Colocalization Of Fos Research Articles

Sex differences in neuronal activation in the cortex and midbrain during quinine-adulterated alcohol intake.

Continued alcohol consumption despite negative consequences is a core symptom of alcohol use disorder. This is modeled in mice by pairing negative stimuli with alcohol, such as adulterating alcohol solution with quinine. Mice consuming alcohol under these conditions are considered to be engaging in aversion-resistant intake. Previously, we have observed sex differences in this behavior, with females more readily expressing aversion-resistant consumption. We also identified three brain regions that exhibited sex differences in neuronal activation during quinine-alcohol drinking: ventromedial prefrontal cortex (vmPFC), posterior insular cortex (PIC), and ventral tegmental area (VTA). Specifically, male mice showed increased activation in vmPFC and PIC, while females exhibited increased activation in VTA. In this study, we aimed to identify what specific type of neurons are activated in these regions during quinine-alcohol drinking. We assessed quinine-adulterated alcohol intake using the two-bottle choice procedure. We also utilized RNAscope in situ hybridization in the three brain regions that previously exhibited a sex difference to examine colocalization of Fos, glutamate, GABA, and dopamine. Females showed increased aversion-resistant alcohol consumption compared to males. We also found that males had higher colocalization of glutamate and Fos in vmPFC and PIC, while females had greater dopamine and Fos colocalization in the VTA. Collectively, these experiments suggest that glutamatergic output from the vmPFC and PIC may have a role in suppressing, and dopaminergic activity in the VTA may promote, aversion-resistant alcohol consumption. Future experiments will examine neuronal circuits that contribute to sex differences in aversion resistant consumption.

Recruitment of Corticotropin-Releasing Hormone (CRH) Neurons in Categorically Distinct Stress Reactions in the Mouse Brain.

Corticotropin-releasing hormone (CRH) neurons in the paraventricular hypothalamic nucleus (PVH) are in the position to integrate stress-related information and initiate adaptive neuroendocrine-, autonomic-, metabolic- and behavioral responses. In addition to hypophyseotropic cells, CRH is widely expressed in the CNS, however its involvement in the organization of the stress response is not fully understood. In these experiments, we took advantage of recently available Crh-IRES-Cre;Ai9 mouse line to study the recruitment of hypothalamic and extrahypothalamic CRH neurons in categorically distinct, acute stress reactions. A total of 95 brain regions in the adult male mouse brain have been identified as containing putative CRH neurons with significant expression of tdTomato marker gene. With comparison of CRH mRNA and tdTomato distribution, we found match and mismatch areas. Reporter mice were then exposed to restraint, ether, high salt, lipopolysaccharide and predator odor stress and neuronal activation was revealed by FOS immunocytochemistry. In addition to a core stress system, stressor-specific areas have been revealed to display activity marker FOS. Finally, activation of CRH neurons was detected by colocalization of FOS in tdTomato expressing cells. All stressors resulted in profound activation of CRH neurons in the hypothalamic paraventricular nucleus; however, a differential activation of pattern was observed in CRH neurons in extrahypothalamic regions. This comprehensive description of stress-related CRH neurons in the mouse brain provides a starting point for a systematic functional analysis of the brain stress system and its relation to stress-induced psychopathologies.

Low Fos expression in newly generated neurons of the main and accessory olfactory bulb following single maternal separation

