Faculty Bibliography

In neonatal brain development there is a period of normal apoptotic cell death that regulates adult neuron number. At approximately the same period, ethanol exposure can cause a dramatic spike in apoptotic cell death. While ethanol-induced apoptosis has been shown to reduce adult neuron number, questions remain about the regional selectivity of the ethanol effect, and whether the brain might have some capacity to overcome the initial neuron loss. The present study used stereological cell counting to compare cumulative neuron loss 8 h after postnatal day 7 (P7) ethanol treatment to that of animals left to mature to adulthood (P70). Across several brain regions we found that the reduction of total neuron number after 8 h was as large as that of adult animals. Comparison between regions revealed that some areas are more vulnerable, with neuron loss in the anterior thalamic nuclei > the medial septum/vertical diagonal band, dorsal subiculum, and dorsal lateral geniculate nucleus > the mammillary bodies and cingulate cortex > whole neocortex. In contrast to estimates of total neuron number, estimates of apoptotic cell number in Nissl-stained sections at 8 h after ethanol treatment provided a less reliable predictor of adult neuron loss. The findings show that ethanol-induced neonatal apoptosis often causes immediate neuron deficits that persist in adulthood, and furthermore suggests that the brain may have limited capacity to compensate for ethanol-induced neuron loss.

Introduction: Transitions between sleep and waking and sleep-dependent cortical oscillations are heavily dependent on GABAergic neurons. Importantly, GABAergic neurons are especially sensitive to developmental ethanol exposure, suggesting a potential unique vulnerability of sleep circuits to early ethanol. In fact, developmental ethanol exposure can produce long-lasting impairments in sleep, including increased sleep fragmentation and decreased delta wave amplitude. Here, we assessed the efficacy of optogenetic manipulations of somatostatin (SST) GABAergic neurons in the neocortex of adult mice exposed to saline or ethanol on P7, to modulate cortical slow-wave physiology. Methods: SST-cre × Ai32 mice, which selectively express channel rhodopsin in SST neurons, were exposed to ethanol or saline on P7. This line expressed similar developmental ethanol induced loss of SST cortical neurons and sleep impairments as C57BL/6By mice. As adults, optical fibers were implanted targeting the prefrontal cortex (PFC) and telemetry electrodes were implanted in the neocortex to monitor slow-wave activity and sleep-wake states. Results: Optical stimulation of PFC SST neurons evoked slow-wave potentials and long-latency single-unit excitation in saline treated mice but not in ethanol mice. Closed-loop optogenetic stimulation of PFC SST neuron activation on spontaneous slow-waves enhanced cortical delta oscillations, and this manipulation was more effective in saline mice than P7 ethanol mice. Discussion: Together, these results suggest that SST cortical neurons may contribute to slow-wave impairment after developmental ethanol.

Developmental exposure to ethanol is a leading cause of cognitive, emotional and behavioral problems, with fetal alcohol spectrum disorder (FASD) affecting more than 1:100 children. Recently, comorbid sleep deficits have been highlighted in these disorders, with sleep repair a potential therapeutic target. Animal models of FASD have shown non-REM (NREM) sleep fragmentation and slow-wave oscillation impairments that predict cognitive performance. Here we use a mouse model of perinatal ethanol exposure to explore whether reduced sleep pressure may contribute to impaired NREM sleep, and compare the function of a brain network reported to be impacted by insomnia-the Salience network-in developmental ethanol-exposed mice with sleep-deprived, saline controls. Mice were exposed to ethanol or saline on postnatal day 7 (P7) and allowed to mature to adulthood for testing. At P90, telemetered cortical recordings were made for assessment of NREM sleep in home cage before and after 4 h of sleep deprivation to assess basal NREM sleep and homeostatic NREM sleep response. To assess Salience network functional connectivity, mice were exposed to the 4 h sleep deprivation period or left alone, then immediately sacrificed for immunohistochemical analysis of c-Fos expression. The results show that developmental ethanol severely impairs both normal rebound NREM sleep and sleep deprivation induced increases in slow-wave activity, consistent with reduced sleep pressure. Furthermore, the Salience network connectome in rested, ethanol-exposed mice was most similar to that of sleep-deprived, saline control mice, suggesting a sleep deprivation-like state of Salience network function after developmental ethanol even without sleep deprivation.

