Characterizing the role of early alcohol reexposure in associations of prenatal alcohol exposure with adolescent alcohol outcomes.

BACKGROUND: Prenatal alcohol exposure has been linked to a host of negative outcomes, although it is largely unknown whether prenatal exposure leads to an earlier age of initiation of alcohol use or exacerbates early alcohol initiation. The current study examined whether adolescents exposed to heavy drinking during gestation began drinking earlier than their nonexposed peers and whether an earlier age of alcohol reexposure in adolescence exacerbated associations with adverse alcohol outcomes. METHODS: Adolescents (17 years of age; 57% female; 96% White) from a longitudinal, population-based cohort study, the Avon Longitudinal Study of Parents and Children, reported on the age they first consumed a whole drink and other alcohol behaviors. Adolescents' mothers also reported on their own heavy drinking during pregnancy (i.e., any consumption of 4+ U.K. units in a drinking day at either 18 or 32 weeks of gestation). RESULTS: Survival analyses indicated that prenatal heavy drinking exposure was not associated with an earlier initiation of alcohol use after controlling for potential demographic and parental mental health and substance use confounds. Generalized negative binomial models demonstrated that prenatal heavy drinking exposure moderated associations of the age of alcohol initiation with alcohol quantity and heavy drinking frequency (but not alcohol frequency or Alcohol Use Disorders Identification Test score), after controlling for the same demographic and parental confounds. Specifically, earlier alcohol initiation was associated with more adverse alcohol outcomes regardless of prenatal exposure. However, the protective associations of delayed alcohol initiation were lower among adolescents exposed to prenatal heavy drinking. CONCLUSIONS: This study provides evidence for the interplay between prenatal and postnatal alcohol exposures. Importantly, adolescents who were prenatally exposed to heavy drinking appeared to be less protected by later alcohol initiation than those who were not exposed in utero.

Gene expression profiling reveals a lingering effect of prenatal alcohol exposure on inflammatory-related genes during adolescence and adulthood.

Prenatal Alcohol Exposure (PAE) exerts devastating effects on the Central Nervous System (CNS), which vary as a function of both ethanol load and gestational age of exposure. A growing body of evidence suggests that alcohol exposure profoundly impacts a wide range of cytokines and other inflammation-related genes in the CNS. The olfactory system serves as a critical interface between infectious/inflammatory signals and other aspects of CNS function, and demonstrates long-lasting plasticity in response to alcohol exposure. We therefore utilized transcriptome profiling to identify gene expression patterns for immune-related gene families in the olfactory bulb of Long Evans rats. Pregnant dams received either an ad libitum liquid diet containing 35% daily calories from ethanol (ET), a pair-fed diet (PF) matched for caloric content, or free choice (FCL) access to the liquid diet and water from Gestational Day (GD) 11-20. Offspring were fostered to dams fed the FCL diet, weaned on P21, and then housed with same-sex littermates until mid-adolescence (P40) or young adulthood (P90). At the target ages of P40 or P90, offspring were euthanized via brief CO2 exposure and brains/blood were collected. Gene expression analysis was performed using a Rat Gene 1.0 ST Array (Affymetrix), and preliminary analyses focused on two moderately overlapping gene clusters, including all immune-related genes and those related to neuroinflammation. A total of 146 genes were significantly affected by prenatal Diet condition, whereas the factor of Age (P40 vs P90) revealed 998 genes significantly changed, and the interaction between Diet and Age yielded 162 significant genes. From this dataset, we applied a threshold of 1.3-fold change (30% increase or decrease in expression) for inclusion in later analyses. Findings indicated that in adolescents, few genes were altered by PAE, whereas adults displayed an increase of a wide range of gene upregulation as a result of PAE. Pathway analysis predicted an increase in Nf-κB activation in adolescence and a decrease in adulthood due to prenatal ethanol exposure, indicating age-specific and long-lasting alterations to immune signaling. These data may provide important insight into the relationship between immune-related signaling cascades and long-term changes in olfactory bulb function after PAE.

Mixtures of Sweeteners and Maltodextrin Enhance Flavor and Intake of Alcohol in Adolescent Rats.

