produce Fos. Other rats had 1 μl of 100% WGA injected into the anterior ethmoidal nerve (AEN; N=4), the main orbital branch of the infraorbital nerve (ION; N=7), terminal branches of the ION that innervate the nasal region (N=9), or supratrochlear nerve (N=8). Results indicate Fos was primarily located within the ventral tip of the superficial MDH and adjacent paratrigeminal nucleus, between the pyramidal decussation caudally and the obex rostrally. The Fos labeling was heaviest when the area postrema was present, especially when Spinal Trigeminal Nucleus Interpolaris displaces the ventral MDH medially. This pattern of Fos labeling within the MDH was similar in rats both with and without intact AENs. Within the MDH WGA central termination sites showed varying degrees of overlap with the location of Fos‐positive neurons. The AEN WGA labeling had the best overlap with Fos‐positive neurons. The main orbital branch of the ION had extensive WGA labeling throughout the MDH, both ventrally and dorsally, and did partially overlap the MDH Fos labeling. The WGA labeling from both nasal branches of the ION and supratrochlear nerve were located more caudally within the MDH near the pyramidal decussation, thus showing poor overlap with the Fos labeling. We conclude that the AEN, and possibly the ION, provides the anatomical pathway that provides the afferent information necessary to initiate the mammalian diving response. However, in the absence of the AEN, it is not clear whether the ION can actually provide this afferent information.Support or Funding InformationSupported by MWU Intramural funding (PFM), CCOM Suarez Summer Research Fellowships (BDP and KAL), and CHS MBS program (EAW).

Repetitive Diving in Trained Rats Still Increases Fos Production in Brainstem Neurons after Bilateral Sectioning of the Anterior Ethmoidal Nerve.

This research was designed to investigate the role of the anterior ethmoidal nerve (AEN) during repetitive trained diving in rats, with specific attention to activation of afferent and efferent brainstem nuclei that are part of this reflexive response. The AEN innervates the nose and nasal passages and is thought to be an important component of the afferent limb of the diving response. Male Sprague-Dawley rats (N = 24) were trained to swim and dive through a 5 m underwater maze. Some rats (N = 12) had bilateral sectioning of the AEN, others a Sham surgery (N = 12). Twelve rats (6 AEN cut and 6 Sham) had 24 post-surgical dive trials over 2 h to activate brainstem neurons to produce Fos, a neuronal activation marker. Remaining rats were non-diving controls. Diving animals had significantly more Fos-positive neurons than non-diving animals in the caudal pressor area, ventral medullary dorsal horn, ventral paratrigeminal nucleus, nucleus tractus solitarius, rostral ventrolateral medulla, Raphe nuclei, A5, Locus Coeruleus, and Kölliker-Fuse area. There were no significant differences in brainstem Fos labeling in rats diving with and without intact AENs. Thus, the AENs are not required for initiation of the diving response. Other nerve(s) that innervate the nose and nasal passages, and/or suprabulbar activation of brainstem neurons, may be responsible for the pattern of neuronal activation observed during repetitive trained diving in rats. These results help define the central neuronal circuitry of the mammalian diving response.

Programing Effect Of Maternal High Fat/High Sucrose Diet On Cardiovascular Responses And Fos Expression following Air Jet And Restraint Stress