Ethanol exposure in neonatal mice induces acute neurodegeneration followed by long-lasting glial activation and GABAergic cell deficits along with behavioral abnormalities, providing a third trimester model of fetal alcohol spectrum disorders (FASD). Retinoic acid (RA), the active form of vitamin A, regulates transcription of RA-responsive genes and plays essential roles in the development of embryos and their CNS. Ethanol has been shown to disturb RA metabolism and signaling in the developing brain, which may be a cause of ethanol toxicity leading to FASD. Using an agonist and an antagonist specific to RA receptor α (RARα), we studied how RA/RARα signaling affects acute and long-lasting neurodegeneration and activation of phagocytic cells and astrocytes caused by ethanol administered to neonatal mice. We found that an RARα antagonist (BT382) administered 30 min before ethanol injection into postnatal day 7 (P7) mice partially blocked acute neurodegeneration as well as elevation of CD68-positive phagocytic cells in the same brain area. While an RARα agonist (BT75) did not affect acute neurodegeneration, BT75 given either before or after ethanol administration ameliorated long-lasting astrocyte activation and GABAergic cell deficits in certain brain regions. Our studies using Nkx2.1-Cre;Ai9 mice, in which major GABAergic neurons and their progenitors in the cortex and the hippocampus are labeled with constitutively expressed tdTomato fluorescent protein, indicate that the long-lasting GABAergic cell deficits are mainly caused by P7 ethanol-induced initial neurodegeneration. However, the partial reduction of prolonged GABAergic cell deficits and glial activation by post-ethanol BT75 treatment suggests that, in addition to the initial cell death, there may be delayed cell death or disturbed development of GABAergic cells, which is partially rescued by BT75. Since RARα agonists including BT75 have been shown to exert anti-inflammatory effects, BT75 may rescue GABAergic cell deficits by reducing glial activation/neuroinflammation.

Animals, including humans, detect odours and use this information to behave efficiently in the environment. Frequently, odours consist of complex mixtures of odorants rather than single odorants, and mixtures are often perceived as configural wholes, i.e. as odour objects (e.g. food, partners). The biological rules governing this 'configural perception' (as opposed to the elemental perception of mixtures through their components) remain weakly understood. Here, we first review examples of configural mixture processing in diverse species involving species-specific biological signals. Then, we present the original hypothesis that at least certain mixtures can be processed configurally across species. Indeed, experiments conducted in human adults, newborn rabbits and, more recently, in rodents and honeybees show that these species process some mixtures in a remarkably similar fashion. Strikingly, a mixture AB (A, ethyl isobutyrate; B, ethyl maltol) induces configural processing in humans, who perceive a mixture odour quality (pineapple) distinct from the component qualities (A, strawberry; B, caramel). The same mixture is weakly configurally processed in rabbit neonates, which perceive a particular odour for the mixture in addition to the component odours. Mice and honeybees also perceive the AB mixture configurally, as they respond differently to the mixture compared with its components. Based on these results and others, including neurophysiological approaches, we propose that certain mixtures are convergently perceived across various species of vertebrates/invertebrates, possibly as a result of a similar anatomical organization of their olfactory systems and the common necessity to simplify the environment's chemical complexity in order to display adaptive behaviours.

Despite major anatomical differences with other mammalian sensory systems, olfaction shares with those systems a modulation by sleep/wake states. Sleep modulates odor sensitivity and serves as an important regulator of both perceptual and associative odor memory. In addition, however, olfaction also has an important modulatory impact on sleep. Odors can affect the latency to sleep onset, as well as the quality and duration of sleep. Olfactory modulation of sleep may be mediated by direct synaptic interaction between the olfactory system and sleep control nuclei, and/or indirectly through odor modulation of arousal and respiration. This reciprocal interaction between sleep and olfaction presents novel opportunities for sleep related modulation of memory and perception, as well as development of non-pharmacological olfactory treatments of simple sleep disorders.

