Multiple areas within the reticular activating system (RAS) can hasten awakening from sleep or light planes of anesthesia. However, stimulation in individual sites has shown limited recovery from deep global suppression of brain activity, such as coma. Here we identify a subset of RAS neurons within the anterior portion of nucleus gigantocellularis (aNGC) capable of producing a high degree of awakening represented by a broad high frequency cortical reactivation associated with organized movements and behavioral reactivity to the environment from two different models of deep pharmacologically-induced coma (PIC): isoflurane (1.25%-1.5%) and induced hypoglycemic coma. Activating aNGC neurons triggered awakening by recruiting cholinergic, noradrenergic, and glutamatergic arousal pathways. In summary, we identify an evolutionarily conserved population of RAS neurons, which broadly restore cerebral cortical activation and motor behavior in rodents through the coordinated activation of multiple arousal-promoting circuits.

The endocannabinoid (eCB) system regulates emotion, stress, memory and cognition through the cannabinoid type 1 (CB1) receptor. To test the role of CB1 signaling in social anxiety and memory, we utilized a genetic knockout (KO) and a pharmacological approach. Specifically, we assessed the effects of a constitutive KO of CB1 receptors (CB1KOs) and systemic administration of a CB1 antagonist (AM251; 5mg/kg) on social anxiety in a social investigation paradigm and social memory in a social discrimination test. Results showed that when compared with wild-type (WT) and vehicle-treated animals, CB1KOs and WT animals that received an acute dose of AM251 displayed anxiety-like behaviors toward a novel male conspecific. When compared with WT animals, KOs showed both active and passive defensive coping behaviors, i.e. elevated avoidance, freezing and risk-assessment behaviors, all consistent with an anxiety-like profile. Animals that received acute doses of AM251 also showed an anxiety-like profile when compared with vehicle-treated animals, yet did not show an active coping strategy, i.e. changes in risk-assessment behaviors. In the social discrimination test, CB1KOs and animals that received the CB1 antagonist showed enhanced levels of social memory relative to their respective controls. These results clearly implicate CB1 receptors in the regulation of social anxiety, memory and arousal. The elevated arousal/anxiety resulting from either total CB1 deletion or an acute CB1 blockade may promote enhanced social discrimination/memory. These findings may emphasize the role of the eCB system in anxiety and memory to affect social behavior.

Expression of the primary female sex behaviour, lordosis, in laboratory animals depends on oestrogen‐induced expression of progesterone receptor (PgR) within a defined cell group in the ventrolateral portion of the ventromedial nucleus of the hypothalamus ( VMH ). The minimal latency from oestradiol administration to lordosis is 18 h. During that time, ligand‐bound oestrogen receptors ( ER ), members of a nuclear receptor superfamily, recruit transcriptional coregulators, which induce covalent modifications of histone proteins, thus leading to transcriptional activation or repression of target genes. The present study aimed to investigate the early molecular epigenetic events underlying oestrogen‐regulated transcriptional activation of the Pgr gene in the VMH of female mice. Oestradiol (E 2 ) administration induced rapid and transient global histone modifications in the VMH of ovariectomised female mice. Histone H3 N‐terminus phosphorylation (H3S10phK14Ac), acetylation (H3Ac) and methylation (H3K4me3) exhibited distinct temporal patterns facilitative to the induction of transcription. A transcriptional repressive (H3K9me3) modification showed a different temporal pattern. Collectively, this should create a permissive environment for the transcriptional activity necessary for lordosis, within 3–6 h after E 2 treatment. In the VMH , changes in the H3Ac and H3K4me3 levels of histone H3 were also detected at the promoter region of the Pgr gene within the same time window, although they were delayed in the preoptic area. Moreover, examination of histone modifications associated with the promoter of another ER‐target gene, oxytocin receptor ( Oxtr ), revealed gene‐ and brain‐region specific effects of E 2 treatment. In the VMH of female mice, E 2 treatment resulted in the recruitment of ERα to the oestrogen‐response‐elements‐containing putative enhancer site of Pgr gene, approximately 200 kb upstream of the transcription start site, although it failed to increase ERα association with the more proximal promoter region. Finally, E 2 administration led to significant changes in the mRNA expression of several ER coregulators in a brain‐region dependent manner. Taken together, these data indicate that, in the hypothalamus and preoptic area of female mice, early responses to E 2 treatment involve highly specific changes in chromatin structure, dependent on cell group, gene, histone modification studied, promoter/enhancer site and time following E 2 .