A high fat/high sucrose diet (HFS) and/or obesity during pregnancy are known stressors for programing hypertension in the adult offspring. Although the exact causative mechanisms are not fully elucidated, exaggerated cardiovascular (CV) and autonomic responses to psychological stress may be a contributing factor. However, the precise nature of the stressor and the central neural pathways via which the programed responses are mediated remain unknown. Our aim was to investigate the effect of a HFS maternal diet on blood pressure (BP) of adult male offspring at rest and in response to 2 psychological stressors of varying intensities. The central pathways involved in mediating these responses were also investigated by quantifying Fos expression in key CV control regions of the brain. The 2 stressors chosen were air jet stress (AJS), considered a moderate stressor, and restraint stress (RS), considered more severe in nature. Sprague Dawley dams were fed a HFS (21% fat, 34% (w/w) sucrose vs control, 4.8% fat, 0% sucrose) diet ad libitum for 4 weeks prior to mating until weaning. Adult offspring (9–12 months) were implanted with radiotelemetry BP transmitters and BP was recorded at rest for 24 hours, in response to the stress tests, and throughout a 2‐hour recovery period. CV parameters, including heart rate (HR) and mean BP (MnBP) were derived from the BP waveform. Following recovery, rats were perfused with 4% paraformaldehyde and brains processed immunohistochemically for Fos expression. Resting MnBP was similar between groups both at night (HFS, 101±2.3 mmHg; control, 97±2.5 mmHg) and day (HFS, 96±0.7 mmHg; control, 93±3.5 mmHg). There was also no difference in HR between groups. Interestingly, MnBP in HFS offspring was approximately 10 mmHg higher than controls in the resting period immediately preceding the stress tests. This suggests that even mild environmental perturbations, such as the presence of researchers in the laboratory, may have an impact on BP responses in HFS offspring. HFS offspring also exhibited greater increases in MnBP upon commencement of AJS (HFS, 25±5.4 mmHg; control, 13±3.1 mmHg) and delayed recovery. At 45 minutes post‐stress, MnBP was at 15% baseline in HFS, compared to 3.5% baseline in controls. RS caused similar increases in MnBP in both groups (HFS, 29±5.0 mmHg, control, 30±8.6 mmHg). Interestingly, initial analysis indicated that although RS produced greater CV responses than AJS in control offspring, this was not observed in HFS. Consistent with this, in control rats, RS evoked a 5‐fold increase in Fos labeling throughout the brainstem compared to baseline, while AJS caused a 3‐fold increase. Future analysis will determine the degree of Fos labeling in HFS offspring. The results suggest that maternal HFS programs a pro‐hypertensive phenotype characterised by exaggerated and extended BP responses to mild to moderate stress. RS evoked greater responses than AJS in control rats only, and this was consistent with greater Fos labelling in key CV nuclei in controls. Fos labelling in HFS is in progress.

Spontaneous seizures in Kcna1-null mice lacking voltage-gated Kv1.1 channels activate Fos expression in select limbic circuits.

Mice lacking voltage-gated Kv1.1 channels as a result of deletion of the Kcna1 gene are an extensively utilized genetic model of human epilepsy and sudden unexpected death in epilepsy because of their frequent seizures and genotypic-phenotypic similarity to the human condition. Ictal behaviors, electrophysiological recordings, and gene expression studies suggest limbic circuits are critical for epilepsy in Kcna1-null mice, but the exact brain networks recruited by seizures remain unknown. In this study, Fos protein expression patterns were used to map limbic brain regions with increased neuronal activity at baseline and during spontaneous seizures in Kcna1-null mice by comparing seizing and non-seizing knockouts and wild-type controls. Basal Fos levels were unchanged in non-seizing knockout mice compared to wild types for all brain regions examined except the dentate gyrus granule cell layer which exhibited a significant decrease in Fos-positive cells. Following seizures, Kcna1-null brains exhibited significantly increased Fos labeling in the basolateral amygdala and the dentate hilus region, but not in other principal cell layers of the hippocampal formation. The selective Fos activation in the amygdala following seizures suggests that extra hippocampal limbic circuits may be critically involved with seizure generation or spread in Kcna1-null mice. Fos protein expression patterns were analyzed using immunohistochemistry to provide the first map of brain regions recruited by spontaneous seizures in mice lacking Kv1.1 channels, an extensively used genetic model of epilepsy. Seizures significantly increased Fos expression in the amygdala and hilus by about fourfold, suggesting an important contribution by extrahippocampal networks to epilepsy in this model.