Sweet flavorants enhance palatability and intake of alcohol in adolescent humans. We asked whether sweet flavorants have similar effects in adolescent rats. The inherent flavor of ethanol in adolescent rats is thought to consist of an aversive odor, bitter/sweet taste, and burning sensation. In Experiment 1, we compared ingestive responses of adolescent rats to 10% ethanol solutions with or without added flavorants using brief-access lick tests. We used 4 flavorants, which contained mixtures of saccharin and sucrose or saccharin, sucrose, and maltodextrin. The rats approached (and initiated licking from) the flavored ethanol solutions more quickly than they did unflavored ethanol, indicating that the flavorants attenuated the aversive odor of ethanol. The rats also licked at higher rates for the flavored than unflavored ethanol solutions, indicating that the flavorants increased the naso-oral acceptability of ethanol. In Experiment 2, we offered rats chow, water, and a flavored or unflavored ethanol solution every other day for 8 days. The rats consistently consumed substantially more of the flavored ethanol solutions than unflavored ethanol across the 8 days. When we switched the rats from the flavored to unflavored ethanol for 3 days, daily intake of ethanol plummeted. We conclude that sweet and sweet/maltodextrin flavorants promote high daily intake of ethanol in adolescent rats (i.e., 6-10 g/kg) and that they do so in large part by improving the naso-oral sensory attributes of ethanol.

Postnatal Exposure to Ethanol Increases Its Oral Acceptability to Adolescent Rats.

The aversive flavor of ethanol limits intake by many consumers. We asked whether intermittent consumption of ethanol increases its oral acceptability, using rats as a model system. We focused on adolescent rats because they (like their human counterparts) have a higher risk for alcohol overconsumption than do adult rats following experience with the drug. We measured the impact of ethanol exposure on 1) the oral acceptability of ethanol and surrogates for its bitter (quinine) and sweet (sucrose) flavor components in brief-access lick tests and 2) responses of the glossopharyngeal (GL) taste nerve to oral stimulation with the same chemical stimuli. During the exposure period, the experimental rats had access to chow, water and 10% ethanol every other day for 16 days; the control rats had access to chow and water over the same time period. The experimental rats consumed 7-14 g/day of 10% ethanol across the exposure period. This ethanol consumption significantly increased the oral acceptability of 3%, 6% and 10% ethanol, but had no impact on the oral acceptability of quinine, sucrose or NaCl. The ethanol exposure also diminished responses of the GL nerve to oral stimulation with ethanol, but not quinine, sucrose or NaCl. Taken together, these findings indicate that ethanol consumption increases the oral acceptability of ethanol in adolescent rats and that this increased oral acceptability is mediated, at least in part, by an exposure-induced reduction in responsiveness of the peripheral taste system to ethanol per se, rather than its bitter and sweet flavor components.

Fetal alcohol exposure reduces responsiveness of taste nerves and trigeminal chemosensory neurons to ethanol and its flavor components.

Fetal alcohol exposure (FAE) leads to increased intake of ethanol in adolescent rats and humans. We asked whether these behavioral changes may be mediated in part by changes in responsiveness of the peripheral taste and oral trigeminal systems. We exposed the experimental rats to ethanol in utero by administering ethanol to dams through a liquid diet; we exposed the control rats to an isocaloric and isonutritive liquid diet. To assess taste responsiveness, we recorded responses of the chorda tympani (CT) and glossopharyngeal (GL) nerves to lingual stimulation with ethanol, quinine, sucrose, and NaCl. To assess trigeminal responsiveness, we measured changes in calcium levels of isolated trigeminal ganglion (TG) neurons during stimulation with ethanol, capsaicin, mustard oil, and KCl. Compared with adolescent control rats, the adolescent experimental rats exhibited diminished CT nerve responses to ethanol, quinine, and sucrose and GL nerve responses to quinine and sucrose. The reductions in taste responsiveness persisted into adulthood for quinine but not for any of the other stimuli. Adolescent experimental rats also exhibited reduced TG neuron responses to ethanol, capsaicin, and mustard oil. The lack of change in responsiveness of the taste nerves to NaCl and the TG neurons to KCl indicates that FAE altered only a subset of the response pathways within each chemosensory system. We propose that FAE reprograms development of the peripheral taste and trigeminal systems in ways that reduce their responsiveness to ethanol and surrogates for its pleasant (i.e., sweet) and unpleasant (i.e., bitterness, oral burning) flavor attributes.NEW & NOTEWORTHY Pregnant mothers are advised to avoid alcohol. This is because even small amounts of alcohol can alter fetal brain development and increase the risk of adolescent alcohol abuse. We asked how fetal alcohol exposure (FAE) produces the latter effect in adolescent rats by measuring responsiveness of taste nerves and trigeminal chemosensory neurons. We found that FAE substantially reduced taste and trigeminal responsiveness to ethanol and its flavor components.

