GABAergic neurons of anterior thalamic reticular nucleus regulate states of consciousness in propofol- and isoflurane-mediated general anesthesia.

BACKGROUND: The thalamus system plays critical roles in the regulation of reversible unconsciousness induced by general anesthetics, especially the arousal stage of general anesthesia (GA). But the function of thalamus in GA-induced loss of consciousness (LOC) is little known. The thalamic reticular nucleus (TRN) is the only GABAergic neurons-composed nucleus in the thalamus, which is composed of parvalbumin (PV) and somatostatin (SST)-expressing GABAergic neurons. The anterior sector of TRN (aTRN) is indicated to participate in the induction of anesthesia, but the roles remain unclear. This study aimed to reveal the role of the aTRN in propofol and isoflurane anesthesia. METHODS: We first set up c-Fos straining to monitor the activity variation of aTRNPV and aTRNSST neurons during propofol and isoflurane anesthesia. Subsequently, optogenetic tools were utilized to activate aTRNPV and aTRNSST neurons to elucidate the roles of aTRNPV and aTRNSST neurons in propofol and isoflurane anesthesia. Electroencephalogram (EEG) recordings and behavioral tests were recorded and analyzed. Lastly, chemogenetic activation of the aTRNPV neurons was applied to confirm the function of the aTRN neurons in propofol and isoflurane anesthesia. RESULTS: c-Fos straining showed that both aTRNPV and aTRNSST neurons are activated during the LOC period of propofol and isoflurane anesthesia. Optogenetic activation of aTRNPV and aTRNSST neurons promoted isoflurane induction and delayed the recovery of consciousness (ROC) after propofol and isoflurane anesthesia, meanwhile chemogenetic activation of the aTRNPV neurons displayed the similar effects. Moreover, optogenetic and chemogenetic activation of the aTRN neurons resulted in the accumulated burst suppression ratio (BSR) during propofol and isoflurane GA, although they represented different effects on the power distribution of EEG frequency. CONCLUSION: Our findings reveal that the aTRN GABAergic neurons play a critical role in promoting the induction of propofol- and isoflurane-mediated GA.

Anterior thalamic NMDA-induced damage impairs extrapolation relying on serial stimulus patterns, in rats.

Extrapolation of serial stimulus patterns seems to depend upon identification and application of patterns relating sequences of stimuli stored in memory, thus allowing prediction of pending events never experienced before. There have been proposals that such a "generator of predictions system" would include the subiculum, mammillary bodies, anteroventral thalamus and cingulate cortex (e.g., Gray, 1982). The anteroventral thalamus (AVT) seems to be in a strategic position, both hodologically and experimentally, to allow testing of this hypothesis. This study investigated the effect of NMDA-induced damage to the anteroventral thalamus [part of the anterodorsal (AD) thalamus was also damaged in some animals], following stereotaxic minute topic microinjections, on the ability of male Wistar rats to extrapolate relying on serial stimulus patterns. Corresponding sham-operated controls received phosphate-saline buffer microinjections at the same stereotaxic coordinates. The subjects were trained to run through a straight alleyway along 31 sessions, one session per day, to get rewarded. Each session included four successive trials. Subjects exposed to the monotonic serial pattern received 14, 7, 3, 1 sunflower seeds along trials. Subjects exposed to the non-monotonic serial pattern received 14, 3, 7, 1 sunflower seeds. On the 32nd testing session, a fifth trial, never experienced before, was included immediately after the fourth trial. Sham-operated control subjects exposed to the monotonic serial pattern were expected to exhibit longer running times, since the content of their prediction in the fifth trial should be "less than 1 sunflower seeds". In contrast, control subjects exposed to the non-monotonic serial pattern were expected to exhibit shorter running times, since the content of their prediction would be "more than 1 sunflower seeds". Confirming these predictions, control subjects exposed to the monotonic serial pattern exhibited longer running times as compared to both, their own running times in previous trials within the same session and control subjects exposed to the non-monotonic schedule, thus indicating the occurrence of extrapolation. In contrast, AVT/AD lesioned subjects exposed to the monotonic schedule did not exhibit this increase in running times on the fifth trial, indicating lack of extrapolation. These results indicate that extrapolation relying on serial stimulus patterns is disrupted following extensive NMDA-induced damage to AVT and part of the AD. This represents the first consistent demonstration that the anterior thalamic nuclei are required for extrapolation of serial stimulus patterns and generation of predictions.