The main and accessory olfactory bulbs (MOB and AOB) are unique in that they produce new neurons throughout adulthood. Despite the recent knowledge about the involvement of postnatally generated cells in several aspects of olfaction, the functional role of these neurons is still not sufficiently understood. The function of newly generated olfactory bulb neurons is primarily investigated in relation to activities related to smell. Stress-induced activation of new olfactory neurons has not yet been studied. Thus, our work was aimed to investigate whether a stressful event, such as maternal separation (MS) can induce Fos expression in postnatally-born neurons in the MOB and AOB. Rat pups were exposed to single maternal separation (SMS) for 2 h at the postnatal days: P7, P14, and P21. Quantification of immunohistochemically labeled Fos + cells revealed that exposure to SMS in different age stages during the first postnatal month stimulates activity in cells of individual MOB/AOB layers in an age-dependent manner. In order to find out whether newly generated cells in the MOB/AOB could express Fos protein as a response to SMS, newborn rats were administrated with the marker of proliferation, bromodeoxyuridine (BrdU) at P0, and three weeks later (at P21) colocalization of Fos and BrdU in the neurons of the MOB and AOB was assessed. Quantitative analysis of BrdU/Fos double-labeled cells showed that Fos is expressed only in a small number of postnatally generated cells within the MOB/AOB. Our results indicate that postnatally generated MOB/AOB neurons are less sensitive to stress caused by MS than preexisting ones. LAY SUMMARY Our results showed that single maternal separation (SMS) is a stressful event that in age-dependent manner stimulates cellular activity in the main and accessory olfactory bulb (AOB) – the structures dedicated to odor information processing. The low level of Fos expression in newborn neurons of the main and accessory bulb indicates that postnatally generated cells are less sensitive to neonatal stress than preexisting neurons.

Spatial exploration induced expression of immediate early genes Fos and Zif268 in adult-born neurons Is reduced after pentylenetetrazole kindling

Seizure activity stimulates adult neurogenesis, the birth of new neurons, in the hippocampus. Many new neurons that develop in the presence of repeatedly induced seizures acquire abnormal morphological and functional characteristics that can promote network hyperexcitability and hippocampal dysfunction. However, the impact of seizure induced neurogenesis on behaviour remains poorly understood. In this study, we investigated whether adult-born neurons generated immediately before and during chronic seizures were capable of integration into behaviorally relevant hippocampal networks. Adult rats underwent pentylenetetrazole (PTZ) kindling for either 1 or 2 weeks. Proliferating cells were labelled with BrdU immediately before kindling commenced. Twenty-four hours after receiving their last kindling treatment, rats were placed in a novel environment and allowed to freely explore for 30 min. The rats were euthanized 90 min later to examine for behaviourally-induced immediate early gene expression (c-fos, Zif268). Using this approach, we found that PTZ kindled rats did not differ from control rats in regards to exploratory behaviour, but there was a marked attenuation in behaviour-induced expression of Fos and Zif268 for rats that received 2 weeks of PTZ kindling. Further examination revealed that PTZ kindled rats showed reduced colocalization of Fos and Zif268 in 2.5 week old BrdU + cells. The proportion of immature granule cells (doublecortin-positive) expressing behaviorally induced Zif268 was also significantly lower for PTZ kindled rats than control rats. These results suggest that chronic seizures can potentially disrupt the ability of adult-born cells to functionally integrate into hippocampal circuits important for the processing of spatial information.

Nucleus Accumbens Drd1-Expressing Neurons Control Aggression Self-Administration and Aggression Seeking in Mice