The Interaction of Ethanol Ingestion and Social Interaction with an Intoxicated Peer on the Odor-Mediated Response to the Drug in Adolescent Rats.

BACKGROUND: Using a social transmission of food preference paradigm in rats, we previously demonstrated that ethanol (EtOH) exposure during adolescence, as either an observer (interaction with an intoxicated conspecific) or demonstrator (intragastric infusion with EtOH), altered the reflexive odor-mediated responses to the drug. The 2 modes of exposure were equivalent in the magnitude of their effects. Human adolescents, however, are likely to experience the drug in a social setting as both an EtOH observer and demonstrator. That is, both interacting with an intoxicated peer and experiencing EtOH's postingestive consequences in conjunction with hematogenic olfaction. Therefore, we tested whether combined adolescent exposure as both an observer and demonstrator differed from either form of individual experience. METHODS: Beginning on postnatal day (P) 29, naïve rats received EtOH or water exposures in a social interaction paradigm as either an observer, a demonstrator, or combined experience (where each animal in the interaction was, itself, an observer and demonstrator). Exposures occurred 4 times, once every 48 hours. On P37, the reflexive behavioral response to EtOH odor was tested, using whole-body plethysmography. RESULTS: The odor-mediated responses of adolescent EtOH observers, demonstrators, and combined exposure animals all significantly differed from controls. Compared to controls, however, the magnitude of the behavioral effect was greatest in the combined exposure animals. Moreover, combined exposure as both an EtOH observer and demonstrator significantly differed from either form of individual EtOH experience. CONCLUSIONS: EtOH's component chemosensory qualities are known to be central contributors to its acceptance and increases in the acceptability of EtOH's odor, resulting from a social transmission experience, are predictive of enhanced EtOH avidity in adolescence. Our findings demonstrate that combined exposure as an observer and demonstrator, within a socially relevant framework, may represent a higher risk scenario for increased EtOH avidity in adolescence (and by extension adult persistence) as compared to the individual effects of direct ingestion or social experience with the drug.

Fetal ethanol exposure increases ethanol intake by making it smell and taste better.

Human epidemiologic studies reveal that fetal ethanol exposure is highly predictive of adolescent ethanol avidity and abuse. Little is known about how fetal exposure produces these effects. It is hypothesized that fetal ethanol exposure results in stimulus-induced chemosensory plasticity. Here, we asked whether gestational ethanol exposure increases postnatal ethanol avidity in rats by altering its taste and odor. Experimental rats were exposed to ethanol in utero via the dam's diet, whereas control rats were either pair-fed an iso-caloric diet or given food ad libitum. We found that fetal ethanol exposure increased the taste-mediated acceptability of both ethanol and quinine hydrochloride (bitter), but not sucrose (sweet). Importantly, a significant proportion of the increased ethanol acceptability could be attributed directly to the attenuated aversion to ethanol's quinine-like taste quality. Fetal ethanol exposure also enhanced ethanol intake and the behavioral response to ethanol odor. Notably, the elevated intake of ethanol was also causally linked to the enhanced odor response. Our results demonstrate that fetal exposure specifically increases ethanol avidity by, in part, making it taste and smell better. More generally, they establish an epigenetic chemosensory mechanism by which maternal patterns of drug use can be transferred to offspring. Given that many licit (e.g., tobacco products) and illicit (e.g., marijuana) drugs have noteworthy chemosensory components, our findings have broad implications for the relationship between maternal patterns of drug use, child development, and postnatal vulnerability.

The interaction of gestational and postnatal ethanol experience on the adolescent and adult odor-mediated responses to ethanol in observer and demonstrator rats.