Estrogen Effects on Neural Activation after Short‐term Vs. Long‐term Furosemide Treatment in Rats

Estrogen influences body Na+ balance, but the central pathways involved in these effects are currently unknown. Studies using immunohistochemical labeling of the fos protein to study central areas involved in body fluid balance report no effect of Na+ loss in male rats. However, standard methods of producing Na+ loss are of comparatively long duration, and it is possible central activation is not sustained. Accordingly, our objective was to assess the effect of estrogen on activation in response to short-term and long-term Na+ depletion using furosemide, a diuretic-natriuretic. Adult female rats were ovariectomized, allowed to recover for 7 days, and then given estradiol benzoate (EB; 10 μg/0.1 ml oil, sc) or oil vehicle (OIL; 0.1 ml, sc). Rats were injected with furosemide (2 X 5 mg/kg, sc) by one of two protocols. For the short term protocol, rats were perfused with paraformaldehyde 2 hrs after furosemide; for the long-term protocol, rats were perfused 18 hrs after furosemide. Brains were removed and cut into 40μ sections and then immunolabeled for fos (Santa Cruz; 1:30,000). Fos immunolabeling was apparent in the supraoptic nucleus of the hypothalamus after short-term, but not long-term, Na+ depletion. Fos labeling was not present in the paraventricular hypothalamic nucleus with either protocol, but was present after both protocols in forebrain circumventricular organs (CVOs). Estrogen did not affect fos immunolabeling in either protocol. Thus, independent of estrogen, acute Na+ loss produces transient neural activation in some central areas involved in body Na+ regulation, and sustained activation in CVOs, which monitor circulating hormones and electrolytes. Supported by OCAST HR12-196

Re-purposing of histological tissue sections for corroborative western blot analysis of hypothalamic metabolic neuropeptide expression following delineation of transactivated structures by Fos immuno-mapping

Fos immunocytochemistry is a valuable anatomical mapping tool for distinguishing cells within complex tissues that undergo genomic activation, but it is seldom paired with corroborative molecular analytical techniques. Due to preparatory requirements that include protein cross-linking for specimen sectioning, histological tissue sections are regarded as unsuitable for those methods. Our studies show that pharmacological activation of the hindbrain energy sensor AMPK by AICAR elicits estradiol (E)-dependent patterns of Fos immunolabeling of hypothalamic metabolic loci. Here, Western blotting was applied to hypothalamic tissue removed from histological sections of E- versus oil (O)-implanted ovariectomized (OVX) female rat brain to measure levels of metabolic transmitters associated with Fos-positive structures. In both E and O rats, AICAR treatment elicited alterations in pro-opiomelanocortin, neuropeptide Y, SF-1, and orexin-A neuropeptide expression that coincided with patterns of Fos labeling of structures containing neurons that synthesize these neurotransmitters, e.g. arcuate and ventromedial nuclei and lateral hypothalamic area. O, but not E animals also exhibited parallel augmentation of tissue corticotropin-releasing hormone neuropeptide levels and paraventricular nucleus Fos staining. Data demonstrate the utility of immunoblot analysis as a follow-through technique to capitalize on Fos mapping of transactivation sites in the brain. Findings that induction of Fos immunoreactivity coincides with adjustments in hypothalamic metabolic neuropeptide expression affirms that this functional indicator reflects changes in neurotransmission in pathways governing metabolic outflow.

Ciproxifan improves working memory through increased prefrontal cortex neural activity in sleep-restricted mice

