ZENK Expression Research Articles

CB1 cannabinoid receptor activation inhibits a neural correlate of song recognition in an auditory/perceptual region of the zebra finch telencephalon.

A notable consequence of CB1 cannabinoid receptor activation in vertebrates is an impairment of cognitive function related to learning and short-term memory. The mechanisms of this impairment remain unclear, but one possibility is that cannabinoids influence encoding of stimuli at sensory and/or perceptual levels. Here, by treating zebra finches with the cannabinoid agonist WIN55212-2 and then measuring expression of the transcription factor zenk following presentation of novel zebra finch song, we show that cannabinoid receptor activation differentially influences zenk expression in sensory versus perceptual regions of the songbird auditory telencephalon. That is, WIN55212-2 dose-dependently inhibited zenk expression in a region for auditory perception (NCM, the caudomedial neostriatum), but had no effect on zenk expression in the primary auditory area, the Field L complex. The inhibitory effects of WIN55212-2 on zenk expression in NCM were reversed by coadministration of the CB1-selective antagonist SR141716A. Moreover, we found that the habituation of the NCM zenk response to repeated presentation of the same song, a well-established neural correlate of song recognition, was blocked when birds were treated with WIN55212-2 during habituation trials. Our data suggest that activation of CB1 cannabinoid receptors can selectively influence perceptual and mnemonic aspects of auditory experience.

Recent experience modulates forebrain gene-expression in response to mate-choice cues in European starlings.

Mate-choice decisions can be experience dependent, but we know little about how the brain processes stimuli that release such decisions. Female European starlings (Sturnus vulgaris) prefer males with long-bout songs over males with short-bout songs, and show higher expression of the immediate early gene (IEG) ZENK in the auditory forebrain when exposed to long-bout songs than when exposed to short-bout songs. We exposed female starlings to a short-day photoperiod for one of three durations and then, on an increased photophase, exposed them to one week of long-bout or short-bout song experience. We then examined their IEG response to novel long-bout versus novel short-bout songs by quantifying ZENK protein in two song-processing areas: the caudo-medial hyperstriatum ventrale and the caudo-medial neostriatum. ZENK expression in both areas increased with tenure on short-day photoperiods, suggesting that short days sensitize females to song. The ZENK response bias toward long-bout songs was greater in females with long-bout experience than in females with short-bout experience, indicating that the forebrain response bias toward a preferred trait depends on recent experience with that category of trait. This surprising level of neuroplasticity is immediately relevant to the natural history and fitness of the organism, and may underlie a mechanism for optimizing mate-choice criteria amidst locally variable distributions of secondary sexual characteristics.

Contact call-driven Zenk protein induction and habituation in telencephalic auditory pathways in the Budgerigar (Melopsittacus undulatus): implications for understanding vocal learning processes.

Expression of the immediate early gene protein Zenk (zif 268, egr-1, NGF1A, Krox24) was induced in forebrain auditory nuclei in a vocal learning parrot species, the budgerigar (Melopsittacus undulatus), when the subjects either listened to playbacks of an unfamiliar contact call or to a contact call with which they had been familiarized previously. Auditory nuclei included the Field L complex (L1, L2a, and L3), the neostriatum intermedium pars ventrolateralis (NIVL), the neostriatum adjacent to caudal nucleus basalis (peri-basalis or pBas), an area in the frontal lateral neostriatum (NFl), the supracentral nucleus of the lateral neostriatum (NLs), and the ventromedial hyperstriatum ventrale (HVvm). The latter three nuclei are main sources of auditory input to the vocal system. Two patterns of nuclear staining were induced by contact call stimulation-staining throughout cell nuclei, which was exhibited by at least some neurons in all areas examined except L2a and perinucleolar staining, which was the only kind of staining exhibited in field L2a. The different patterns of Zenk staining indicate that auditory stimulation may regulate the Zenk-dependent transcription of different subsets of genes in different auditory nuclei. The numbers of neurons expressing Zenk staining increased from seven- to 43-fold over control levels when the birds listened to a repeating unfamiliar call. Familiarization of the subjects with the call stimulus, through repeated playbacks, greatly reduced the induction of Zenk expression to the call when it was presented again after an intervening 24-h interval. To determine if neurons exhibiting contact call-driven Zenk expression project to the vocal control system, call stimulation was coupled with dextran amines pathway tracing. The results indicated that tracer injections in the vocal nucleus HVo (oval nucleus of the hyperstriatum ventrale), in fields lateral to HVo and in NLs labeled many Zenk-positive neurons in HVvm, NFl, and NLs. These results support the idea that, in these neurons, egr-1 couples auditory stimulation to the synthesis of proteins involved in either the storing of new perceptual engrams for vocal learning or the processing of novel and/or meaningful acoustic stimuli related to vocal learning or the context in which it occurs.