We recently developed a mouse model of appetitive operant aggression and reported that adult male outbred CD-1 mice lever-press for the opportunity to attack subordinate male mice and relapse to aggression seeking during abstinence. Here we studied the role of nucleus accumbens (NAc) dopamine receptor (Drd)1- and Drd2-expressing neurons in aggression self-administration and aggression seeking. We trained CD-1 mice to self-administer intruders (9 d, 12 trials/d) and tested them for aggression self-administration and aggression seeking on abstinence Day 1. We used immunohistochemistry and in situ hybridization to measure the neuronal activity marker Fos in the NAc, and cell-type-specific colocalization of Fos with Drd1- and Drd2-expressing neurons. To test the causal role of Drd1- and Drd2-expressing neurons, we validated a transgenic hybrid breeding strategy crossing inbred Drd1-Cre and Drd2-Cre transgenic mice with outbred CD-1 mice and used cell-type-specific Cre-DREADD (hM4Di) to inhibit NAc Drd1- and Drd2-expressing neuron activity. We found that aggression self-administration and aggression seeking induced higher Fos expression in NAc shell than in core, that Fos colocalized with Drd1 and Drd2 in both subregions, and that chemogenetic inhibition of Drd1-, but not Drd2-, expressing neurons decreased aggression self-administration and aggression seeking. Results indicate a cell-type-specific role of Drd1-expressing neurons that is critical for both aggression self-administration and aggression seeking. Our study also validates a simple breeding strategy between outbred CD-1 mice and inbred C57-based Cre lines that can be used to study cell-type and circuit mechanisms of aggression reward and relapse.SIGNIFICANCE STATEMENT Aggression is often comorbid with neuropsychiatric diseases, including drug addiction. One form, appetitive aggression, exhibits symptomatology that mimics that of drug addiction and is hypothesized to be due to dysregulation of addiction-related reward circuits. However, our mechanistic understanding of the circuitry modulating appetitive operant aggression is limited. Here we used a novel mouse model of aggression self-administration and relapse, in combination with immunohistochemistry, in situ hybridization, and chemogenetic manipulations to examine how cell types in the nucleus accumbens are recruited for, and control, operant aggression self-administration and aggression seeking on abstinence Day 1. We found that one population, dopamine receptor 1-expressing neurons, act as a critical modulator of operant aggression reward and aggression seeking.

Sex-dependent regulation of social reward by oxytocin receptors in the ventral tegmental area.

Social reward is critical for social relationships, and yet we know little about the characteristics of social interactions that are rewarding or the neural mechanisms underlying that reward. Here, we investigate the sex-dependent role of oxytocin receptors within the ventral tegmental area (VTA) in mediating the magnitude and valence of social reward. Operant and classical conditioning tests were used to measure social reward associated with same-sex social interactions. The effects of oxytocin, selective oxytocin receptor agonists, antagonists, and vehicle injected into the VTA on social reward was determined in male and female Syrian hamsters. The colocalization of FOS and oxytocin in sites that project to the VTA following social interaction was also determined. Females find same-sex social interactions more rewarding than males and activation of oxytocin receptors in the VTA is critical for social reward in females, as well as males. These studies provide support for the hypothesis that there is an inverted U relationship between the duration of social interaction and social reward, mediated by oxytocin; and that in females the dose-response relationship is initiated at lower doses compared with males. Same-sex social interaction is more rewarding in females than in males, and an inverted U relationship mediated by oxytocin may have a critical role in assigning positive and negative valence to social stimuli. Understanding these sex differences in social reward processing may be essential for understanding the sex differences in the prevalence of many psychiatric disorders and the development of gender-specific treatments of neuropsychiatric disorders.

Rapid nonapeptide synthesis during a critical period of development in the prairie vole: plasticity of the paraventricular nucleus of the hypothalamus.

Vasopressin (VP) and oxytocin (OT) are involved in modulating basic physiology and numerous social behaviors. Although the anatomical distributions of nonapeptide neurons throughout development have been described, the functional roles of VP and OT neurons during development are surprisingly understudied, and it is unknown whether they exhibit functional changes throughout early development. We utilized an acute social isolation paradigm to determine if VP and OT neural responses in eight nonapeptide cell groups differ at three different stages of early development in prairie voles. We tested pups at ages that are representative of the three rapid growth stages of the developing brain: postnatal day (PND)2 (closed eyes; poor locomotion), PND9 (eye opening; locomotion; peak brain growth spurt), and PND21 (weaning). Neural responses were examined in pups that (1) were under normal family conditions with their parents and siblings, (2) were isolated from their parents and siblings and then reunited, and (3) were isolated from their parents and siblings. We found that VP and OT neural activity (as assessed via Fos co-localization) did not differ in response to social condition across development. However, remarkably rapid VP and OT synthesis in response to social isolation was observed only in the paraventricular nucleus of the hypothalamus (PVN) and only in PND9 pups. These results suggest that PVN nonapeptide neurons exhibit distinct cellular properties during a critical period of development, allowing nonapeptide neurons to rapidly upregulate peptide production in response to stressors on a much shorter timescale than has been observed in adult animals.