Histamine receptor type 3 (H3) antagonists are promising awakening drugs for treatment of sleep disorders. However, few works have tried to identify their cognitive effects after sleep restriction and their impact on associated neural networks. To that aim, Bl/6J male mice were submitted to acute sleep restriction in a shaker apparatus that prevents sleep by transient (20–40 ms) up and down movements. Number of stimulations (2–4), and delay between 2 stimulations (100–200 ms) were randomized. Each sequence of stimulation was also randomly administered (10–30 s interval) for 20 consecutive hours during light (8 h) and dark (12 h) phases. Immediately after 20 h-sleep restriction, mice were injected with H3 antagonist (ciproxifan 3 mg/kg ip) and submitted 30-min later to a working memory (WM) task using spatial spontaneous alternation behaviour. After behavioural testing, brains were perfused for Fos immunohistochemistry to assess neuronal brain activation in the dorsal dentate gyrus (dDG) and the prefrontal cortex. Results showed that sleep restriction decreased slow wave sleep (from 35.8 ± 1.4% to 9.2 ± 2.7%, p < 0.001) and was followed by sleep rebound (58.2 ± 5.9%, p < 0.05). Sleep restriction did not modify anxiety-like reactivity and significantly decreased WM at long (30 s) but not short (5 s) inter-trial intervals. Whereas sleep restriction failed to significantly modify immunopositive cells in vehicles, ciproxifan administration prevented WM deficits in sleep restricted mice through significant increases of Fos labelling in prelimbic, infralimbic and cingulate 2 cortex.In conclusion, ciproxifan at 3 mg/kg enhanced WM in sleep restricted mice through specific modulation of prefrontal cortex areas.

Role of dorsal vagal complex A2 noradrenergic neurons in hindbrain glucoprivic inhibition of the luteinizing hormone surge in the steroid-primed ovariectomized female rat: Effects of 5-thioglucose on A2 functional biomarker and AMPK activity

Neuro-glucostasis is required for normal expression of the steroid positive-feedback-induced preovulatory pituitary luteinizing hormone (LH) surge, a critical element of female reproduction. Glucoprivic signals from the caudal hindbrain restrain this surge, but the cellular source of this stimulus is unclear. Norepinephrine (NE) exerts well-defined stimulatory effects on the reproductive neuroendocrine axis. Our studies show that medullary A2 noradrenergic neurons are both estrogen- and glucoprivic-sensitive. Here, we investigated the premise that the LH surge is inhibited by A2 cell reactivity to hindbrain glucopenia and diminished preoptic NE neurotransmission. Estradiol- and progesterone-primed ovariectomized (OVX) female rats were injected into the caudal fourth ventricle (CV4) with the glucose anti-metabolite, 5-thioglucose (5TG) or saline (SAL) prior to onset of the LH surge. Pretreatment by intra-CV4 delivery of the selective catecholamine neurotoxin, 6-OHDA, attenuated LH output, but prevented inhibition by 5TG. 5TG modified patterns of steroid feedback-associated Fos staining of A2, but not other medullary catecholamine cell groups. Intra-preoptic administration of the alpha1-adrenergic receptor agonist, methoxamine, elicited site-specific reversal of hindbrain glucoprivic suppression of gonadotropin-releasing hormone (GnRH) neuron Fos labeling and LH release. Western blotting of laser-microdissected A2 neurons revealed glucoprivic stimulation of Fos, but inhibition of the catecholamine synthetic enzyme, dopamine-β-hydroxylase; 5TG also diminished A2 estrogen receptor (ER)-α and progesterone receptor profiles, but augmented ER-β protein. Intriguingly, A2 AMPK activity was decreased in 5TG-treated rats, despite down-regulation of GLUT3 and no change in MCT2 protein expression. Rostral preoptic GnRH neurons also exhibited decreased AMPK activation simultaneous with apparent reduction of neuropeptide signaling to the pituitary. The present studies demonstrate that hindbrain glucoprivation inhibits the LH surge, in part, by reducing preoptic noradrenergic input, and furthermore implicate A2 neurons as a source of this altered signal. Results also suggest that AMPK sensor deactivation does not supersede the impact of pharmacological inhibition of glucose catabolism on A2 cell function nor afferent signaling of hindbrain glucopenia on GnRH neurons. Further studies are needed to determine if decreased AMPK activation in these cell populations reflect compensatory gain in positive energy balance and/or direct effects of estrogen on AMPK.