Expression of Transcription FactorZENK(zif/268) in Telencephalon of Quail Chicks after Induced Seizure and Passive Avoidance Training

Functional significance of an immediate early gene ZENK (zif/268) was examined in telencephalic regions (homologues of neocortex and basal ganglia) of newly-hatched quail chicks; hyperstriatum accessorium (HA), hyperstriatum ventrale (HV), neostriatum (N) and lobus parolfactorius (LPO). Chicks were trained by a green bead soaked either in a strong aversant (methylanthranilate, MeA), in a weak aversant (MeA diluted by ethanol, 1/3MeA), or in water. Chicks were then tested at 45–50 min post-training, and immediately processed for ZENK immunostaining. Neither the training condition (MeA, 1/3MeA, or water) nor the responses at test (recall or amnesia) significantly contributed to the immunopositive cell densities in all of these regions. On the other hand, single intraperitoneal injection of metrazole (CNS convulsant) induced a transient epileptiform seizure, and caused significantly enhanced ZENK expression in HV and LPO but not in HA and N. However, the metrazol-induced seizure did not interfere with the following passive avoidance training, and chicks successfully learned to avoid the aversive bead when tested at 24 hr subsequently. Among three groups of chicks (metrazol-treated, saline control, and untreated chicks), no significant differences were found in their responses at test (recall, generalized avoidance, or amnesia). These results suggest that enhanced ZENK expression may represent lasting neural activities, but may not be involved specifically in the passive avoidance memory formation.

Post-transcriptional regulation of zenk expression associated with zebra finch vocal development

In the male zebra finch, highly variable juvenile song and stereotyped adult song induce mRNA expression of the immediate early gene zenk in telencephalon. However, the functional consequences of this behavior-driven gene expression remain unknown. Here we characterize the developmental expression of zenk mRNA and protein in two forebrain song regions (HVC, the higher vocal center, and RA, the robust nucleus of the archistriatum). In HVC, singing results in similar percentages of cells producing zenk mRNA and zenk protein at different stages of vocal development. Similarly, song behavior at all stages of vocal development induces a comparable percentage of RA cells expressing zenk mRNA. However, the percentage of RA zenk immunoreactive cells is low during early vocal learning, increasing only as the vocal pattern matures. Early induction of a stereotyped vocal pattern in juvenile birds is associated with increased zenk immunoreactivity in RA, indicating that it is the form of the behavior (and not the age of the bird) that correlates with changes in zenk immunoreactivity. Together, our findings reveal a previously unrecognized relationship between behavioral development and post-transcriptional gene regulation.

Response biases in auditory forebrain regions of female songbirds following exposure to sexually relevant variation in male song.

In many species of songbirds, individual variation between the songs of competing males is correlated with female behavioral preferences. The neural mechanisms of song based female preference in songbirds are not known. Working with female European starlings (Sturnus vulgaris), we used immunocytochemistry for ZENK protein to localize forebrain regions that respond to sexually relevant variation in conspecific male song. The number of ZENK-ir cells in ventral caudo-medial neostriatum [NCMv] was significantly higher in females exposed to longer songs than in those exposed to shorter songs, whereas variation in the total duration of song exposure yielded no significant differences in ZENK expression. ZENK expression in caudo-medial ventral hyperstriatum [cmHV] was uniformly high in all subjects, and did not vary significantly among the three groups. These results suggest that subregions of NCM in female starlings are tuned to variation in male song length, or to song features correlated therewith. Female starlings exhibit robust behavioral preferences for longer over shorter male songs (Gentner and Hulse; Anim Behav 59:443-458, 2000). Therefore, the results of this study strongly implicate NCM in at least a portion of the perceptual processes underlying the complex natural behavior of female choice.

Does sex or photoperiodic condition influence ZENK induction in response to song in European starlings?