Deep brain stimulation (DBS) has shown promise in the treatment of many neurological and psychiatric disorders as well as a disorder of consciousness, the minimally conscious state (MCS). In the clinic, DBS is always monotonic standard pulses; however, we have hypothesized that temporally patterned pulses might be more efficient in achieving desired behavioral responses. Here we present two experiments on DBS of the central thalamus to increase arousal, as measured by motor activity, and to affect the electroencephalogram (EEG). In the first, we optimized amplitude and frequency in standard stimulation of the central thalamus in intact mice. In the second, the optimized fixed frequency was compared to two alternative temporal patterns, chaotic and random, which were physically identical to each other and fixed frequency in all ways except temporal pattern. In both experiments and with all types of stimulation, DBS of the central thalamus increased arousal as measured by motor activity. These data also revealed that temporal patterning of pulses can modulate response to stimulation. That temporal patterns in DBS of the central thalamus were found to alter motor activity response implies possible usefulness of temporal patterns in DBS of other contexts. More investigation into exactly how temporally patterned stimulation may affect neuronal circuit dynamics is necessary.

Medullary gigantocellular reticular nucleus (mGi) neurons have been ascribed a variety of behaviors, many of which may fall under the concepts of either arousal or motivation. Despite this, many details of the connectivity of mGi neurons, particularly in reference to those neurons with ascending axons, remain unknown. To provide a neuroanatomical and molecular characterization of these cells, with reference to arousal and level-setting systems, large medullary reticular neurons were characterized with retrograde dye techniques and with real-time reverse transcriptase PCR (RT-PCR) analyses of single-neuron mRNA expression in the mouse. We have shown that receptors consistent with participation in generalized arousal are expressed by single mGi neurons and that receptors from different families of arousal-related neurotransmitters are rarely coexpressed. Through retrograde labeling, we have shown that neurons with ascending axons and neurons with descending axons tend to form like-with-like clusters, a finding that is consistent across age and gender. In comparing the two groups of retrogradely labeled neurons in neonatal animals, those neurons with axons that ascend to the midbrain show markers for GABAergic or coincident GABAergic and glutamatergic function; in contrast, approximately 60% of the neurons with axons that descend to the spinal cord are glutamatergic. We discuss the mGi's relationship to the voluntary and emotional motor systems and speculate that neurons in the mGi may represent a mammalian analogue to Mauthner cells, with a separation of function for neurons with ascending and descending axons.

A theory is put forward that emphasises differences in neuronal responses to fluctuations in steroid hormone levels compared to constant hormone levels. We propose that neuronal functions that regulate gonadotrophin release from the anterior pituitary tend to be more sensitive to rapid increases in the levels of oestrogens than they are to constant oestrogen levels. By contrast, neurones that control certain behavioral functions are affected just as well by constant oestrogen levels as by positively accelerating levels of oestrogen. In addition to providing examples of data from recent experiments that examine actions of the long-term effects of oestrogen on mouse behaviour, we illustrate the behavioural effects of microinjections of adeno-associated viral vectors of small interfering RNA directed against the mRNA for oestrogen receptor-alpha (ER alpha). This manipulation provides for a long-term loss of ER alpha function in a neuranatomically specific manner. The theoretical distinction between temporal features of oestrogen sensitivity of neuroendocrine versus behavioural function is not absolute, but is intended to stimulate new experimentation that examines temporal features of oestrogen administration.