Nesting behavior is associated with VIP expression and VIP–Fos colocalization in a network-wide manner

Many species, including humans, engage in a series of behaviors that are preparatory to the arrival of offspring. Such “nesting behaviors” are of obvious importance, but relevant neuroendocrine mechanisms remain little studied. We here focus on the potential roles of vasoactive intestinal polypeptide (VIP) in the performance of appetitive and consummatory nesting behaviors in male and female zebra finches (Taeniopygia guttata). Using combined immunocytochemistry for Fos and in situ hybridization for VIP, we now show that many VIP cell groups show increased transcriptional activity in response to nest building in male and female zebra finches. Particularly strong data come from the preoptic area (medial preoptic area and medial preoptic nucleus), where VIP–Fos co-expression correlates positively with three different measures of nesting behavior, as does the number of VIP-expressing cells. Remarkably, we find that VIP mRNA and/or VIP–Fos co-expression is correlated with nesting behavior in virtually every brain area that we examined, including the medial amygdala (anterior and posterior), medial bed nucleus of the stria terminalis, medial preoptic area, medial preoptic nucleus, anterior hypothalamus, ventromedial hypothalamus, periaqueductal gray complex (central gray and nucleus intercollicularis), and ventral tegmental area. Near-significant effects are also obtained in the tuberoinfundibular hypothalamus. Although most correlations are positive, negative correlations are observed for the VIP cell group of the anterior hypothalamus, a population that selectively promotes aggression, and also the periaqueductal gray complex. These data demonstrate a network-wide relationship between peptide production and social behavior that is, to our knowledge, unparalleled by other peptidergic modulators.

Involvement of different mesotocin (oxytocin homologue) populations in sexual and aggressive behaviours of the brown anole.

The oxytocin (OT) family of neuropeptides are known to modulate social behaviours and anxiety in mammals and birds. We investigated cell numbers and neural activity, assessed as Fos induction, within magnocellular and parvocellular populations of neurons producing the OT homologue mesotocin (MT, Ile(8)-oxytocin). This was conducted within the male brown anole lizard, Anolis sagrei, following agonistic or courtship encounters with a conspecific. Both neurons colocalizing and not colocalizing corticotropin-releasing factor (CRF) were examined. Parvocellular neurons of the paraventricular nucleus exhibited a positive correlation between courtship frequency and Fos colocalization, regardless of whether they produce just MT or MT + CRF. Magnocellular populations showed only trends towards positive relationships with courtship and no cell populations showed aggression-related Fos induction. These findings are novel because they demonstrate the involvement of MT neurons in male social behaviour, especially in reptiles for whom the involvement of MT in social behaviour was previously unknown.

The role of the area postrema in the anorectic effects of amylin and salmon calcitonin: behavioral and neuronal phenotyping

Amylin reduces meal size by activating noradrenergic neurons in the area postrema (AP). Neurons in the AP also mediate the eating-inhibitory effects of salmon calcitonin (sCT), a potent amylin agonist, but the phenotypes of the neurons mediating its effect are unknown. Here we investigated whether sCT activates similar neuronal populations to amylin, and if its anorectic properties also depend on AP function. Male rats underwent AP lesion (APX) or sham surgery. Meal patterns were analysed under ad libitum and post-deprivation conditions. The importance of the AP in mediating the anorectic action of sCT was examined in feeding experiments of dose-response effects of sCT in APX vs. sham rats. The effect of sCT to induce Fos expression was compared between surgery groups, and relative to amylin. The phenotype of Fos-expressing neurons in the brainstem was examined by testing for the co-expression of dopamine beta hydroxylase (DBH) or tryptophan hydroxylase (TPH). By measuring the apposition of vesicular glutamate transporter-2 (VGLUT2)-positive boutons, potential glutamatergic input to amylin- and sCT-activated AP neurons was compared. Similar to amylin, an intact AP was necessary for sCT to reduce eating. Further, co-expression between Fos activation and DBH after amylin or sCT did not differ markedly, while co-localization of Fos and TPH was minor. Approximately 95% of neurons expressing Fos and DBH after amylin or sCT treatment were closely apposed to VGLUT2-positive boutons. Our study suggests that the hindbrain pathways engaged by amylin and sCT share many similarities, including the mediation by AP neurons.

