Internal State: Dynamic, Interconnected Communication Loops Distributed Across Body, Brain, and Time.

Internal state profoundly alters perception and behavior. For example, a starved fly may approach and consume foods that it would otherwise find undesirable. A socially engaged newt may remain engaged in the presence of a predator, whereas a solitary newt would otherwise attempt to escape. Yet, the definition of internal state is fluid and ill-defined. As an interdisciplinary group of scholars spanning five career stages (from undergraduate to full professor) and six academic institutions, we came together in an attempt to provide an operational definition of internal state that could be useful in understanding the behavior and the function of nervous systems, at timescales relevant to the individual. In this perspective, we propose to define internal state through an integrative framework centered on dynamic and interconnected communication loops within and between the body and the brain. This framework is informed by a synthesis of historical and contemporary paradigms used by neurobiologists, ethologists, physiologists, and endocrinologists. We view internal state as composed of both spatially distributed networks (body-brain communication loops), and temporally distributed mechanisms that weave together neural circuits, physiology, and behavior. Given the wide spatial and temporal scales at which internal state operates-and therefore the broad range of scales at which it could be defined-we choose to anchor our definition in the body. Here we focus on studies that highlight body-to-brain signaling; body represented in endocrine signaling, and brain represented in sensory signaling. This integrative framework of internal state potentially unites the disparate paradigms often used by scientists grappling with body-brain interactions. We invite others to join us as we examine approaches and question assumptions to study the underlying mechanisms and temporal dynamics of internal state.

Calcium-induced calcium release activates spontaneous miniature outward currents in newt medullary reticular formation neurons.

Neurons in the medullary reticular formation are involved in the control of postural and locomotor behaviors in all vertebrates. Reticulospinal neurons in this brain region provide one of the major descending projections to the spinal cord. Although neurons in the newt medullary reticular formation have been extensively studied using in vivo extracellular recordings, little is known of their intrinsic biophysical properties or of the underlying circuitry of this region. Using whole cell patch-clamp recordings in brain slices containing the rostromedial reticular formation from adult male newts, we observed spontaneous miniature outward currents (SMOCs) in ~2/3 of neurons. Although SMOCs superficially resembled inhibitory postsynaptic currents (IPSCs), they had slower risetimes and decay times than spontaneous IPSCs. SMOCs required intracellular Ca2+ release from ryanodine receptors and were also dependent on the influx of extracellular Ca2+. SMOCs were unaffected by apamin but were partially blocked by iberiotoxin and charybdotoxin, indicating that SMOCs were mediated by big-conductance Ca2+-activated K+ channels. Application of the sarco/endoplasmic Ca2+ ATPase inhibitor cyclopiazonic acid blocked the generation of SMOCs and also increased neural excitability. Neurons with SMOCs had significantly broader action potentials, slower membrane time constants, and higher input resistance than neurons without SMOCs. Thus, SMOCs may serve as a mechanism to regulate action potential threshold in a majority of neurons within the newt medullary reticular formation. NEW & NOTEWORTHY The medullary reticular formation exerts a powerful influence on sensorimotor integration and subsequent motor behavior, yet little is known about the neurons involved. In this study, we identify a transient potassium current that regulates action potential threshold in a majority of medullary reticular neurons.

Suppression of sex behavior by kappa opiates and stress steroids occurs via independent neuroendocrine pathways.

Endocannabinoids and their receptors are found throughout the brain of all vertebrates. By virtue of their wide distribution, endocannabinoids have the potential to affect many behaviors. Prior research has shown that cannabinoids inhibit courtship-clasping and mediate behavioral responses to stress in male rough-skinned newts, Taricha granulosa, and cannabinoid signaling is initiated by rapid actions of the steroid corticosterone (CORT) at its specific membrane receptor (mCR). This same mCR also recognizes κ-opioid receptor agonists and antagonists. Prior behavioral studies show that κ-opioid agonists suppress clasping behavior in a dose dependent manner. Combined, these studies suggest that κ-opioid agonists might suppress clasping behavior via the same pathway initiated by CORT: up-regulation of endocannabinoid signaling. We examined whether pretreatment with a CB1 antagonist, AM281, would block κ-opioid-mediated suppression of clasping. We found that the CB1 antagonist did not reverse κ-opioid-induced suppression of clasping, revealing that while endocannabinoids mediate CORT-induced suppression of clasping, endocannabinoids do not mediate the κ-opioid-induced suppression of clasping.