BACKGROUND: Gestational ethanol exposure enhances the adolescent reflexive sniffing response to ethanol odor. Postnatal exposures of naïve animals as either an observer (i.e., conspecific) or demonstrator (i.e., intoxicated peer) using a social transmission of food odor preference paradigm also yields enhanced odor-mediated responses. Studies on the interaction of fetal and postnatal exposures using the social transmission paradigm have been limited to the responses of observers. When combined, the enhanced response is greater than either form of exposure alone and, in observer females, yields adult persistence. The absence of a male effect is noteworthy, given that chemosensory mechanisms are suggested to be an important antecedent factor in the progression of ethanol preference. Observers gain odor information on the breath of the demonstrator through social interaction. Demonstrators experience the pharmacologic properties of ethanol along with retronasal and hematogenic olfaction. Thus, we tested whether augmentation of the fetal ethanol-induced behavioral response with postnatal exposure as a demonstrator differed from that as an observer. We also examined whether re-exposure as a demonstrator yields persistence in both sexes. METHODS: Pregnant dams were fed an ethanol containing or control liquid diet throughout gestation. Progeny received four ethanol or water exposures: one every 48 hours through either intragastric infusion or social interaction with the infused peer beginning on P29. The reflexive behavioral sniffing response to ethanol odor was tested at postnatal (P) day 37 or P90, using whole-body plethysmography. RESULTS: When tested in either adolescence or adulthood - fetal ethanol exposed adolescent ethanol observers and demonstrators significantly differed in their odor-mediated response to ethanol odor both between themselves and from their respective water controls. Nonetheless, adolescent ethanol re-exposure as a demonstrator, like an observer, enhanced the reflexive sniffing response to ethanol odor at both testing ages by augmenting the known effects of prior fetal ethanol experience. At each age, the magnitude of the enhanced odor response in demonstrators was similar to that of observers. Interestingly, only re-exposure as a demonstrator resulted in persistence of the behavioral response into adulthood in both sexes. CONCLUSIONS: The method of ethanol re-exposure plays an important role in prolonging the odor-mediated effects of fetal exposure. While ethanol odor-specific exposure through social interaction is important, additional factors such as the pairing of retronasal and hematogenic olfaction with ethanol's intoxicating properties appear necessary to achieve persistence in both sexes.

Ontogeny of the enhanced fetal-ethanol-induced behavioral and neurophysiologic olfactory response to ethanol odor.

BACKGROUND: Studies report a fundamental relationship between chemosensory function and the responsiveness to ethanol, its component orosensory qualities, and its odor as a consequence of fetal ethanol exposure. Regarding odor, fetal exposed rats display enhanced olfactory neural and behavioral responses to ethanol odor at postnatal (P) day 15. Although these consequences are absent in adults (P90), the behavioral effect has been shown to persist into adolescence (P37). Given the developmental timing of these observations, we explored the decay in the response to ethanol odor by examining ages between P37 and young adulthood. Moreover, we sought to determine whether the P15 neurophysiologic effect persists, at least, to P40. METHODS: Behavioral and olfactory epithelial (OE) responses of fetal ethanol exposed and control rats were tested at P40, P50, P60, or P70. Whole-body plethysmography was used to quantify each animal's innate behavioral response to ethanol odor. We then mapped the odorant-induced activity across the OE in response to different odorants, including ethanol, using optical recording methods. RESULTS: Relative to controls, ethanol exposed animals showed an enhanced behavioral response to ethanol odor that, while significant at each age, decreased in magnitude. These results, in conjunction with previous findings, permitted the development of an ontologic odor response model of fetal exposure. The fitted model exemplifies that odor-mediated effects exist at birth, peak in adolescence and then decline, becoming absent by P90. There was no evidence of an effect on the odor response of the OE at any age tested. CONCLUSIONS: Fetal exposure yields an enhanced behavioral response to ethanol odor that peaks in adolescence and wanes through young adulthood. This occurs absent an enhanced response of the OE. This latter finding suggests that by P40 the OE returns to an ethanol "neutral" status and that central mechanisms, such as ethanol-induced alterations in olfactory bulb circuitry, underlie the enhanced behavioral response. Our study provides a more comprehensive understanding of the ontogeny of fetal-ethanol-induced olfactory functional plasticity and the behavioral response to ethanol odor.

Lingering Effects of Prenatal Alcohol Exposure on Basal and Ethanol-Evoked Expression of Inflammatory-Related Genes in the CNS of Adolescent and Adult Rats.