Neural activity in catecholaminergic populations following sexual and aggressive interactions in the brown anole, Anolis sagrei

Social behaviors in vertebrates are modulated by catecholamine (CA; dopamine, norepinephrine, epinephrine) release within the social behavior neural network. Few studies have examined activity across CA populations in relation to social behaviors. The involvement of CAs in social behavior regulation is especially underexplored in reptiles, relative to other amniotes. In this study, we mapped CA populations throughout the brain (excluding retina and olfactory bulb) of the male brown anole lizard, Anolis sagrei, via immunofluorescent visualization of the rate-limiting enzyme for CA synthesis, tyrosine hydroxylase (TH). Colocalization of TH with the immediate early gene product Fos, an indirect marker of neural activity, also enabled us to relate activity in TH-immunoreactive (TH-ir) neurons to appetitive and consummatory sexual and aggressive behaviors. We detected most major TH-ir cell populations that are present in other amniotes (within the hypothalamus, midbrain, and hindbrain), although the A15 population was entirely absent. We also detected a few novel or rare cell clusters within the amygdala, medial septum, and inferior raphe. Many CA populations, especially dopaminergic groups, showed increased TH–Fos colocalization in association with appetitive and consummatory sexual behavior expression, while a small number of regions showed increased colocalization in relation to solely consummatory aggression (biting of an opponent). In conclusion, we here map CA populations throughout the brown anole brain and demonstrate evidence for catecholaminergic involvement in appetitive and consummatory sexual behaviors and consummatory aggressive behaviors in this species.

Brain neuronal activation induced by flibanserin treatment in female rats

Flibanserin, a 5-HT1A agonist and 5-HT2A antagonist, is developed for the treatment of hypoactive sexual desire disorder in women, and its efficacy has been evidenced in several clinical studies. Flibanserin prosexual effects have been also evidenced in preclinical animal models. However, the mechanism of action of flibanserin remains not fully understood. The aim of the present study was to examine brain neuronal activation in female rats treated with flibanserin, using single immunocytochemical labeling of Fos protein, a marker of neuronal activation, and co-localization of Fos and catecholaminergic marker. Six groups of female rats received either acute or chronic administrations of vehicle, flibanserin 15mg/kg or flibanserin 45mg/kg. The brains were collected and processed for immunocytochemical labeling. Acute flibanserin increased levels of Fos immunoreactivity in the nucleus accumbens, arcuate hypothalamic nucleus, locus coeruleus, lateral paragigantocellular nucleus, and nucleus of the solitary tract. Chronic 22-day treatment with flibanserin increased Fos expression in the medial preoptic area and arcuate nucleus of the hypothalamus, ventral tegmental area, locus coeruleus, and lateral paragigantocellular nucleus. Both acute and chronic flibanserin increased the density of activated catecholaminergic neurons in the ventral tegmental area but not in the locus coeruleus. Altogether, our results showed that flibanserin, at the dose known to enhance female sexual motivation, preferentially activated the brain regions belonging to the mesolimbic dopaminergic pathway and hypothalamic structures involved in the integration of sexual cues related to sexual motivation.