Identification of Neural Networks That Contribute to Motion Sickness through Principal Components Analysis of Fos Labeling Induced by Galvanic Vestibular Stimulation

Motion sickness is a complex condition that includes both overt signs (e.g., vomiting) and more covert symptoms (e.g., anxiety and foreboding). The neural pathways that mediate these signs and symptoms are yet to identified. This study mapped the distribution of c-fos protein (Fos)-like immunoreactivity elicited during a galvanic vestibular stimulation paradigm that is known to induce motion sickness in felines. A principal components analysis was used to identify networks of neurons activated during this stimulus paradigm from functional correlations between Fos labeling in different nuclei. This analysis identified five principal components (neural networks) that accounted for greater than 95% of the variance in Fos labeling. Two of the components were correlated with the severity of motion sickness symptoms, and likely participated in generating the overt signs of the condition. One of these networks included neurons in locus coeruleus, medial, inferior and lateral vestibular nuclei, lateral nucleus tractus solitarius, medial parabrachial nucleus and periaqueductal gray. The second included neurons in the superior vestibular nucleus, precerebellar nuclei, periaqueductal gray, and parabrachial nuclei, with weaker associations of raphe nuclei. Three additional components (networks) were also identified that were not correlated with the severity of motion sickness symptoms. These networks likely mediated the covert aspects of motion sickness, such as affective components. The identification of five statistically independent component networks associated with the development of motion sickness provides an opportunity to consider, in network activation dimensions, the complex progression of signs and symptoms that are precipitated in provocative environments. Similar methodology can be used to parse the neural networks that mediate other complex responses to environmental stimuli.

Brain Activity Mapping inMecp2Mutant Mice Reveals Functional Deficits in Forebrain Circuits, Including Key Nodes in the Default Mode Network, that are Reversed with Ketamine Treatment

Excitatory-inhibitory imbalance has been identified within specific brain microcircuits in models of Rett syndrome (RTT) and other autism spectrum disorders (ASDs). However, macrocircuit dysfunction across the RTT brain as a whole has not been defined. To approach this issue, we mapped expression of the activity-dependent, immediate-early gene product Fos in the brains of wild-type (Wt) and methyl-CpG-binding protein 2 (Mecp2)-null (Null) mice, a model of RTT, before and after the appearance of overt symptoms (3 and 6 weeks of age, respectively). At 6 weeks, Null mice exhibit significantly less Fos labeling than Wt in limbic cortices and subcortical structures, including key nodes in the default mode network. In contrast, Null mice exhibit significantly more Fos labeling than Wt in the hindbrain, most notably in cardiorespiratory regions of the nucleus tractus solitarius (nTS). Using nTS as a model, whole-cell recordings demonstrated that increased Fos expression in Nulls at 6 weeks of age is associated with synaptic hyperexcitability, including increased frequency of spontaneous and miniature EPSCs and increased amplitude of evoked EPSCs in Nulls. No such effect of genotype on Fos or synaptic function was seen at 3 weeks. In the mutant forebrain, reduced Fos expression, as well as abnormal sensorimotor function, were reversed by the NMDA receptor antagonist ketamine. In light of recent findings that the default mode network is hypoactive in autism, our data raise the possibility that hypofunction within this meta-circuit is a shared feature of RTT and other ASDs and is reversible.

Blunted Behavioral and C Fos Responses to Acidic Fumes in the African Naked Mole-Rat

Acidosis in the skin triggers activation of pain pathways and behaviors indicative of pain in vertebrates. The exception is the naked mole-rat, the only known vertebrate to show physiological and behavioral insensitivity to acid pain in the skin. The goal of the present study was to determine behavioral and physiological responses of this species to airborne acidic fumes, which would be expected to affect the trigeminal pain pathway in other species. Behaviorally, naked mole-rats did not avoid fumes from moderately high concentrations of acetic acid (10 and 20%), and c Fos labeling showed no increase in activity in the trigeminal nuclei and nucleus tractus solitarius. In contrast, these concentrations triggered behavioral aversion and increased Fos activity in other laboratory rodents. For a very high concentration of acetic acid (50%), naked mole-rats showed significant avoidance behavior and increased Fos labeling in the nucleus tractus solitarius caudal region, which receives vagal chemosensory information. However, there was no increase in trigeminal labeling, and in fact, activity significantly decreased. This pattern is opposite of that associated with another irritant, ammonia fumes, which elicited an increase in trigeminal but not nucleus tractus solitarius Fos labeling, and no behavioral avoidance. Behavioral avoidance of acidic fumes, but no increased labeling in the trigeminal pain nucleus is consistent with the notion of adaptations to blunt acid pain, which would be advantageous for naked mole-rats as they normally live under chronically high levels of acidosis-inducing CO2.