Chemogenetics Reveal an Anterior Cingulate-Thalamic Pathway for Attending to Task-Relevant Information.

In a changing environment, organisms need to decide when to select items that resemble previously rewarded stimuli and when it is best to switch to other stimulus types. Here, we used chemogenetic techniques to provide causal evidence that activity in the rodent anterior cingulate cortex and its efferents to the anterior thalamic nuclei modulate the ability to attend to reliable predictors of important outcomes. Rats completed an attentional set-shifting paradigm that first measures the ability to master serial discriminations involving a constant stimulus dimension that reliably predicts reinforcement (intradimensional-shift), followed by the ability to shift attention to a previously irrelevant class of stimuli when reinforcement contingencies change (extradimensional-shift). Chemogenetic disruption of the anterior cingulate cortex (Experiment 1) as well as selective disruption of anterior cingulate efferents to the anterior thalamic nuclei (Experiment 2) impaired intradimensional learning but facilitated 2 sets of extradimensional-shifts. This pattern of results signals the loss of a corticothalamic system for cognitive control that preferentially processes stimuli resembling those previously associated with reward. Previous studies highlight a separate medial prefrontal system that promotes the converse pattern, that is, switching to hitherto inconsistent predictors of reward when contingencies change. Competition between these 2 systems regulates cognitive flexibility and choice.

The Role of Hippocampal Neurogenesis in ANT-DBS for LiCl-Pilocarpine-Induced Epileptic Rats.

PURPOSE: Abnormal neurogenesis in the hippocampus after status epilepticus (SE) has been suggested as a key pathogeny of temporal lobe epilepsy. This study aimed to investigate the effect of deep brain stimulation of the anterior thalamic nucleus (ANT-DBS) on hippocampal neurogenesis in LiCl-pilocarpine-induced epileptic rats and to analyze its relationship with postoperative spontaneous recurrent seizures (SRS) and anxiety. METHOD: SE was induced by a systemic LiCl-pilocarpine injection in adult male rats. Rats in the DBS group underwent ANT-DBS immediately after successful SE induction. SRS was only recorded during the chronic stage. An elevated plus maze was used to evaluate the level of anxiety in rats 7, 28, and 60 days after SE onset. After the elevated plus-maze experiment, rats were sacrificed under anesthesia in order to evaluate hippocampal neurogenesis. Doublecortin (DCX) was used as a marker for neurogenesis. RESULTS: During the chronic stage, SRS in rats in the DBS group were significantly decreased. The level of anxiety was increased significantly in rats in the DBS group 28 days after SE, while no significant differences in anxiety levels were found 7 and 60 days after SE. The number of DCX-positive cells in the hippocampus was significantly increased 7 days after SE and was significantly decreased 60 days after SE in all rats in which SE was induced. However, the number of DCX-positive cells in the DBS group was significantly lower than that in the other groups 28 days after SE. CONCLUSIONS: ANT-DBS may suppress SRS and increase the postoperative anxiety of epileptic rats by influencing hippocampal neurogenesis.

Dissociable contributions of mediodorsal and anterior thalamic nuclei in visual attentional performance: A comparison using nicotinic and muscarinic cholinergic receptor antagonists.

BACKGROUND: Thalamic subregions mediate various cognitive functions, including attention, inhibitory response control and decision making. Such neuronal activity is modulated by cholinergic thalamic afferents and deterioration of such modulatory signaling has been theorised to contribute to cognitive decline in neurodegenerative disorders. However, the thalamic subnuclei and cholinergic receptors involved in cognitive functioning remain largely unknown. AIMS: We investigated whether muscarinic or nicotinic receptors in the mediodorsal thalamus and anterior thalamus contribute to rats' performance in the five-choice serial reaction time task, which measures sustained visual attention and impulsive action. METHODS: Male Long-Evans rats were trained in the five-choice serial reaction time task then surgically implanted with guide cannulae targeting either the mediodorsal thalamus or anterior thalamus. Reversible inactivation of either the mediodorsal thalamus or anterior thalamus were achieved with infusions of the γ-aminobutyric acid-ergic agonists muscimol and baclofen prior to behavioural assessment. To investigate cholinergic mechanisms, we also assessed the behavioural effects of locally administered nicotinic (mecamylamine) and muscarinic (scopolamine) receptor antagonists. RESULTS: Reversible inactivation of the mediodorsal thalamus severely impaired discriminative accuracy and response speed and increased omissions. Inactivation of the anterior thalamus produced less profound effects, with impaired accuracy at the highest dose. In contrast, blocking cholinergic transmission in these regions did not significantly affect five-choice serial reaction time task performance. CONCLUSIONS/INTERPRETATIONS: These findings show the mediodorsal thalamus plays a key role in visuospatial attentional performance that is independent of local cholinergic neurotransmission.