Influence of ERβ selective agonism during the neonatal period on the sexual differentiation of the rat hypothalamic-pituitary-gonadal (HPG) axis

BackgroundIt is well established that sexual differentiation of the rodent hypothalamic-pituitary-gonadal (HPG) axis is principally orchestrated by estrogen during the perinatal period. Here we sought to better characterize the mechanistic role the beta form of the estrogen receptor (ERβ) plays in this process.MethodsTo achieve this, we exposed neonatal female rats to three doses (0.5, 1 and 2 mg/kg) of the ERβ selective agonist diarylpropionitrile (DPN) using estradiol benzoate (EB) as a positive control. Measures included day of vaginal opening, estrous cycle quality, GnRH and Fos co-localization following ovariectomy and hormone priming, circulating luteinizing hormone (LH) levels and quantification of hypothalamic kisspeptin immunoreactivity. A second set of females was then neonatally exposed to DPN, the ERα agonist propyl-pyrazole-triol (PPT), DPN+PPT, or EB to compare the impact of ERα and ERβ selective agonism on kisspeptin gene expression in pre- and post-pubescent females.ResultsAll three DPN doses significantly advanced the day of vaginal opening and induced premature anestrus. GnRH and Fos co-labeling, a marker of GnRH activation, following ovariectomy and hormone priming was reduced by approximately half at all doses; the magnitude of which was not as large as with EB or what we have previously observed with the ERα agonist PPT. LH levels were also correspondingly lower, compared to control females. No impact of DPN was observed on the density of kisspeptin immunoreactive (-ir) fibers or cell bodies in the arcuate (ARC) nucleus, and kisspeptin-ir was only significantly reduced by the middle (1 mg/kg) DPN dose in the preoptic region. The second experiment revealed that EB, PPT and the combination of DPN+PPT significantly abrogated preoptic Kiss1 expression at both ages but ARC expression was only reduced by EB.ConclusionOur results indicate that selective agonism of ERβ is not sufficient to completely achieve male-typical HPG organization observed with EB or an ERα agonist.

Nitric oxidergic cells related to ejaculation in gerbil forebrain contain androgen receptor and respond to testosterone.

Two clusters of forebrain neurons-one in the posterodorsal preoptic nucleus (PdPN) and one in the lateral part of the posterodorsal medial amygdala (MeApd)-are activated at ejaculation in male rats and gerbils as seen with Fos immunocytochemistry. To understand the functions of these cells and how they respond synchronously, it may be useful to identify their neurotransmitters. Nitric oxide (NO) was of interest because its levels in the preoptic area affect ejaculation, and it could synchronize clustered neurons through paracrine/volume transmission. Thus, we determined whether the ejaculation-related cells produce NO by assessing Fos co-localization with NO synthase (NOS) in recently mated male gerbils. We also studied NOS-Fos co-localization in the medial part of the medial preoptic nucleus (MPNm), where half of the neurons that express Fos after mating reflect ejaculation. We also quantified NOS co-localization with androgen receptor (AR) and NOS sensitivity to androgens at these sites. Without quantification, we extended these analyses throughout the hypothalamus and amygdala. Many mating-activated PdPN, lateral MeApd, and MPNm cells contained NOS (32-54%), and many NOS neurons at these sites expressed Fos (34-51%) or AR (25-69%). PdPN and MPNm NOS cells were sensitive to testosterone but not its androgenic metabolite dihydrotestosterone. The overall distribution of NOS and NOS-AR cells was similar to that in rats. These data suggest that NO may help to synchronize the activation of PdPN and lateral MeApd neurons at ejaculation and that NOS in PdPN and MPNm cells is regulated by testosterone acting via estradiol or without undergoing metabolism.

A forebrain-retrorubral pathway involved in male sex behavior is GABAergic and activated with mating in gerbils