We present a novel microfluidic device for transdermal delivery of small quantities of drugs in brief time pulses for neurobiological studies. The device consists of dispensing and delivery channels connected to drug and buffer lines, respectively, and coupled by a check valve. The planar check valve and the microchannels are within a single flow layer which allows for simple fabrication and easy system integration. Drug and buffer lines are connected to separate pneumatic lines for briefly pulsatile driving to enable drug dispensing and delivery consecutively in a stage-shifting working principle. Experimental results demonstrate that our microfluidic device is capable of continuously delivering well-defined nanoliter-scale volumes of drug solution within time pulses tens of milliseconds in duration. Based on the investigation of the characteristics of the check valve, we have systematically characterized the drug delivery device in terms of the dispensed and delivered drug volumes under varying pneumatic driving pressures and pulse durations, showing that the device has virtually zero diffusive leakage and good controllability. Furthermore, in vitro pulsatile delivery into mouse brain tissue using this device has also been performed at controlled drug volumes, demonstrating the potentially wide application of this microfluidic device in various neurobiological studies.

The sagittalis nucleus (SGN) of the hypothalamus is a newly‐identified nucleus that is located in the interstitial area between the arcuate and ventromedial nuclei of the rat hypothalamus and for which the long axis of the nucleus is oriented sagittally. Interestingly, the SGN exhibits structural and physiological sex differences, as defined by Nissl staining and oestrogen receptor (ER)α immunoreactivity (‐ir), being larger in males than females. The structural sex difference is established by sex steroid action in neonates because the treatment of female pups with testosterone propionate masculinised the SGN. The phenotypical sex difference in ERα‐ir is mediated hormonally in adulthood. Ovariectomy of female rats caused a significant increase in ERα‐ir in the SGN, and eliminated the physiological sex difference, but with recovery to the level of gonad‐intact females when given oestradiol replacement. Adult females have oestrous cycle‐related variations in ERα‐ir in the SGN, with levels at a nadir during the evening of pro‐oestrous. The discovery of the SGN, a target of sex steroid action, provides a new opportunity for explaining hormonal regulation of sexually‐differentiated behavioural and endocrine functions.

Oestradiol actions in the hypothalamus play an important role in reproductive behaviour. Oestradiol treatment in vivo induces α 1b ‐adrenoceptor mRNA and increases the density of α 1B ‐adrenoceptor binding in the hypothalamus. Oestradiol is also known to modulate neuronal excitability, in some cases by modulating calcium channels. We assessed the effects of phenylephrine, an α 1 ‐adrenergic agonist, on low‐voltage‐activated (LVA) and high‐voltage‐activated (HVA) calcium channels in ventromedial hypothalamic (VMN) neurones from vehicle‐ and oestradiol‐treated female rats. Whole‐cell and gramicidin perforated‐patch recordings were obtained, with barium as the charge carrier. In the absence of phenylephrine, oestradiol treatment increased the magnitude of LVA currents compared to controls, but had no effect on HVA currents. Phenylephrine enhanced HVA currents in a significantly greater proportion of neurones from oestradiol‐treated rats (76%) than from vehicle‐treated (41%) rats. The L‐channel blocker nifedipine abolished this oestradiol effect on phenylephrine‐enhanced HVA currents. Preincubating slices with the N‐type channel blocker omega‐conotoxin GVIA completely blocked the phenylephrine response, suggesting that the N‐type channel is essential. Phenylephrine also stimulated LVA currents in approximately two‐thirds of neurones in slices from both vehicle‐ and oestradiol‐treated rats. Our data show that oestradiol increases LVA currents in the VMN. Oestradiol also amplifies α 1 ‐adrenergic signalling by increasing the proportion of neurones showing phenylephrine‐stimulated HVA currents mediated by N‐ and L‐type calcium channels. In this way, oestradiol may increase excitatory responses to arousing adrenergic inputs to VMN neurones governing oestradiol‐dependent reproductive behaviour.