Neuro-behavioral effects after systemic administration of MK-801 and disinhibition of the anterior thalamic nucleus in rats: Potential relevance in schizophrenia.

Non-competitive N-methyl-d-aspartate receptor (NMDA-R) antagonists have been suggested to evoke psychotomimetic-like behaviors by selectively targeting GABAergic elements in cortical and thalamic circuits. In previous studies, we had reported the involvement of the reticular and anterior thalamic nuclei (ATN) in the MK-801-evoked hyperactivity and other motor alterations. Consistent with the possibility that these responses were mediated by thalamic disinhibition, we examined the participation of cortical and hippocampal areas innervated by ATN in the responses elicited by the systemic administration of MK-801 (0.2 mg/kg) and compared them to the effects produced by the microinjection of a subconvulsive dose of bicuculline (GABAA receptor antagonist) in the ATN. We used the expression of Fos related antigen 2 (Fra-2) as a neuronal activity marker in the ATN and its projection areas such as hippocampus (HPC), retrosplenial cortex (RS), entorhinal cortex (EC) and medial prefrontal cortex (mPFC). Dorsal (caudate-putamen, CPu) and ventral striatum (nucleus accumbens, core and shell, NAc,co and NAc,sh) were also studied. Behavioral and brain activation results suggest a partial overlap after the effect of MK-801 administration and ATN disinhibition. MK-801 and ATN disinhibition increases locomotor activity and disorganized movements, while ATN disinhibition also reduces rearing behavior. A significant increase in Fra-2 immunoreactivity (Fra-2-IR) in the ATN, mPFC (prelimbic area, PrL) and NAc,sh was observed after MK-801, while a different pattern of Fra-2-IR was detected following ATN disinhibition (e.g., increase in DG and NAc,sh, and decrease in PrL cortex). Overall, our data may contribute to the understanding of dysfunctional neural circuits involved in schizophrenia.

Anterior thalamic nuclei, but not retrosplenial cortex, lesions abolish latent inhibition in rats.

The present study examined the effects of excitotoxic lesions in 2 closely related structures, the anterior thalamic nuclei and the retrosplenial cortex, on latent inhibition. Latent inhibition occurs when nonreinforced preexposure to a stimulus retards the subsequent acquisition of conditioned responding to that stimulus. Latent inhibition was assessed in a within-subject procedure with auditory stimuli and food reinforcement. As expected, sham-operated animals were slower to acquire conditioned responding to a stimulus that had previously been experienced without consequence, relative to a non-preexposed stimulus. This latent inhibition effect was absent in rats with excitotoxic lesions in the anterior thalamic nuclei, as these animals conditioned to both stimuli at equivalent rates. The retrosplenial lesions appeared to spare latent inhibition, as these animals displayed a robust stimulus preexposure effect. The demonstration here that anterior thalamic nuclei lesions abolish latent inhibition is consistent with emerging evidence of the importance of these thalamic nuclei for attentional control. (PsycINFO Database Record (c) 2018 APA, all rights reserved).

Differential role of the anterior and intralaminar/lateral thalamic nuclei in systems consolidation and reconsolidation.

The anterior thalamic nuclei (ATN) and the intralaminar/lateral thalamic nuclei (ILN/LT) play different roles in memory processes. The ATN are believed to be part of an extended hippocampal system, and the ILN/LT have strong connections with the medial prefrontal cortex. It was shown that the ILN/LT are involved in systems consolidation. However, whether they are necessary for memory retrieval as well remains unclear. We, therefore, used c-Fos immunohistochemistry and reversible inactivations to investigate the role of the ATN and ILN/LT in recent and remote contextual fear memory retrieval in rats. The results confirm a differential role of the ATN and ILN/LT in systems consolidation, showing the involvement of the ATN in recent but not remote memory retrieval. This study also pinpoints which specific nuclei are involved in retrieval: the anterodorsal nucleus for recent memories, and the lateral mediodorsal nucleus for remote memories. Lastly, we also show that the ATN are not involved in reconsolidation. Together, the results suggest that these nuclei provide critical feedback for successful memory retrieval and systems consolidation.