Variables such as the photoperiod a bird experiences, or its sex, affect behavioral responses to song. The present experiment investigated whether song-induced expression of the immediate-early gene ZENK is also influenced by sex or photoperiod. We examined the expression of the protein product of ZENK in wild-caught male and female European starlings ( Sturnus vulgaris) in different photoperiodic conditions. In the first experiment, adult reproductively active male and female starlings were presented with either conspecific male song or no song in experimental chambers. In the second experiment, conspecific male song was presented to reproductively active and reproductively inactive adult female starlings. Localization of the ZENK protein product revealed that song stimulation resulted in a significant increase in the number of ZENK immunoreactive (-ir) cells in the caudomedial neostriatum (NCM) and the caudomedial hyperstriatum ventrale (cmHV) compared with unstimulated birds. No differences in the number of ZENK-ir neurons were observed between males and females or between reproductively active and inactive females. Thus, the present data indicate that the number of cells expressing ZENK in NCM and cmHV following song playback does not vary with sex or photoperiod in starlings.

Light- and focus-dependent expression of the transcription factor ZENK in the chick retina.

Ocular growth and refraction are regulated by visual processing in the retina. We identified candidate regulatory neurons by immunocytochemistry for immediate-early gene products, ZENK (zif268, Egr-1) and Fos, after appropriate visual stimulation. ZENK synthesis was enhanced by conditions that suppress ocular elongation (plus defocus, termination of form deprivation) and suppressed by conditions that enhance ocular elongation (minus defocus, form deprivation), particularly in glucagon-containing amacrine cells. Fos synthesis was enhanced by termination of visual deprivation, but not by defocus and not in glucagon-containing amacrine cells. We conclude that glucagon-containing amacrine cells respond differentially to the sign of defocus and may mediate lens-induced changes in ocular growth and refraction.

ZENK protein regulation by song in the brain of songbirds.

When songbirds hear the song of another individual of the same species or when they sing, the mRNA levels of the ZENK gene increase rapidly in forebrain areas involved in vocal communication. This gene induction is thought to be related to long-term neuronal change and possibly the formation of song-related memories. We used immunocytochemistry to study the levels and distribution of ZENK protein in the brain of zebra finches and canaries after presentation of song playbacks. Birds that heard the playbacks and did not sing in response showed increased ZENK protein levels in auditory brain areas, including the caudomedial neostriatum and hyperstriatum ventrale, fields L1 and L3, the shelf adjacent to the high vocal center (HVC), the cup adjacent to the nucleus robustus archistriatalis (RA), and the nucleus mesencephalicus lateralis pars dorsalis (MLd). No ZENK expression was seen in song nuclei in these birds. Males that sang in response to the playbacks showed, in addition to auditory areas, increased ZENK protein in several song control nuclei, most prominently in HVC, RA, area X, and the dorsomedial nucleus (DN) of the intercollicular complex. The rise in ZENK protein followed that described previously for ZENK mRNA by a short lag, and the distribution of ZENK-labeled cells was in agreement with previous analysis of mRNA distribution. Thus, ZENK protein regulation can be used to assess activation of brain areas involved in perceptual and motor aspects of song. Possible implications of ZENK induction in these areas are discussed.

Zenk expression in the brain of bengalese finch

Bird species with vocal learning possess a projection from the telencephalic nucleus to the nucleus nervi hypoglossi, pars tracheosyringealis (XIIts) in the medulla, where a final common pathway that controls the vocal organ, i.e., the synrinx, originates. The anatomical basis of this projection has not been well investigated in one species of songbird, the Bengalese finch (Lonchura striata var. domestica). The present study used anterograde and retrograde tracing experiments to examine and describe this projection in Bengalese finches. Following iontophoretic injections of biotinylated dextran amine into the telencephalic nucleus robustus arcopallialis (RA), we detected anterograde-labeled terminations in the XIIts. In addition, labeled terminals were seen in other vocal–respiratory-related nuclei, such as the dorsomedial nucleus of the nucleus intercollicularis, nucleus infraolivaris superior, nucleus of the rostral ventrolateral medulla, nucleus parambigualis, nucleus ambiguous, and nucleus retroambigualis. Furthermore, following injections into the XIIts, we detected retrograde-labeled cell bodies scattered throughout the ipsilateral RA. The present results revealed that the direct projections of the RA to the XIIts in male Bengalese finches are similar to those in other songbirds with vocal learning abilities.