Estrogens act upon ventromedial hypothalamic (VMH) neurons, and their effects on female arousal and sexual behaviors mediated by VMH neurons involve several neurotransmitters and neuromodulators. Among these are opioid peptides which might be predicted to oppose estrogenic action on VMH because they tend to decrease CNS arousal. Spontaneous excitatory postsynaptic currents were recorded from VMH neurons from 17β-estradiol- (E, 10 μg/0.1 ml) or oil-treated control ovariectomized (OVX) mice using whole-cell patch-clamp techniques. To examine the impact of opioidergic inputs, recordings of neurons from both treatment groups were obtained in the presence of the general opioid receptor agonist methionine enkephalin-Arg-Phe (MERF, 3 μM), or μ-receptor specific agonist [d-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO, 1 μM). Compared with oil, E treatment for 48 h significantly increased the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) without affecting their amplitude. MERF and DAMGO each abolished this E effect, causing significant reductions in sEPSCs. The effect of MERF was abolished by naltrexone (general opioid receptor antagonist, 3 μM) and the effect of DAMGO by d-Phe-Cys-Tyr-d-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) (μ-opioid receptor selective antagonist, 1 μM); in contrast, κ- and δ-opioid receptor agonists, U69593 (300 nM) and [d-Pen2,d-Pen5]-enkephalin (DPDPE, 1 μM) respectively, had little effect on the sEPSCs compared with DAMGO. To consider presynaptic vs. postsynaptic effects of opioids, miniature excitatory postsynaptic currents (mEPSCs) were investigated in E- and oil-treated VMH neurons and opioid receptor antagonist effects on mEPSCs were observed. Both MERF and DAMGO reduced the frequency of mEPSCs, but had no effect on their amplitude. Our findings indicate that opioids suppress excitatory synaptic transmissions in VMH neurons primarily through μ-receptors and could thereby decrease sexual arousal in mice.

Reverse engineering takes the facts we know about a device or a process and reasons backwards to infer the principles underlying the structure-function relations. The goal of this review is to apply this approach to a well-studied hormone-controlled behavior, namely the reproductive stance of female rodents, lordosis. We first provide a brief overview on the considerable amount of progress in the analysis of female reproductive behavior. Then, we propose an analysis of the mechanisms of this behavior from a reverse-engineering perspective with the goal of generating novel hypotheses about the properties of the circuitry elements. In particular, the previously proposed neuronal circuit modules, feedback signals, and genomic mechanisms are considered to make predictions in this manner. The lordosis behavior itself appears to proceed ballistically once initiated, but negative and positive hormonal feedback relations are evident in its endocrine controls. Both rapid membrane-initiated and slow genomic hormone effects contribute to the behavior's control. We propose that the value of the reverse-engineering approach is based on its ability to provide testable, mechanistic hypotheses that do not emerge from either traditional evolutionary or simple reductionistic perspectives, and several are proposed in this review. These novel hypotheses may generalize to brain functions beyond female reproductive behavior. In this way, the reverse-engineering perspective can further develop our conceptual frameworks for behavioral and systems neuroscience.

Methadone is widely used in treatment of short-acting opiate addiction. The on-off effects of opioids have been documented to have profound differences from steady-state opioids. The authors hypothesize that opioids play important roles in either generalized arousal (GA) or aversive state of arousal during opioid withdrawal. Both male and female C57BL6 mice received steady-state methadone (SSM) through osmotic pumps at 10 or 20 mg/kg/day, and GA was measured in voluntary motor activity, sensory responsivity, and contextual fear conditioning. SSM did not have any effect on those GA behaviors in either sex. Females had higher activity and less fear conditioning than males. The effects of SSM on stress-responsive orexin gene expression in the lateral hypothalamus (LH) and medial hypothalamus (MH, including perifornical and dorsomedial areas) were measured after the behavioral tests. Females showed significantly lower basal LH (but not MH) orexin mRNA levels than males. A panel of GA stressors increased LH orexin mRNA levels in females only; these increases were blunted by SSM at 20 mg/kg. In summary, SSM had no effect on GA behaviors. In females, SSM blunted the GA stress-induced LH orexin gene expression.