The effect of pharmacological inactivation of the mammillary body and anterior thalamic nuclei on hippocampal theta rhythm in urethane-anesthetized rats.

The mammillary body (MB) and the anterior thalamic nuclei (ATN) are closely related structures, which take part in learning and memory processes. However, the exact role of these structures has remained unclear. In both structures neurons firing according to hippocampal theta rhythm have been found, mainly in the medial mammillary nucleus (MM) and anteroventral thalamic nucleus (AV). These neurons are driven by descending projections from the hippocampal formation and are thought to convey theta rhythm back to the hippocampus (HP). We argue that the MB-ATN axis not only relays theta signal, but may also modulate it. To examine it, we performed a pharmacological inactivation of the MM and AV by local infusion of procaine, and measured changes in theta activity in selected structures of the extended hippocampal system in urethane-anesthetized rats. The inactivation of the MM resulted in decrease in EEG power in the HP and AV, the most evidently in the lower theta frequency bands, i.e. 3-5Hz in the HP (down to 9.2% in 3- to 4-Hz band and 37.6% in 4- to 5-Hz band, in comparison to the power in the control conditions) and 3-4Hz in the AV (down to 24.9%). After the AV inactivation, hippocampal EEG power decreased in theta frequency bands of 3-8Hz (down to 61.6% in 6- to 7-Hz band and 69.4% in 7- to 8-Hz band). Our results suggest that the role of the MB-ATN axis in regulating theta rhythm signaling may be much more important than has been speculated so far.

A role for the anteromedial thalamic nucleus in the acquisition of contextual fear memory to predatory threats.

Previous studies from our group have shown that cytotoxic lesions in the ventral portion of the anteromedial thalamic nucleus (AMv), one of the main targets of the hypothalamic predator-responsive circuit, strongly impairs contextual fear responses to an environment previously associated with a predator. The AMv is in a position to convey information to cortico-hippocampal-amygdalar circuits involved in the processing of fear memory. However, it remains to be determined whether the nucleus is involved in the acquisition or subsequent expression of contextual fear. In the present investigation, we addressed this question by inactivating the rat AMv with muscimol either prior to cat exposure or prior to exposure to the cat-related context. Accordingly, AMv pharmacological inactivation prior to cat exposure did not interfere with innate fear responses, but it drastically reduced contextual conditioning to the predator-associated environment. On the other hand, AMv inactivation prior to exposure to the environment associated with the predator threat did not affect contextual fear responses. The behavioral results were further supported by the demonstration that AMv inactivation prior to cat exposure also blocked the activation of sites critically involved in the expression of anti-predatory contextual defensive responses (i.e., the dorsal premammillary nucleus and the dorsolateral periaqueductal gray) in animals exposed to the predator-associated context. The AMv projections were also examined, and the results of this investigation outline important paths that can influence hippocampal circuitry and raise new ideas for anterior thalamic-hippocampal paths involved in emotional learning.

Impaired spatial working memory after anterior thalamic lesions: recovery with cerebrolysin and enrichment.

Lesions to the anterior thalamic nuclei (ATN) in rats produce robust spatial memory deficits that reflect their influence as part of an extended hippocampal system. Recovery of spatial working memory after ATN lesions was examined using a 30-day administration of the neurotrophin cerebrolysin and/or an enriched housing environment. As expected, ATN lesions in standard-housed rats given saline produced severely impaired reinforced spatial alternation when compared to standard-housed rats with sham lesions. Both cerebrolysin and enrichment substantially improved this working memory deficit, including accuracy on trials that required attention to distal cues for successful performance. The combination of cerebrolysin and enrichment was more effective than either treatment alone when the delay between successive runs in a trial was increased to 40 s. Compared to the intact rats, ATN lesions in standard-housed groups produced substantial reduction in c-Fos expression in the retrosplenial cortex, which remained low after cerebrolysin and enrichment treatments. Evidence that multiple treatment strategies restore some memory functions in the current lesion model reinforces the prospect for treatments in human diencephalic amnesia.