Corticosterone suppresses vasotocin-enhanced clasping behavior in male rough-skinned newts by novel mechanisms interfering with V1a receptor availability and receptor-mediated endocytosis.

In rough-skinned newts, Taricha granulosa, exposure to an acute stressor results in the rapid release of corticosterone (CORT), which suppresses the ability of vasotocin (VT) to enhance clasping behavior. CORT also suppresses VT-induced spontaneous activity and sensory responsiveness of clasp-controlling neurons in the rostromedial reticular formation (Rf). The cellular mechanisms underlying this interaction remain unclear. We hypothesized that CORT blocks VT-enhanced clasping by interfering with V1a receptor availability and/or VT-induced endocytosis. We administered a physiologically active fluorescent VT conjugated to Oregon Green (VT-OG) to the fourth ventricle 9 min after an intraperitoneal injection of CORT (0, 10, 40 µg/0.1mL amphibian Ringers). The brains were collected 30 min post-VT-OG, fixed, and imaged with confocal microscopy. CORT diminished the number of endocytosed vesicles, percent area containing VT-OG, sum intensity of VT-OG, and the amount of VT-V1a within each vesicle; indicating that CORT was interfering with V1a receptor availability and VT-V1a receptor-mediated endocytosis. CORT actions were brain location-specific and season-dependent in a manner that is consistent with the natural and context-dependent expression of clasping behavior. Furthermore, the sensitivity of the Rf to CORT was much higher in animals during the breeding season, arguing for ethologically appropriate seasonal variation in CORT's ability to prevent VT-induced endocytosis. Our data are consistent with the time course and interaction effects of CORT and VT on clasping behavior and neurophysiology. CORT interference with VT-induced endocytosis may be a common mechanism employed by hormones across taxa for mediating rapid context- and season-specific behavioral responses.

Lithium modulates cortical excitability in vitro.

The sometimes devastating mood swings of bipolar disorder are prevented by treatment with selected antiepileptic drugs, or with lithium. Abnormal membrane ion channel expression and excitability in brain neurons likely underlie bipolar disorder, but explaining therapeutic effects in these terms has faced an unresolved paradox: the antiepileptic drugs effective in bipolar disorder reduce Na(+) entry through voltage-gated channels, but lithium freely enters neurons through them. Here we show that lithium increases the excitability of output neurons in brain slices of the mouse olfactory bulb, an archetypical cortical structure. Treatment in vitro with lithium (1 to 10mM) depolarizes mitral cells, blocks action potential hyperpolarization, and modulates their responses to synaptic input. We suggest that Na(+) entry through voltage-gated channels normally directly activates K(+) channels regulating neuron excitability, but that at therapeutic concentrations, lithium entry and accumulation reduces this K(+) channel activation. The antiepileptic drugs effective in bipolar disorder and lithium may thus share a membrane target consisting of functionally coupled Na(+) and K(+) channels that together control brain neuron excitability.

All-or-none population bursts temporally constrain surround inhibition between mouse olfactory glomeruli.

With each sniff, the olfactory bulbs of the brain generate a neural activity pattern representing the odour environment, transmitting this to higher brain centres in the form of mitral cell output. Inhibitory circuits in the olfactory bulb glomerular and external plexiform layers may amplify contrast in these patterns, through surround inhibition of mitral cells. These circuits may operate in series, but their respective roles are unclear. A single sniff is sufficient for odour discrimination, but is not clear that the inhibitory circuits act within this timeframe. We used microdissected slices of mouse olfactory bulb to study each circuit in isolation. We found that unlike surround inhibition mediated in the external plexiform layer, surround inhibition mediated in the glomerular layer was activated by sensory synaptic input, but not by mitral cell output. The results also suggest that interactions between olfactory glomeruli are exclusively inhibitory, unlike in antennal lobe, and that surround inhibition mediated within the external plexiform layer may involve neural circuit elements not preserved in slice preparations. Surround inhibition was effective only after an interval corresponding to a single sniff in vivo. Surplus excitation, initiated by sensory input but generated by collective all-or-none responses of mitral cells, may delay surround inhibition and allow the synchronous activation of multiple glomeruli without each suppressing the other. Surround inhibition in the glomerular layer may subsequently allow a fresh representation of the odour environment to be generated with each sniff. These findings are consistent with combinatorial odour coding based on all-or-none glomerular responses.

Endocannabinoids mediate the effects of acute stress and corticosterone on sex behavior.