Social interaction deficits seen in psychiatric disorders emerge in early-life and are most closely linked to aberrant neural circuit function. Due to technical limitations, we have limited understanding of how typical versus pathological social behavior circuits develop. Using a suite of invasive procedures in awake, behaving infant rats, including optogenetics, microdialysis, and microinfusions, we dissected the circuits controlling the gradual increase in social behavior deficits following two complementary procedures-naturalistic harsh maternal care and repeated shock alone or with an anesthetized mother. Whether the mother was the source of the adversity (naturalistic Scarcity-Adversity) or merely present during the adversity (repeated shock with mom), both conditions elevated basolateral amygdala (BLA) dopamine, which was necessary and sufficient in initiating social behavior pathology. This did not occur when pups experienced adversity alone. These data highlight the unique impact of social adversity as causal in producing mesolimbic dopamine circuit dysfunction and aberrant social behavior.

Odor perception can both evoke emotional states and be shaped by emotional or hedonic states. The amygdala complex plays an important role in recognition of, and response to, hedonically valenced stimuli, and has strong, reciprocal connectivity with the primary olfactory (piriform) cortex. Here, we used differential odor-threat conditioning in rats to test the role of basolateral amygdala (BLA) input to the piriform cortex in acquisition and expression of learned olfactory threat responses. Using local field potential recordings, we demonstrated that functional connectivity (high gamma band coherence) between the BLA and posterior piriform cortex (pPCX) is enhanced after differential threat conditioning. Optogenetic suppression of activity within the BLA prevents learned threat acquisition, as do lesions of the pPCX prior to threat conditioning (without inducing anosmia), suggesting that both regions are critical for acquisition of learned odor threat responses. However, optogenetic BLA suppression during testing did not impair threat response to the CS+ , but did induce generalization to the CS-. A similar loss of stimulus control and threat generalization was induced by selective optogenetic suppression of BLA input to pPCX. These results suggest an important role for amygdala-sensory cortical connectivity in shaping responses to threatening stimuli.

Determining the valence of an odor to guide rapid approach-avoidance behavior is thought to be one of the core tasks of the olfactory system, and yet little is known of the initial neural mechanisms supporting this process or of its subsequent behavioral manifestation in humans. In two experiments, we measured the functional processing of odor valence perception in the human olfactory bulb (OB)-the first processing stage of the olfactory system-using a noninvasive method as well as assessed the subsequent motor avoidance response. We demonstrate that odor valence perception is associated with both gamma and beta activity in the human OB. Moreover, we show that negative, but not positive, odors initiate an early beta response in the OB, a response that is linked to a preparatory neural motor response in the motor cortex. Finally, in a separate experiment, we show that negative odors trigger a full-body motor avoidance response, manifested as a rapid leaning away from the odor, within the time period predicted by the OB results. Taken together, these results demonstrate that the human OB processes odor valence in a sequential manner in both the gamma and beta frequency bands and suggest that rapid processing of unpleasant odors in the OB might underlie rapid approach-avoidance decisions.

In animal models that mimic human third-trimester fetal development, ethanol causes substantial cellular apoptosis in the brain, but for most brain structures the extent of permanent neuron loss that persists into adulthood is unknown. We injected ethanol into C57BL/6J mouse pups at postnatal day 7 (P7) to model human late-gestation ethanol toxicity, and then used stereological methods to investigate adult cell numbers in several subcortical neurotransmitter systems that project extensively in the forebrain to regulate arousal states. Ethanol treatment caused especially large reductions (34-42%) in the cholinergic cells of the basal forebrain, including cholinergic cells in the medial septal/vertical diagonal band (Ch1/Ch2) and in the horizontal diagonal band/substantia innominata/nucleus basalis (Ch3/Ch4) nuclei. Cell loss was also present in non-cholinergic basal forebrain cells, as demonstrated by 34% reduction of parvalbumin immunolabeled GABA cells and 25% reduction of total Nissl-stained neurons in the Ch1/Ch2 region. In contrast, cholinergic cells in the striatum were reduced only 12% by ethanol, and those of the brainstem pedunculopontine/lateral dorsal tegmental nuclei (Ch5/Ch6) were not significantly reduced. Similarly, ethanol did not significantly reduce dopamine cells of the ventral tegmental area/substantia nigra or serotonin cells in the in the dorsal raphe nucleus. Orexin (hypocretin) cells in the hypothalamus showed a modest reduction (14%). Our findings indicate that the basal forebrain is especially vulnerable to alcohol exposure in the late gestational period. Reduction of cholinergic and GABAergic projection neurons from the basal forebrain that regulate forebrain arousal may contribute to the behavioral and cognitive deficits associated with neonatal ethanol exposure.