The ventral bed nuclei of the stria terminalis (BST) and medial preoptic nucleus (MPN) of gerbils contain cells that regulate male sex behavior via a largely uncrossed pathway to the retrorubral field (RRF). Our goal was to learn more about cells at the pathway source and target. To determine if the pathway uses GABA as its transmitter, we used immunocytochemistry (ICC) to study glutamic acid decarboxlyase67 (GAD67) colocalization with fluoro-gold (FG) in the ventral BST and MPN after applying FG to the RRF. To determine if the pathway is activated with mating, we studied FG-Fos colocalization in the ventral BST of recently mated males. The ventral BST expresses Fos with mating and is the major pathway source. To determine to what extent other GABAergic cells in the ventral BST are activated with mating, we studied Fos colocalization with GAD67 mRNA visualized by in situ hybridization (ISH). We also looked for GAD67 mRNA in RRF cells. Almost all ventral BST and MPNm cells projecting to the RRF (95–97%) and most ventral BST cells activated with mating (89%), were GABAergic. GABAergic cells were also seen in the RRF. RRF-projecting cells represented 37% of ventral BST cells activated with mating. Their activation may reflect arousal and anticipation of sexual reward. Among ventral BST cells that project to the RRF, 14% were activated with mating, consistent with how much of this pathway is needed for mating. The activated GABAergic cells that do not project to the RRF may release GABA locally and inhibit ejaculation.

Detection of conspecific pheromones elicits fos expression in GABA and calcium-binding cells of the rat vomeronasal system–medial extended amygdala

The olfactory accessory system is specialized in the detection of pheromones, being an afferent to medial extended amygdala. In spite of the fact that numerous phenotypes are found in these structures, in the current literature, there are no detailed descriptions about the phenotype of neurons in the vomeronasal system-medial extended amygdala after their activation by pheromonal stimuli. Using immunohistochemistry for fos and dual immunohistochemistry for fos and phenotypes, here we show that females have a greater number of activated neurons by the pheromonal stimulus. Likewise, a great colocalization of fos with GABA, calretinin, and calbindin was observed in the vomeronasal system-medial extended amygdala. These data suggest that in amygdaloid areas, neuronal excitability is controlled by GABAergic neurons that contain different calcium-binding proteins, indicating the important role of inhibitory control on the incoming sensory pheromonal and olfactory inputs controlled and processed by the vomeronasal system.

Systemic hypertension and hypotension produce a similar distribution of Fos expressing neurons in nucleus tractus solitarii (NTS).

The NTS is a highly integrative region receiving afferent projections from the arterial baroreceptors. Neurons in the NTS are activated by both increasing and decreasing baroreceptor input by changing arterial pressure (AP). We have begun testing the hypothesis that different populations of NTS neurons are activated by hypertension vs. hypotension. The retrograde tracer Fluoro-Gold (FG, 2%, 15–30 nL) was microinjected unilaterally into the caudal ventrolateral medulla (CVLM) of male rats to label NTS output neurons, and arterial and venous catheters were implanted. Five days later, conscious rats were subjected to sustained (90 min) increases (phenylephrine, PE, +44 ± 4.4 mmHg) or decreases (diazoxide, DZ, −52±6.5 mmHg) in AP. Rats were then transcardially perfused and 30 μm medullary sections throughout the NTS were stained for Fos, to evaluate neuronal activation, and GAD67 to identify GABA synthesizing neurons. The total number of Fos expressing neurons was similar for both PE (415 ± 16) and DZ (502 ± 21) treated rats throughout the rostral-caudal extent of the NTS, with the greatest expression at the level of the area postrema (PE, 45%; DZ, 52%). PE and DZ also resulted in colocalization of Fos+GAD67 and Fos+FG positive cells in NTS. These findings suggest that despite differences in baroreceptor input the rostral-caudal pattern of Fos expression in the NTS was similar. In addition, both GABAergic and CVLM-projecting neurons are activated in response to hypertension and hypotension. Support by HL54669 (EMH) and HL059167 (JTP)

Role of Amygdalo-Nigral Circuitry in Conditioning of a Visual Stimulus Paired with Food