Emerging data suggest that alcohol's effects on central inflammatory factors are not uniform across the lifespan. In particular, prenatal alcohol exposure (PAE) significantly alters steady-state levels of neuroimmune factors, as well as subsequent reactivity to later immune challenge. Thus, the current experiment investigated developmental sensitivities to, and long-lasting consequences of, PAE on ethanol-evoked cytokine expression in male and female adolescent and adult rats. Pregnant dams received either an ad libitum ethanol liquid diet (2.2% GD 6-8; 4.5% GD 9-10; 6.7% GD11-20; 35% daily calories from ethanol) or free-choice access to a control liquid diet and water. At birth, offspring were fostered to dams given free-choice access to the control liquid diet. Pups then matured until mid-adolescence [postnatal day (PD) 35] or adulthood (PD90), at which time they were challenged with either a binge-like dose of ethanol (4 g/kg; intragastrically) or tap water. During intoxication (3 h post-ethanol challenge), brains and blood were collected for assessment of neuroimmune gene expression (reverse transcription-polymerase chain reaction; RT-PCR) in the hippocampus, amygdala, and PVN, as well as for blood ethanol concentrations (BEC) and plasma corticosterone levels. Results revealed that rats challenged with ethanol at either PD35 or PD90 generally exhibited a characteristic cytokine signature of acute intoxication that we have previously reported: increased Il-6 and IkBα expression, with decreased Il-1ß and Tnfα gene expression. With a few exceptions, this pattern of gene changes was observed in all three structures examined, at both ages of postnatal ethanol challenge, and in both sexes. While few significant effects of PAE were observed for ethanol-induced alterations in cytokine expression, there was a consistent (but nonsignificant) trend for PAE to potentiate the expression of Il-6 and IkBα in all groups except adult females. Although these data suggest that later-life ethanol challenge was a far greater driver of inflammatory signaling than PAE, the current results demonstrate PAE resulted in subtle long-term alterations in the expression of many key neuroinflammatory factors associated with NF-κB signaling. Such long-lasting impacts of PAE that may engender vulnerability to later environmental events triggering neuroinflammatory processes, such as chronic ethanol exposure or stress, could contribute to heightened vulnerability for PAE-related alterations and deficits.

Adolescent ethanol experience alters immediate and long-term behavioral responses to ethanol odor in observer and demonstrator rats.

BACKGROUND: The social transmission of food preference paradigm centers on the finding that observers obtain dietary information through olfactory cues on the breath of a demonstrator peer that has ingested a novel substance. This phenomenon plays a role in ethanol acceptability. Historically, studies using this technique have focused on observer animals in order to study the social transmission process. With respect to ethanol, studies of acute intoxication have shown that the pharmacologic properties of ethanol and hematogenic olfaction can influence the subsequent ethanol odor-mediated responses of the intoxicated animals. These acute studies, however, demonstrate odor aversion. The present study compared the effect of adolescent ethanol exposure, via the social transmission paradigm, on the behavioral response to ethanol odor in both observer and demonstrator animals in adolescence (postnatal day (P) 37) and the persistence of these effects into adulthood (P90). METHODS: Beginning on P29, naïve rats received four ethanol or water exposures: one every 48 hours through either direct intragastric infusion or social interaction with an infused peer. The reflexive sniffing response of observers and demonstrators to ethanol odor was tested at P37 or P90 using whole-body plethysmography. RESULTS: The behavioral response of adolescent ethanol observers and demonstrators significantly differed between themselves and from their respective water controls. Ethanol and water observers both displayed a greater response to ethanol odor than their respective demonstrator counterparts. Compared to controls, both modes of ethanol exposure produced similar magnitudes of enhancement. At P90, both forms of exposure displayed similar responses to ethanol odor and similar magnitudes of enhancement. Only demonstrators displayed equivalent enhanced responses in both sexes. CONCLUSION: In contrast to previous studies showing odor aversion following acute ethanol intoxication, within the context of the social transmission paradigm, adolescent demonstrators like observers showed an enhanced behavioral response to ethanol odor. The differential enhanced odor response between observers and demonstrators, despite similar net enhancements relative to controls, suggests the presence of a stress effect from the infusion technique. This finding contrasts previous suggestions that intragastric infusions create minimal stress: an important consideration when conducting ethanol research. This stress effect appears to ameliorate by adulthood.