To assess how early can estrogens induce female mating behaviors, rat pups 8-29 days old (D8-D29, respectively) were injected twice daily with estradiol benzoate (E) or oil (O) followed by progesterone (P) or oil, and then observed for the estrogen-dependent ear wiggling (EW) and lordosis in response to natural stimulation from male rats. In female pups treated with E + E + P, the incidence of EW appeared as early as D13 and increased gradually to reach maximum at D18, when all pups tested showed EW. EW also occurred in E + E + O females, but never in O + O + P females or in any E + E + P male. Lordosis in E + E + P, as well as E + E + O, female pups occurred later, starting at D15. O + O + P females or E + E + P males never display lordosis. To explore the possibilities that the age and gender differences are due to distribution and/or function of estrogen receptor-alpha (ER alpha) or progesterone receptor (PR), separate pups were used for immunocytochemical (ICC) staining of these receptors in the hypothalamic ventromedial nucleus (VMN). There was no age difference in female pups in the density of ER alpha or the induction of PR between D11/D12, when no sexual behavior was observed, and D19/D20, when almost all pups tested performed the behaviors. There were gender differences: male pups had less ER alpha than females at D19/D20, though not at D11/D12, and did not respond to E in the induction of PR in the VMN. These results show that ERs and their signaling systems in the VMN of rat pups are functional at least after D11 but only in females, and that the gender differences appeared to be due to differences in the molecular biology of ER alpha.

This chapter discusses hormonal, neural, and genomic mechanisms for female reproductive behaviors, sexual motivation, and arousal through mammalian researches. The female reproductive behavior depends on estrogen and progestins. The behavioral actions of estrogen affect neurons directly rather than through an obligatory pituitary mechanism because estrogen and progesterone can support female reproductive behavior in hypophysectomized ovariectomized rats. In female monkeys, attractivity and proceptivity clearly change according to the stage of the menstrual cycle or the steroid injected, but there is less agreement among results from different laboratories on the effects of steroid hormones on receptivity. The primary sex behavior of female quadrupeds, lordosis, depends on defined physical signals: cutaneous stimuli and estrogens plus progestins. The neural circuit has been worked out; estrogen-dependent transcription in ventromedial hypothalamic cells allows permissive signals to the midbrain central grey, thus enabling the rest of the circuit. In the absence of fear or anxiety-provoking conditions, females under the influence of estrogens plus progestins demonstrate courtship and then mating behaviors. During the normal female cycle, these behavioral components of reproduction are synchronized with ovulation. Thus, with the mediation of estrogens and progestins, the neural, behavioral, and endocrine preparations for reproduction are harmonized.

RATIONALE:Progesterone (P) and its 5alpha-reduced metabolite, 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-THP), facilitate sexual behavior of rodents via agonist-like actions at intracellular progestin receptors (PRs) and membrane GABA(A)/benzodiazepine receptor complexes (GBRs), respectively.OBJECTIVE:Given that ovarian secretion of progestins declines with aging, whether or not senescent mice are responsive to progestins was of interest.METHODS:Homozygous PR knockout (PRKO) or wild-type mice that were between 10-12 (mid-aged) or 20-24 (aged) months of age were administered P or 3alpha,5alpha-THP, and the effect on lordosis were examined. Effects of a progestin-priming regimen that enhances PR-mediated (experiment 1) or more rapid, PR-independent effects of progestins (experiments 2 and 3) on sexual behavior were examined. Levels of P, 3alpha,5alpha-THP, and muscimol binding were examined in tissues from aged mice (experiment 4).RESULTS:Wild-type, but not PRKO, mice were responsive when primed with 17beta-estradiol (E(2); 0.5 microg) and administered P (500 microg, subcutaneously). Mid-aged wild-type mice demonstrated greater increases in lordosis 6 h later compared to their pre-P, baseline test than did aged wild-type mice (experiment 1). Lordosis of younger and older wild-type, but not PRKO, mice was significantly increased within 5 min of intravenous (IV) administration of P (100 ng), compared with E(2)-priming alone (experiment 2). However, wild-type and PRKO mice demonstrated significant increases in lordosis 5 min after IV administration of 3alpha,5alpha-THP, an effect which was more pronounced in mid-aged than in aged animals (100 ng-experiment 3). In tissues from aged wild-type and PRKO mice, levels of P, 3alpha,5alpha-THP, and muscimol binding were increased by P administration (experiment 4). PR binding was lower in the cortex of PRKO than that of wild-type mice.CONCLUSIONS:Mid-aged and aged PRKO and wild-type mice demonstrated rapid P or 3alpha,5alpha-THP-facilitated lordosis that may be, in part, independent of activity at PRs.