The amygdala central nucleus (CeA) plays an important part in associative learning. Although most research has focused on functions of its descending projections to brainstem areas involved in autonomic and somatomotor responses, the ascending projections of CeA also play critical roles in learning. For example, a CeA-nigrostriatal pathway is important for acquiring orienting responses (ORs) to conditioned stimuli (CSs) that signal food delivery. In this study, the function of this CeA-nigrostriatal pathway in appetitive conditioning of rats was considered in more detail. In experiment 1, we combined anatomical tracing and methods for detecting neuronal activation to examine whether CeA neurons that project to the substantia nigra pars compacta (SNc) are activated by a visual CS for food. After injection of the retrograde tracer Fluoro-Gold (FG) into SNc, the rats received pairings of a visual CS with food. After a test with the CS alone, the brains were prepared to assess FG labeling and CS-induced Fos expression in CeA with immunohistochemical procedures. Colocalization of Fos and FG in CeA neurons was visualized with confocal-fluorescence microscopy. The CS induced Fos expression in CeA, and a majority of these Fos-positive neurons were also FG positive, indicating activation of the CeA-SNc pathway by the CS. In experiment 2, lesions that disconnected CeA and SNc prevented the acquisition of conditioned ORs but did not affect the acquisition of conditioned food-related responses or the display of unconditioned ORs. These experiments demonstrate a role for amygdalo-nigral circuitry in learned modulation of attention to signals for biologically significant events.

GABA and glutamate in mating-activated cells in the preoptic area and medial amygdala of male gerbils.

The posterodorsal medial amygdala (MeApd), the posterodorsal preoptic nucleus (PdPN), and the medial cell group of the sexually dimorphic preoptic area (mSDA) contain cells that are activated specifically at ejaculation as assessed by Fos expression. The mSDA also expresses Fos early in the mating context. Because little is known about the neurotransmitters of these activated cells, the possibility that they use gamma-aminobutyric acid (GABA) or glutamate was assessed. Putative glutamatergic cells were visualized with immunocytochemistry (ICC) for glutamate and its neuron-specific transporter. Their distributions were compared with those of GABAergic cells visualized with ICC for the 67-kDa form of glutamic acid decarboxylase (GAD(67)) and in situ hybridization for GAD(67) messenger RNA (mRNA). Colocalization of Fos and GAD(67) mRNA in recently mated males indicated that half of the activated cells in the PdPN, mSDA, and lateral MeApd are GABAergic. Colocalization of Fos and glutamate suggested that a quarter of the activated mSDA and lateral MeApd cells are glutamatergic. The PdPN does not appear to have glutamatergic cells. In the lateral MeApd, the percentage of activated cells that are GABAergic (45%) matches the percentage that project to the principal part of the bed nucleus of the stria terminalis (BST; 43%), and the percentage likely to be glutamatergic (27%) matches the percentage projecting to the mSDA (27%). The latter could help to trigger ejaculation. The distribution of GABAergic and putative glutamatergic cells in the caudal preoptic area, caudal BST, and medial amygdala of male gerbils is also described.

Differential expression of c-fos in subtypes of GABAergic cells following sensory stimulation in the cat primary visual cortex.

Recent immunocytochemical stainings on cat visual cortex, visually stimulated for 1 h, showed a strong induction of Fos expression in cortical neurons. We initiated immunocytochemical double staining experiments with different cytochemical markers to investigate the neurochemical and morphological character of these activated neurons showing Fos induction after sensory stimulation. Double staining with Fos and glutamic acid decarboxylase (GAD) demonstrated the presence of Fos in the nuclei of GABAergic neurons of the primary visual cortex. To further subdivide this Fos/GABAergic cell population we investigated whether Fos colocalized with parvalbumin, calbindin or calretinin. Colocalization of Fos with these calcium-binding proteins delineated distinct neuronal subclasses of Fos-immunoreactive neurons in supra- and infragranular layers of cat area 17. Quantitative analysis of the proportion of immunoreactive local circuit neurons revealed that 35% of the GABAergic neurons showed Fos induction in supragranular layers, whereas in infragranular layers a mere 10% of the GABAergic cells revealed Fos expression. Fos coexisted in about 24% of the calbindin-immunopositive cells within supra- and infragranular layers, but only a minority of the parvalbumin and the calretinin neuronal subgroups were immunopositive for Fos in the corresponding layers of area 17. These findings suggest that visual stimulation induces Fos expression in distinct subsets of inhibitory neurons in cat primary visual cortex.