For animals in the wild, survival depends on being able to detect and respond rapidly to danger by switching from risky (e.g. conspicuous courtship) to survival-oriented behaviors. Very little is known about the hormonal or neuroendocrine mechanisms that control the rapid switch in behavioral state that occurs when an animal detects threats or other stressors. Prior studies with rough-skinned newts (Taricha granulosa), an amphibian model, found that stress-induced suppression of male sexual behaviors (amplectic clasping) involves corticosterone (CORT) and that this steroid hormone uses a novel membrane receptor and modulates the responsiveness of medullary neurons in clasp-controlling neural circuits. We provide evidence that this rapid suppression of male sex behaviors, when induced by either acute stress or CORT administration, involves activation of endocannabinoids signaling in the hindbrain. In a series of behavioral studies, administration of a cannabinoid antagonist, AM281, blocked the suppressive effects of exposure to acute stress or an injection of CORT on the performance of clasping behaviors in sexually active males. Similarly, in electrophysiological studies, prior treatment with AM281 blocked CORT-induced suppression of spontaneous neuronal activity and sensory responsiveness of hindbrain neurons in clasp-controlling neural circuits. These data suggest that, in response to acute stress, elevated CORT concentration increases endocannabinoid signaling in the hindbrain and alters sexual behaviors by modulating the excitability of medullary circuits.

Neuroanatomical distribution of cannabinoid receptor gene expression in the brain of the rough-skinned newt, Taricha granulosa.

Type I cannabinoid receptor (CB1) is a G-protein coupled receptor with a widespread distribution in the central nervous system in mammals. In a urodele amphibian, the rough-skinned newt (Taricha granulosa), recent evidence indicates that endogenous cannabinoids (endocannabinoids) mediate behavioral responses to acute stress and electrophysiological responses to corticosterone. To identify possible sites of action for endocannabinoids, in situ hybridization using a gene and species specific cRNA probe was used to label CB1 mRNA in brains of male T. granulosa. Labeling of CB1 mRNA in the telencephalon was observed in the olfactory bulb and all areas of the pallium, as well as the bed nucleus of the stria terminalis and nucleus amygdalae dorsolateralis. The labeling of CB1 mRNA was also found in regions of the preoptic area, thalamus, midbrain tegmentum and tectum, cerebellum, and the stratum griseum of the hindbrain. A notable difference in CB1 labeling between this amphibian and mammals is the abundance of labeling in areas associated with olfaction (anterior olfactory nuclei, nucleus amygdalae dorsolateralis, and lateral pallium), which hints that endocannabinoids might modulate responses to odors as well as pheromones. This widespread distribution of CB1 labeling, particularly in sensory and motor control centers, fits with prior results showing that endocannabinoids modulate sensorimotor processing and behavioral output in this species. The distribution of CB1 in the brain of T. granulosa was in many of the same sites previously observed in the brain of the anuran amphibian, Xenopus laevis, as well as those of different species of mammals, suggesting that endocannabinoid signaling pathways are conserved.

Neuroendocrinology of context-dependent stress responses: vasotocin alters the effect of corticosterone on amphibian behaviors.

The ability of an animal to respond with appropriate defensive behaviors when confronted with an immediate threat can affect its survival and reproductive success. In the roughskin newt (Taricha granulosa), exogenous corticosterone (CORT) rapidly blocks and vasotocin (VT) enhances reproductive behaviors (mainly clasping behavior). Electrophysiological studies have shown that pretreatment of male Taricha with VT counteracts the inhibitory effects of CORT on neuronal activity in the medulla. To test whether similar interactions between VT and CORT influence reproductive behaviors in Taricha, we recorded the time spent and incidence of clasping in males injected with VT or vehicle at 60 min and then CORT or vehicle at 5 min before presentation of a female. This study found that clasping behavior is suppressed in males that received vehicle and then CORT, but is not suppressed in males that received VT and then CORT. Considering these results and the possibility that the performance of clasping behaviors might cause increases in endogenous VT activity, we tested whether the suppressive effects of CORT administration on clasping behavior would occur in males that had recently clasped females. The study found that, in contrast to males that had been isolated from females, CORT administration did not suppress clasping behavior in males that had been allowed to clasp females for 60 min prior to the hormone injection. Our results suggest that, at least in this amphibian and perhaps in other animals, the neuroendocrine regulation of alternative behavioral responses to threats involves functional interactions between corticosteroids and VT-like peptides.