Mediodorsal but not anterior thalamic nuclei lesions impair acquisition of a conditional discrimination task.

The limbic thalamus is a heterogeneous structure with distinctive cortical connectivity. A recent review suggests that the mediodorsal thalamic nucleus (MD), unlike the anterior thalamic nuclei (ATN), may be involved in selecting relevant information in tasks relying on executive functions. We compared the effects of excitotoxic lesions of the MD or the ATN on the acquisition of a simple conditional discrimination in rats. When required to choose from two levers according to auditory or visual cues, ATN rats and sham-lesioned rats performed to the same levels and displayed similar acquisition curves. Under the same conditions, MD rats' acquisition of the task was markedly delayed. This group nevertheless attained nearly normal performances after more extensive training. Furthermore, all rats learned reversal of the original discrimination at the same rate. These results highlight functional specialization within the limbic thalamus and support the notion that MD contributes to the identification of relevant dimensions in conditional tasks during the initial stages of acquisition.

NMDA blockade inhibits experience-dependent modification of anterior thalamic head direction cells.

Head Direction (HD) cells of the rodent Papez circuit are thought to reflect the spatial orientation of the animal. Because NMDA transmission is important for spatial behavior, we sought to determine the effects of NMDA blockade on the basic directional signal carried by HD cells and on experience-dependent modification of this system. In Experiment 1, HD cells were recorded from the anterior dorsal thalamus in female Long-Evans rats while they foraged in a familiar enclosure following administration of the NMDA antagonist CPP or saline. While the drug produced a significant decrease in peak firing rates, it failed to affect the overall directional specificity and landmark control of HD cells. Experiment 2 took place over 2 days and assessed whether the NMDA antagonist would interfere with the stabilization of the HD network in a novel environment. On Day 1 the animal was administered CPP or saline and placed in a novel enclosure to allow the stabilization of the HD signal relative to the new environmental landmarks. On Day 2 the animal was returned to the formerly novel enclosure to determine if the enclosure specific direction-dependent activity established on Day 1 was maintained. In contrast to HD cells from control animals, cells from animals receiving CPP during the initial exposure to the novel enclosure did not maintain the same direction-dependent activity relative to the enclosure in the subsequent drug-free exposure. These findings demonstrate that plasticity in the HD system is dependent on NMDA transmission similar to many other forms of spatial learning.

Anterior thalamic nuclei lesions and recovery of function: Relevance to cognitive thalamus.

Injury to the anterior thalamic nuclei (ATN) and their neural connections is the most consistent neuropathology associated with diencephalic amnesia. ATN lesions in rats produce memory impairments that support a key role for this region within an extended hippocampal system of complex overlapping neural connections. Environmental enrichment is a therapeutic tool that produces substantial, although incomplete, recovery of memory function after ATN lesions, even after the lesion-induced deficit has become established. Similarly, the neurotrophic agent cerebrolysin, also counters the negative effects of ATN lesions. ATN lesions substantially reduce c-Fos expression and spine density in the retrosplenial cortex, and reduce spine density on CA1 neurons; only the latter is reversed by enrichment. We discuss the implications of this evidence for the cognitive thalamus, with a proposal that there are genuine interactions among different but allied thalamo-cortical systems that go beyond a simple summation of their separate effects.

Spatial navigation. Disruption of the head direction cell network impairs the parahippocampal grid cell signal.

Navigation depends on multiple neural systems that encode the moment-to-moment changes in an animal's direction and location in space. These include head direction (HD) cells representing the orientation of the head and grid cells that fire at multiple locations, forming a repeating hexagonal grid pattern. Computational models hypothesize that generation of the grid cell signal relies upon HD information that ascends to the hippocampal network via the anterior thalamic nuclei (ATN). We inactivated or lesioned the ATN and subsequently recorded single units in the entorhinal cortex and parasubiculum. ATN manipulation significantly disrupted grid and HD cell characteristics while sparing theta rhythmicity in these regions. These results indicate that the HD signal via the ATN is necessary for the generation and function of grid cell activity.

