Acute Regulation of Habituation Learning via Posttranslational Palmitoylation

Abstract

Habituation is an adaptive learning process that enables animals to adjust innate behaviors to changes in their environment. Despite its well-documented implications for a wide diversity of behaviors, the molecular and cellular basis of habituation learning is not well understood. Using whole-genome sequencing of zebrafish mutants isolated in an unbiased genetic screen, we identified the palmitoyltransferase Huntingtin interacting protein 14 (Hip14) as a critical regulator of habituation learning. We demonstrate that Hip14 regulates depression of sensory inputs onto an identified hindbrain neuron and provide evidence that Hip14 palmitoylates the Shaker-like K⁺ voltage-gated channel subunit (Kv1.1), thereby regulating Kv1.1 subcellular localization. Furthermore, we show that, like for Hip14, loss of Kv1.1 leads to habituation deficits and that Hip14 is dispensable in development and instead acts acutely to promote habituation. Combined, these results uncover a previously unappreciated role for acute posttranslational palmitoylation at defined circuit components to regulate learning.

Keywords: Hip14; Kv1.1; Mauthner; Zdhhc17; axon cap; behavior; genetic screen; habituation learning; startle; zebrafish.

Figure 1.. Hip14 is required for habituation learning in the larval zebrafish.

(A) Acoustic stimulation protocol used to induce habituation learning. Vertical lines indicate acoustic stimuli. 10 stimuli are presented with 20-second ISI to assess baseline startle responsiveness. 30 stimuli are then presented with 1-second ISI to induce habituation learning. (B) Habituation curves for sibling and p174 mutant animals. Startle responses are averaged across bins of 10 stimuli. (Baseline = 10 stimuli at 20 second ISI, 1–10 = 1st bin of 10 stimuli at 1-second ISI, 11–20 = 2nd bin of 10 stimuli at 1-second ISI, 21–30 = 3rd bin of 10 stimuli at 1-second ISI). Mean response frequency within each bin ± SD are depicted, n≥19 larvae per genotype. (C) Predicted polypeptide domain structure of Hip14 and Hip14 mutant alleles. Gray indicates ankyrin repeat domains, green indicates transmembrane domains, pink indicates DHHC catalytic domain. Inset depicts domain conformations. (D) Complementation testing to confirm mapping of habituation phenotype to hip14 nonsense mutation. % Habituation = (1−[response frequency 21–30] $÷$ [response frequency baseline])*100. Mean ± SD, n ≥18 larvae per genotype. *** indicates Kruskall-Wallis test with Dunn’s multiple comparisons test for multiple comparisons p<0.0001.

Figure 2.. hip14 mutants fail to exhibit synaptic depression at the Mauthner lateral dendrite.

(A) Diagram of auditory nerve inputs onto the Mauthner cell. The lateral dendrite, the site of these inputs, is one known locus for acoustic startle habituation. (B) Single frames showing GCaMP6s expressed in the Mauthner cell in an unstimulated animal and (C) following a high-intensity acoustic stimulus. (D) Habituation curves for head-embedded hip14 mutant and sibling animals. Baseline = average of 5 responses at 2-minute ISI, 1–8 = 1st bin of 8 responses at 5-second ISI, 9–16 = 2nd bin of 8 responses at 5-second ISI, 17–24 = 3rd bin of 8 responses at 5-second ISI. Error bars indicate standard deviation. Mean ± SD, n=4 larvae per genotype. (E) Final 4 Mauthner soma responses (stimuli #21–24). By this point, sibling animals are fully habituated and no longer perform startle responses. Concomitantly, Ca²⁺ activity in the Mauthner soma has subsided. hip14 mutants show no/minimal behavioral habituation and exhibit robust Ca²⁺ responses in the soma. Mean ± shading indicates SEM, n=4 larvae per genotype. (F) Lateral dendrite responses before and after habituation in sibling and hip14 mutant animals. Note the significant reduction in lateral dendrite Ca²⁺ activity in siblings (black), whereas hip14 mutants show little/no such reduction (red) Mean lateral dendrite response, n=4 larvae per genotype. Mutant lateral dendrite responses undergo significantly less depression than WT responses, unpaired t test Habituated Responses $÷$ Baseline Responses, p=0.0021. Scale bar 10μm. See also Figure S1.

Figure 3.. Hip14 acts independently of previously established habituation pathways.

(A) Pathway diagram indicating cellular mechanisms of action for PAPP-AA and NF1. Red text denotes drugs that impinge on individual pathway elements. Dotted lines indicate biochemical interactions between proteins that may or may not play a functional role in regulating habituation learning. (B-D) Habituation rates in hip14 mutants and sibling larvae are unaffected by pharmacological manipulation of known habituation pathways: (B) 10μM Rolipram (or DMSO control) applied 30 minutes prior to and throughout behavior testing. (C) 1μM U0126 (or DMSO control) applied 30 minutes prior to and throughout behavior testing. (D) 1μM SC-79 (or DMSO control) applied from 3dpf-5dpf and throughout behavior testing. See also Figure S2.