Enhanced ZENK ( zif-268) expression after passive avoidance task does not correlate with memory formation in neonatal chicks striata

WEST is a full metallic tokamak with an extensive set of diagnostics for heat load measurements. In this paper, heat loads on the lower divertor of WEST are investigated using two independent methods. A first method relies on the thermal inversion of temperature measurements from arrays of thermal sensors embedded a few millimeters below the surface, while the second consists in the inversion of black body surface temperatures measured by infra-red (IR) thermography. The challenge of IR based temperature measurements in the full metal environment of WEST is addressed through a simplified model, allowing to correct for global reflections and low surface emissivities of tungsten surfaces. A large database (>100 L-mode discharges) is investigated. It is found that the energy absorbed by an outer divertor tile during a plasma discharge is closely estimated by the two diagnostics, over a large set of experimental conditions. A similar match is also found for the peak heat flux value on the outer target. The toroidal modulation of target heat loads by magnetic ripple is found to be consistent with the geometrical projection of a parallel heat flux component. Additionally, the heat flux channel width at the target is found to scale linearly with the magnetic flux expansion as expected. These observations give confidence in the robustness of the data from both diagnostics, and confirm the simple geometrical rules at use in the description of heat flux deposition on divertor targets. However, it is shown that the heat flux channel width estimated from infra-red thermography is about three times lower than the width estimated from embedded measurements, which is still under investigation.

Brain gene regulation by territorial singing behavior in freely ranging songbirds.

To investigate the ecological relevance of brain gene regulation associated with singing behavior in songbirds, we challenged freely ranging song sparrows with conspecific song playbacks within their breeding territories. Males responded by approaching the speaker, searching for an intruder and actively singing. In situ hybridization of brain sections revealed significantly higher expression of the transcriptional regulator ZENK in challenged birds than in unstimulated controls in several auditory structures and song control nuclei. We conclude that singing behavior in the context of territorial defense is associated with gene regulation in brain centers that control song perception and production, and that behaviorally regulated gene expression can be used to investigate brain areas involved in the natural behaviors of freely ranging animals.

Motor-driven gene expression.

There is increased neuronal firing in the high vocal center (a motor nucleus) and other song nuclei of canaries, Serinus canaria, and zebra finches, Taeniopygia guttata, whenever these songbirds sing or hear song. These observations suggested that song perception involved sensory and motor pathways. We now show that the act of singing, but not hearing song, induces a rapid and striking increase (up to 60-fold) in expression of the transcriptional regulator ZENK in the high vocal center and other song nuclei. This motor-driven gene expression is independent of auditory feedback, since it occurs in deafened birds when they sing and in muted birds when they produce silent song. Conversely, hearing song, but not the act of singing, induces ZENK expression in parts of the auditory forebrain. Our observations show that even though the same auditory stimulus activates sensory and motor pathways, perception and production of song are accompanied by anatomically distinct patterns of gene expression.

Decrements in auditory responses to a repeated conspecific song are long-lasting and require two periods of protein synthesis in the songbird forebrain.

Earlier work showed that playbacks of conspecific song induce expression of the immediate early gene ZENK in the caudo-medial neostriatum (NCM) of awake male zebra finches and that this response disappears with repeated presentations of the same stimulus. In the present study, we investigated whether repetitions of a song stimulus also elicited a decrement in the electrophysiological responses in the NCM neurons of these birds. Multiunit auditory responses in NCM were initially vigorous, but their amplitude decreased (habituated) rapidly to repeated stimulation, declining to about 40% of the initial response during the first 50 iterations. A similar time course of change was seen at the single unit level. This habituation occurred specifically for each song presented but did not occur when pure tones were used as a stimulus. Habituation to conspecific, but not heterospecific, song was retained for 20 h or longer. Injections of inhibitors of protein or RNA synthesis at the recording site did not affect the initial habituation to a novel stimulus, but these drugs blocked the long-term habituation when injected at 0.5-3 h and at 5.5-7 h after the first exposure to the stimulus. Thus, at least two waves of gene induction appear to be necessary for long-lasting habituation to a particular song.

Associative learning and stimulus novelty influence the song-induced expression of an immediate early gene in the canary forebrain.

To identify variables that affect immediate early gene (IEG) expression in the auditory telencephalon of songbirds, we developed a conditioning paradigm that trained adult male canaries to associate song with a mild shock. Learning of the association was measured by a bird's fear and avoidance responses. Birds exposed to paired song and shock were compared to yoked controls exposed to each stimulus alone or to both unpaired. Additional groups examined the effects of attention and stress, and of the novelty of the stimulus situation. In situ hybridization analysis of brain sections revealed an enhancement of ZENK expression in birds learning the association between song and shock above levels induced by song alone or yoked-unpaired song and shock. This effect was specifically seen in the caudomedial auditory telencephalon (NCM-HVCM). A comparison of the several control groups indicated that novelty of the song stimulus or of its pairing with shock were the main variables that predicted ZENK levels in NCM-HVCM. These observations are compatible with ZENK playing a role in the formation of song perceptual memories.