The effects of lesions to the postsubiculum or the anterior dorsal nucleus of the thalamus on spatial learning in rats.

To investigate the role of the head direction (HD) cell circuit in spatial navigation, rats with bilateral, neurotoxic lesions to the postsubiculum (PoS; Experiment 1) or the anterior dorsal nucleus of the thalamus (ADN; Experiment 2) were compared to sham controls on 2 tasks that could be solved using directional heading. Rats were first trained on a direction problem in a water T maze where they learned to travel either east or west from 2 locations in the experimental room. ADN lesioned rats were impaired relative to sham controls on the first block of 8 trials, but not on the total trials taken to reach criterion. This transient deficit was not observed in rats with lesions to the PoS. In the food-foraging task, rats were trained to leave a home cage at the periphery of a circular table, find food in the center of the table, and return to the home cage. Both PoS and ADN lesioned rats showed impairments on this task relative to sham rats, making more errors on the return component of the foraging trip. The spatial deficits produced by lesions to the PoS and the ADN, downstream structures in the HD cell circuit, are not as severe as those observed in earlier studies in rats with lesions to the dorsal tegmental nucleus.

High-frequency stimulation of anterior nucleus thalamus improves impaired cognitive function induced by intra-hippocampal injection of Aß1-40 in rats.

BACKGROUND: The advent of brain stimulation techniques to treat movement disorders and psychiatric diseases has shown potential to decode the neural mechanism that underlies the cognitive process by modulating the interrupted circuit. Here, the present investigation aimed at evaluating the influence of deep brain stimulation of the anterior nucleus thalamus (ANT-DBS) on memory. METHODS: Thirty-two rats were randomized into phosphate buffer saline (PBS) group (n = 8, rats received PBS injections without implantation of electrodes into the ANT), Alzheimer's dementia (AD) group (n = 8, rats received Aß1-40 injections without implantation of electrodes into the ANT), ANT sham stimulation group (n = 8, rats received Aß1-40 injections with implantation of electrodes into the ANT but without stimulation) and ANT stimulation group (n = 8, rats received Aß1-40 injections with implantation of electrodes into the ANT and stimulation). A Morris maze test was used for determining the effect of electrical stimulation on cognitive function in rats. The data were assessed statistically with one-way analysis of variance (ANOVA) followed by Tukey's tests for multiple post hoc comparisons. RESULTS: The data showed that in the training test, PBS group and AD group managed to learn the hidden-platform faster and faster while AD group needed a significantly longer time to reach the platform than PBS group (P < 0.05). Meanwhile, ANT stimulation group demonstrated a significantly shorter time to reach the platform (P < 0.05) compared to the AD group, while there was no significant difference between the ANT sham stimulation group and the AD group (P > 0.05). On the probe test, the AD group spent less time ((10.15 ± 2.34) seconds) in the target quadrant than the PBS group ((28.20 ± 2.75) seconds) (P < 0.05). And the times of platform-traversing of the AD group (3.35 ± 1.12) significantly decreased compared with the PBS group (8.69 ± 2.87) (P < 0.05). However, the times of platform-traversing and the time spent in the target quadrant of the ANT stimulation group significantly increased compared to the AD group (P < 0.05), while times of platform-traversing or the time spent in the target quadrant was not significantly different between the ANT sham stimulation group and the AD group (P > 0.05). CONCLUSION: Bilateral high-frequency stimulation of the ANT may be useful as a potential therapeutic modality for cognitive dysfunction in AD.

Differential control of sex differences in estrogen receptor α in the bed nucleus of the stria terminalis and anteroventral periventricular nucleus.

The principal nucleus of the bed nucleus of the stria terminalis (BNSTp) and anteroventral periventricular nucleus of the hypothalamus (AVPV) are sexually dimorphic, hormone-sensitive forebrain regions. Here we report a profound sex difference in estrogen receptor-α (ERα) immunoreactivity (IR) in the BNSTp, with robust ERα IR in females and the near absence of labeling in males. This sex difference is due to the suppression of ERα IR by testicular hormones in adulthood: it was not present at birth and was not altered by neonatal treatment of females with estradiol; gonadectomy of adult males increased ERα IR to that of females, whereas gonadectomy of adult females had no effect. Treating gonadally intact males with an aromatase inhibitor partially feminized ERα IR in the BNSTp, suggesting that testicular suppression required aromatization. By contrast, in AVPV we found a modest sex difference in ERα IR that was relatively insensitive to steroid manipulations in adulthood. ERα IR in AVPV was, however, masculinized in females treated with estradiol at birth, suggesting that the sex difference is due to organizational effects of estrogens. The difference in ERα IR in the BNSTp of males and females appears to be at least in part due to greater expression of mRNA of the ERα gene (Esr1) in females. The sex difference in message is smaller than the difference in immunoreactivity, however, suggesting that posttranscriptional mechanisms also contribute to the pronounced suppression of ERα IR and presumably to functions mediated by ERα in the male BNSTp.