Figure 4.. Kv1.1 is required for habituation learning in the larval zebrafish.

(A) Habituation curves for p181 mutant animals and siblings. Startle responses are averaged across bins of 10 stimuli (as in Figure 1A) Mean response frequency within each bin ± SD are depicted n≥57 per genotype (B) Predicted polypeptide domain structure of kcna1a mutant alleles, (C) domain structure of Kv1.1. Transmembrane domains are in green and pore helix domain is shown in pink. Orange arrow indicates N250K missense mutation identified in Kv1.1 encoded by kcna1a^p181. (D) Alignment demonstrating strong conservation of Kv1.1 S2-S3 linker and S3 domain sequences. Note that the N250K missense mutation identified in our screen is equivalent to the N255K missense mutation associated with human PKD disease. (E-G) Representative whole-cell K⁺ family currents evoked by 200-ms voltage pulses from −80 to +90mV in 10mV increments from a holding potential of −80mV as in Figure 1E-top in N2a cells transfected with (E) vector only (control); cell capacitance of 15.5 pF, (F) wild type Kv1.1; cell capacitance of 18.2 pF, (G) KV1.1^N250K; cell capacitance of 14.9 pF. For details of solutions see the Methods section. Dashed lines indicate 0-current level. (H) Steady-state current-voltage (I-V) relations for Vector (control), wild type Kv1.1 and KV1.1^N250K, obtained by measurements of the currents at the end of 200-ms pulses, normalized by the cell capacitance. e.g., the normalized currents at +90 mV are 226.9 ± 54.5 (pA/pF) for wild type (n=6), 12.8 ± 1.0 (pA/pF) for KV1.1^N250K (n =7), and 11.2 ± 1.2 (pA/pF) for vector (n = 5). Two-tailed Student’s unpaired t-test: P=0.003, t₉ = 3.89 for wild type and P = 0.315 and t₁₀=1.06 for KV1.1^N250K, compared to vector, indicating KV1.1^N250K is a nonfunctional channel. The cells used in these measurements had the whole-cell capacitance of 14.8 ± 1.6 (pF) (n = 5) for vector, 15.4 ± 0.8 (pF) (n = 6) for wild type, and 14.9 ± 1.3 (pF) (n = 7) for KV1.1^N250K, respectively. (I) Complementation testing to confirm mapping of habituation phenotype to kcna1a missense mutation. Mean ± SD, n ≥9 larvae per genotype. P values for Kruskall-Wallis test with Dunn’s multiple comparisons test are indicated.

Figure 5.. Hip14 palmitoylates Kv1.1 and regulates its localization in vivo.

(A-B) Acyl Biotin Exchange assays, in which Kv1.1-Flag +/− Hip14-HA are transfected into HEK cells. Immunoblot indicates palmitoylation of WT Kv1.1-Flag in lanes with functional co-transfected Hip14-HA, and effects of various mutations in Kv1.1 and Hip14. Only in the presence of WT Hip14, WT Kv1.1 and hydroxylamine (+HAM) does robust palmitoylation of Kv1.1 occur. Samples without hydroxylamine (−HAM) are negative controls for the ABE reactions. (C) Immunohistochemical labeling of the Mauthner cell (green) and Kv1.1 (magenta) in gffDMC130a; uas:gap43:citrine sibling, using Chicken anti-GFP, and Rabbit anti-Kv1.1 antibodies. Note the bright localization of Kv1.1 to the axon cap: synaptic inputs from spiral fiber neurons onto Mauthner cell AIS. (D) Immunohistochemical labeling of a hip14 mutant animal as in (C). Note the reduction in Kv1.1 signal at the axon cap. (E) Quantification of Kv1.1 signal in mutants as compared to WT. Mean signal intensity = pixel intensity for axon cap region of interest (inset) divided by axon cap area. Unpaired t-test p=0.0021. Scale bar 10μm. Mean ± SD, n=18 axon caps per genotype (n≤2 axon caps per animal). See also Figure S3.

Figure 6.. Hip14 acutely regulates habituation learning.

(A)Timeline depicting startle circuit development and heat shock timing. Numbers indicate hours post-fertilization. Red vertical lines indicate start times for heat-shock (96 hours in (B) and 117 hours in (D)) (B) Rates of habituation in animals heat-shocked at 37°C at 96 hours post-fertilization (4dpf). (C) Rates of habituation in animals without heat-shock. (D) Rates of habituation in animals heat-shocked at 37°C at 117 hours post-fertilization (5dpf). In B-D, testing is always performed at 120hpf (5dpf). Transgene = hsp70:hip14:p2a:mkate; Mean ± SD, n ≥12 per genotype; *** indicates p<0.0001; NS = Not Significant. See also Figure S4.