Spontaneous behavioral responses in the orofacial region: a model of trigeminal pain in mouse.

To develop a translational mouse model for the study and measurement of non-evoked pain in the orofacial region by establishing markers of nociceptive-specific grooming behaviors in the mouse. Some of the most prevalent and debilitating conditions involve pain in the trigeminal distribution. Although there are current therapies for these pain conditions, for many patients, they are far from optimal. Understanding the pathophysiology of pain disorders arising from structures innervated by the trigeminal nerve is still limited, and most animal behavioral models focus on the measurement of evoked pain. In patients, spontaneous (non-evoked) pain responses provide a more accurate representation of the pain experience than do responses that are evoked by an artificial stimulus. Therefore, the development of animal models that measure spontaneous nociceptive behaviors may provide a significant translational tool for a better understanding of pain neurobiology. C57BL/6 mice received either an injection of 0.9% saline solution or complete Freund's adjuvant into the right masseter muscle. Animals were video-recorded and then analyzed by an observer blind to the experiment group. The duration of different facial grooming patterns performed in the area of injection were measured. After 2 hours, mice were euthanized and perfused, and the brainstem was removed. Fos protein expression in the trigeminal nucleus caudalis was quantified using immunohistochemistry to investigate nociceptive-specific neuronal activation. A separate group of animals was treated with morphine sulfate to determine the nociceptive-specific nature of their behaviors. We characterized and quantified 3 distinct patterns of acute grooming behaviors: forepaw rubbing, lower lip skin/cheek rubbing against enclosure floor, and hindpaw scratching. These behaviors occurred with a reproducible frequency and time course, and were inhibited by the analgesic morphine. Complete Freund's adjuvant-injected animals also showed Fos labeling consistent with neuronal activation in nociceptive-specific pathways of the trigeminal nucleus after 2 hours. These behaviors and their correlated cellular responses represent a model of trigeminal pain that can be used to better understand basic mechanisms of orofacial pain and identify new therapeutic approaches to this common and challenging condition.

Estradiol selectively reduces central neural activation induced by hypertonic NaCl infusion in ovariectomized rats

We recently reported that the latency to begin drinking water during slow, intravenous infusion of a concentrated NaCl solution was shorter in estradiol-treated ovariectomized rats compared to oil vehicle-treated rats, despite comparably elevated plasma osmolality. To test the hypothesis that the decreased latency to begin drinking is attributable to enhanced detection of increased plasma osmolality by osmoreceptors located in the CNS, the present study used immunocytochemical methods to label fos, a marker of neural activation. Increased plasma osmolality did not activate the subfornical organ (SFO), organum vasculosum of the lamina terminalis (OVLT), or the nucleus of the solitary tract (NTS) in either oil vehicle-treated rats or estradiol-treated rats. In contrast, hyperosmolality increased fos labeling in the area postrema (AP), the paraventricular nucleus of the hypothalamus (PVN) and the rostral ventrolateral medulla (RVLM) in both groups; however, the increase was blunted in estradiol-treated rats. These results suggest that estradiol has selective effects on the sensitivity of a population of osmo-/Na+-receptors located in the AP, which, in turn, alters activity in other central areas associated with responses to increased osmolality. In conjunction with previous reports that hyperosmolality increases blood pressure and that elevated blood pressure inhibits drinking, the current findings of reduced activation in AP, PVN, and RVLM—areas involved in sympathetic nerve activity—raise the possibility that estradiol blunts HS-induced blood pressure changes. Thus, estradiol may eliminate or reduce the initial inhibition of water intake that occurs during increased osmolality, and facilitate a more rapid behavioral response, as we observed in our recent study.