Developmental GnRH signaling is not required for sexual differentiation of kisspeptin neurons but is needed for maximal Kiss1 gene expression in adult females.

Kisspeptin, encoded by Kiss1, stimulates reproduction. In rodents, one Kiss1 population resides in the hypothalamic anterior ventral periventricular nucleus and neighboring rostral periventricular nucleus (AVPV/PeN). AVPV/PeN Kiss1 neurons are sexually dimorphic (greater in females), yet the mechanisms regulating their development and sexual differentiation remain poorly understood. Neonatal estradiol (E2) normally defeminizes AVPV/PeN kisspeptin neurons, but emerging evidence suggests that developmental E2 may also influence feminization of kisspeptin, although exactly when in development this process occurs is unknown. In addition, the obligatory role of GnRH signaling in governing sexual differentiation of Kiss1 or other sexually dimorphic traits remains untested. Here, we assessed whether AVPV/PeN Kiss1 expression is permanently impaired in adult hpg (no GnRH or E2) or C57BL6 mice under different E2 removal or replacement paradigms. We determined that 1) despite lacking GnRH signaling in development, marked sexual differentiation of Kiss1 still occurs in hpg mice; 2) adult hpg females, who lack lifetime GnRH and E2 exposure, have reduced AVPV/PeN Kiss1 expression compared to wild-type females, even after chronic adulthood E2 treatment; 3) E2 exposure to hpg females during the pubertal period does not rescue their submaximal adult Kiss1 levels; and 4) in C57BL6 females, removal of ovarian E2 before the pubertal or juvenile periods does not impair feminization and maximal adult AVPV/PeN Kiss1 expression nor the ability to generate LH surges, indicating that puberty is not a critical period for Kiss1 development. Thus, sexual differentiation still occurs without GnRH, but GnRH or downstream E2 signaling is needed sometime before juvenile development for complete feminization and maximal Kiss1 expression in adult females.

Comparative analysis of kisspeptin-immunoreactivity reveals genuine differences in the hypothalamic Kiss1 systems between rats and mice.

Kiss1 mRNA and its corresponding peptide products, kisspeptins, are expressed in two restricted brain areas of rodents, the anteroventral periventricular nucleus (AVPV) and the arcuate nucleus (ARC). The concentration of mature kisspeptins may not directly correlate with Kiss1 mRNA levels, because mRNA translation and/or posttranslational modification, degradation, transportation and release of kisspeptins could be regulated independently of gene expression, and there may thus be differences in kisspeptin expression even in species with similar Kiss1 mRNA profiles. We measured and compared kisspeptin-immunoreactivity in both nuclei and both sexes of rats and mice and quantified kisspeptin-immunoreactive nerve fibers. We also determined Kiss1 mRNA levels and measured kisspeptin-immunoreactivity in colchicine pretreated rats. Overall, we find higher levels of kisspeptin-immunoreactivity in the mouse compared to the rat, independently of brain region and gender. In the female mouse AVPV high numbers of kisspeptin-immunoreactive neurons were present, while in the rat, the female AVPV displays a similar number of kisspeptin-immunoreactive neurons compared to the level of Kiss1 mRNA expressing cells, only after axonal transport inhibition. Interestingly, the density of kisspeptin innervation in the anterior periventricular area was higher in female compared to male in both species. Species differences in the ARC were evident, with the mouse ARC containing dense fibers, while the rat ARC contains clearly discernable cells. In addition, we show a marked sex difference in the ARC, with higher kisspeptin levels in females. These findings show that the translation of Kiss1 mRNA and/or the degradation/transportation/release of kisspeptins are different in mice and rats.