The consequence of fetal ethanol exposure and adolescent odor re-exposure on the response to ethanol odor in adolescent and adult rats.

BACKGROUND: An epidemiologic predictive relationship exists between fetal ethanol exposure and the likelihood for adolescent use. Further, an inverse relationship exists between the age of first experience and the probability of adult abuse. Whether and how the combined effects of prenatal and adolescent ethanol experiences contribute to this progressive pattern remains unknown. Fetal ethanol exposure directly changes the odor attributes of ethanol important for both ethanol odor preference behavior and ethanol flavor perception. These effects persist only to adolescence. Here we tested whether adolescent ethanol odor re-exposure: (Experiment 1) augments the fetal effect on the adolescent behavioral response to ethanol odor; and/or (Experiment 2) perpetuates previously observed adolescent behavioral and neurophysiological responses into adulthood. METHODS: Pregnant rats received either an ethanol or control liquid diet. Progeny (observers) experienced ethanol odor in adolescence via social interaction with a peer (demonstrators) that received an intragastric infusion of either 1.5 g/kg ethanol or water. Social interactions were scored for the frequency that observers followed their demonstrator. Whole-body plethysmography evaluated the unconditioned behavioral response of observers to ethanol odor in adolescence (P37) or adulthood (P90). The olfactory epithelium of adults was also examined for its neural response to five odorants, including ethanol. RESULTS: Experiment 1: Relative to fetal or adolescent exposure alone, adolescent re-exposure enhanced the behavioral response to ethanol odor in P37 animals. Compared to animals with no ethanol experience, rats receiving a single experience (fetal or adolescent) show an enhanced, yet equivalent, ethanol odor response. Fetal ethanol experience also increased olfactory-guided following of an intoxicated peer. Experiment 2: Combined exposure yielded persistence of the behavioral effects only in adult females. We found no evidence for persistence of neurophysiological effects in either sex. CONCLUSION: Fetal ethanol exposure influences adolescent re-exposure, in part, by promoting interactions with intoxicated peers. Re-exposure subsequently enhances ethanol odor responsivity during a key developmental transition point for emergent abuse patterns. While persistence of behavioral effects occurred in females, the level of re-exposure necessary to uniformly yield persistence in both sexes remains unknown. Nonetheless, these results highlight an important relationship between fetal and adolescent experiences that appears essential to the progressive pattern of developing ethanol abuse.

The effect of gestational ethanol exposure on voluntary ethanol intake in early postnatal and adult rats.

Clinical and epidemiological studies provide strong data for a relationship between prenatal ethanol exposure and the risk for abuse in adolescent and young adult humans. However, drug-acceptance results in response to fetal exposure have differed by study, age at evaluation, and experimental animal. In the present study, the authors tested whether voluntary ethanol intake was enhanced in both the infantile and adult rat (15 and 90 days of age, respectively), as a consequence of chronic fetal drug experience. Experimental rats were exposed in utero by administering ethanol to a pregnant dam in a liquid diet during gestational Days 6-20. Compared with those for isocaloric pair-fed and ad lib chow control animals, the results for experimental animals demonstrated that fetal exposure significantly increased infantile affinity for ethanol ingestion without affecting intake patterns of an alternative fluid (water). Heightened affinity for ethanol was absent in adulthood. Moreover, the results argue against malnutrition as a principal factor underlying the infantile phenomenon. These data add to a growing literature indicative of heightened early postnatal acceptance patterns resulting from maternal use or abuse of ethanol during pregnancy.

Experience-induced fetal plasticity: the effect of gestational ethanol exposure on the behavioral and neurophysiologic olfactory response to ethanol odor in early postnatal and adult rats.

Human fetal ethanol exposure is strongly associated with ethanol avidity during adolescence. Evidence that intrauterine olfactory experience influences chemosensory-guided postnatal behaviors suggests that an altered response to ethanol odor resulting from fetal exposure may contribute to later abuse risk. Using behavioral and neurophysiological methods, the authors tested whether ethanol exposure via the dam's diet resulted in an altered responsiveness to ethanol odor in infant and adult rats. Compared with controls, (a) fetal exposure tuned the neurophysiologic response of the olfactory epithelium to ethanol odor at some expense to its responsiveness to other odorants in infantile rats--this effect was absent in adults; (b) the neural effect in infantile rats was paralleled by an altered behavioral response to ethanol odor that was specific to this odorant--this effect was also absent in adults; and (c) a significant component of the infantile behavioral effect was attributable to ethanol's effect on the olfactory neural modality. These data provide evidence for an important relationship between prenatal ethanol experience and postnatal behavioral responsiveness to the drug that is modulated or determined by olfactory function.

Odorant identification and quality perception following methyl bromide-induced lesions of the olfactory epithelium.

Using a 5-odorant identification confusion matrix task, the authors assessed the consequence of olfactory epithelial damage on odorant quality perception in the rat. After establishing prelesion identification performance, each rat's epithelium was subjected to 330 ppm methyl bromide gas for 6 hr. Comparison of prelesion and 3-day postlesion performance demonstrated a significant decrease in identification as a consequence of 95%-98% epithelial destruction. Further, there was a differential effect of lesion on the ability of different animals to identify the different individual odorants. Evaluation of the anatomical state of the epithelium relative to performance on the identification task demonstrated a significant relationship between the extent and location of anatomical sparing and changes in individual odorant identifications. Assessment of pre- and postlesion quality perception for the individual rats demonstrated a highly significant shift in quality perception that was independent of any decrease in performance. These results provide strong support for the proposition that the regional variations in mucosal sensitivities within and across olfactory receptor gene expression zones are fundamentally important for the encoding of odorant quality.

Predicting odorant quality perceptions from multidimensional scaling of olfactory bulb glomerular activity patterns.

Odorants and their perceptions differ along multiple dimensions, requiring that a critical examination of any putative neural code directly assess the multidimensional nature of the encoding process. Previous work has examined simple, systematic odorant differences that, regardless of coding strategy, would be expected to produce simple, systematic predictions in neural and behavioral responses. In the present study, an odorant identification confusion matrix task that extracts precise quality relationships across odorants was used to determine whether spatially specific glomerular activity patterns predict perceptual quality relationships for odorants that cannot easily be classified a priori along a single chemical dimension. Multidimensional scaling (MDS) analysis of odorant pattern similarity measures derived from the comparison of [14C]-2-deoxyglucose glomerular activity pattern data yielded a two-dimensional odorant activity space that was highly significantly predictive of similarly obtained odorant perceptual spaces, uniformly across animals. These results strongly support the relevance of global spatial patterns in the olfactory bulb to the encoding of odor quality.

Odorant receptor expression patterns are restored in lesion-recovered rat olfactory epithelium.

Lesions of the olfactory periphery provide a means for examining the reconstitution of a diverse and highly regulated population of sensory neurons and the growth, en masse, of nascent axons to the bulb. The olfactory epithelium and its projection onto the bulb are reconstituted after ablation by methyl bromide gas, and some measure of olfactory function is restored. The extent to which the system regenerates the full repertoire of odorant receptor-expressing neurons, particularly their spatially restricted distribution across the epithelial sheet, is unknown, however, and altered odorant receptor expression might contribute to the persistent distortion of odorant quality that is observed in the lesioned-recovered animals. To address the question of receptor expression in the recovered epithelium, we performed in situ hybridization with digoxigenin-labeled riboprobes for eight odorant receptors on the olfactory epithelium from unilaterally methyl bromide-lesioned and control rats. The data demonstrate that the distribution of sensory neuron types, as identified and defined by odorant receptor expression, is restored to normal or nearly so by 3 months after lesion. Likewise, the numbers of probe-labeled neurons in the lesioned-recovered epithelium are nearly equivalent to the unlesioned side at this time. Finally, our evidence suggests that odorant receptors are distributed in multiple overlapping bands in the normal, unlesioned, and lesioned-recovered epithelium rather than in the conventionally accepted three or four zones. Thus, the primary sensory elements required for functional recovery of the olfactory system after damage are restored, and altered function implies the persistence of a more central failure in regeneration.

Globose basal cells are required for reconstitution of olfactory epithelium after methyl bromide lesion.

Despite a remarkable regenerative capacity, recovery of the mammalian olfactory epithelium can fail in severely injured areas, which subsequently reconstitute as aneuronal respiratory epithelium (metaplasia). We contrasted the cellular response of areas of the rat epithelium that recover as olfactory after methyl bromide lesion with those undergoing respiratory metaplasia in order to identify stem cells that restore lesioned epithelium as olfactory. Ventral olfactory epithelium is at particular risk for metaplasia after lesion and patches of it are rendered acellular by methyl bromide exposure. In contrast, globose basal cells (GBCs, marked by staining with GBC-2) are preserved in surrounding ventral areas and uniformly throughout dorsal epithelium, which consistently and completely recovers as olfactory after lesion. Over the next few days, neurons reappear, but only in those areas in which GBCs are preserved and multiply. In contrast, parts of the epithelium in which GBCs are destroyed are repopulated in part by Bowman's gland cells, which pile up above the basal lamina. Electron microscopy confirms the reciprocity between gland cells and globose basal cells. By 14 days after lesion, the areas that are undergoing metaplasia are repopulated by typical respiratory epithelial cells. As horizontal basal cells are eliminated from all parts of the ventral epithelium, the data suggest that GBC-2(+) cells are ultimately responsible for regenerating olfactory neuroepithelium. In contrast, GLA-13(+) cells may give rise to respiratory metaplastic epithelium where GBCs are eliminated. Thus, we support the idea that a subpopulation of GBCs is the neural stem cell of the olfactory epithelium.

Expression patterns of basic helix-loop-helix transcription factors define subsets of olfactory progenitor cells.

Direct damage to the olfactory epithelium by inhalation of the olfactotoxin methyl bromide activates a population of multipotent globose basal cells, which reconstitute all depleted cell populations. Because members of the basic helix-loop-helix family of transcription factors are known to regulate neurogenesis and cell production, we performed in situ hybridization to examine the expression of several members of that family during the recovery of the rat olfactory epithelium after methyl bromide lesion. The numbers of basal cells expressing the proneural transcriptional activators Mash1, Neurogenin1, and NeuroD all fall precipitously 1 day after lesion. Mash1 levels begin to recover by 2 days, Neurogenin1 and NeuroD by 3 days, and substantial numbers of neurons reappear by 4 days. The antineurogenic factor Hes1 is limited to the sustentacular cells of the unlesioned olfactory epithelium and to the adjoining respiratory epithelium. Immediately after methyl bromide lesion, but not at any time after bulbectomy, a large fraction of residual, marker-confirmed globose basal cells initiate expression of Hes1. Subsequently, the Hes1-positive cells lose their association with the basal lamina, shift apically, and differentiate into sustentacular cells. In contrast, Hes5 is expressed by a small subset of globose basal cells and by olfactory ensheathing glia in the normal mucosa; Hes5 label disappears from both transiently after lesion. In sum, the recovery of the neuronal population after peripheral lesion recapitulates the sequence of transcription factor expression observed during embryonic development of the epithelium. Moreover, expression of Hes1 marks that population of globose basal cells committed to making sustentacular cells after methyl bromide lesion.

Altered epithelial density and expansion of bulbar projections of a discrete HSP70 immunoreactive subpopulation of rat olfactory receptor neurons in reconstituting olfactory epithelium following exposure to methyl bromide.

A previously described subpopulation of rat olfactory receptor neurons, the 2A4(+)ORNs, is 1) distinguished by intense constitutive cytoplasmic immunoreactivity to antibodies to the 70-kD heat shock protein (HSP70); 2) occurs sparsely but consistently through ventral and lateral olfactory epithelium (OE); and 3) projects to just two to three consistently located glomeruli in each olfactory bulb (OB) (Carr et al. [1994] J Comp Neurol 348:150-160). Immunoreactivity appears not to be stress-related. To examine the persistence of these features following destruction and reconstitution of the OE, rats were subjected to methyl bromide-induced OE lesion (Schwob et al. [1995] J Comp Neurol 59:15-37; Schwob et al. [1999] J Comp Neurol 412:439-457] and their OE and OBs examined with antibodies to HSP70 6-10.5 weeks postlesion. Lesioned OE showed significantly increased 2A4(+)ORN densities but no alteration of 2A4(+)ORN zonal distribution. The OBs of lesioned animals showed marked expansions of 2A4(+)ORN bulbar projections, with 2-15-fold increases in numbers of glomeruli showing 2A4(+)axons, and projection expansions were greater in animals maintained on chronic food restriction prior to lesioning. Examination of archival 5-month post-MeBr lesion material indicates that altered projection patterns are maintained.