Mice lacking the transcription factor Ikaros display behavioral alterations of an anti-depressive phenotype

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Experimental Neurology 211 (2008) 107 – 114 www.elsevier.com/locate/yexnr

Mice lacking the transcription factor Ikaros display behavioral alterations of an anti-depressive phenotype Tim-Rasmus Kiehl a,⁎, Sandra E. Fischer a , Shereen Ezzat b,c , Sylvia L. Asa a a

c

Department of Pathology, University Health Network, 610 University Avenue, Toronto, ON, Canada M5G 2M9 b Department of Medicine, Mount Sinai Hospital, 610 University Avenue, Toronto, ON, Canada M5G 2M9 The Freeman Centre for Endocrine Oncology and Ontario Cancer Institute, University of Toronto, 610 University Avenue, Toronto, ON, Canada M5G 2M9 Received 16 October 2007; accepted 11 January 2008 Available online 5 February 2008

Abstract The Ikaros (Ik) family of transcription factors has critical functions in immune regulation, lymphohematopoiesis and the hypothalamicpituitary axis. Ik influences cell fate decisions through transcriptional activation of target genes and its interaction with chromatin remodeling complexes. While Ik is well-described in the lymphoid system and pituitary, its presence and function in the brain has received limited attention to date. This study describes the transient spatio-temporal expression of Ik in striatal medium spiny neurons of the developing murine CNS. To determine the impact of Ik deficiency, standardized behavioral tests were performed. In the elevated plus-maze and contextual fear conditioning tests, homozygous Ik-deficient mice performed similarly to wild-type or heterozygote mice. However, significant differences were observed in Iknull mice in several behavioral tests. Pinch-induced catalepsy was markedly extended. In the Porsolt forced swim test, Ik-null mice showed reduced immobility, consistent with an anti-depressive effect. The acoustic startle response of Ik-null mice was also markedly diminished. Our findings extend the role of the Ikaros zinc-finger protein to the maturation and differentiation of striatal medium spiny neurons and indicate important actions for Ik in the development of neurocognitive functions and affecting depressive behaviors. © 2008 Elsevier Inc. All rights reserved. Keywords: Striatum; Brain; Ikaros; Depression; Medium spiny neuron

Introduction The striatum is a central component of the basal ganglia and performs critical functions in the integration of information, including motor control, cognition, and emotion (Gerfen, 1992; Graybiel 1995). Its importance is also reflected by a number of neurological disorders, including Parkinson's disease, Huntington's disease, schizophrenia and bipolar disorder. Despite major advances in the understanding of basal ganglia circuitry, relatively little is known about the developmental programs that specify striatal neurochemistry and connectivity. Over 90% of striatal neurons are the GABAergic medium spiny neurons (MSN). During development, the ventricular

⁎ Corresponding author. University Health Network, 200 Elizabeth St., E11444, Toronto, Ontario, Canada M5G 2C4. Fax: +1 416 340 4626. E-mail address: [email protected] (T.-R. Kiehl). 0014-4886/$ - see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2008.01.014

zone (VZ) and subventricular zone (SVZ) give rise to all neurons and glia that will populate the cerebral hemispheres. After passing through the cell cycle in the VZ or SVZ, cells migrate outward, where they undergo terminal differentiation. In contrast to the cerebral cortex, the mechanisms governing cell migration and maturation in the basal ganglia are still poorly understood. A transcription factor that has previously been observed in the striatum during development is Ikaros (Georgopoulos et al., 1992; Agoston et al., 2007). The Ikaros (Ik) family of zinc-finger transcription factors is essential in the development and function of leukocytes, including all classes of lymphocytes (NK, T, and B cells), monocytes/macrophages, and dendritic cells (Georgopoulos et al., 1994). This has been confirmed in studies of mice homozygous for a targeted deletion in the Ik gene, which develop a variety of defects in their lymphoid compartments (Wang et al., 1996). After a period of differentiation, Ik expression is down-regulated in most of the cell types involved (Klug et al.,

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1998). It was previously shown that Ik is a component of a transcriptional complex that is capable of recruiting multiple corepressors with histone deacetylase complex (HDAC) activity (Yu et al., 2002). The transient expression, which disappears once a mature cellular phenotype is reached, as well as the chromatin remodeling activity of Ik, point to a role in cell fate specification. Recently, we showed that corticomelanotrophs in the anterior pituitary also express Ik (Ezzat et al., 2005), and that Ik plays an essential role in hypothalamic-mediated somatic growth (Ezzat et al., 2006). These findings suggest that Ik facilitates the differentiation and maturation of cells required for immune and endocrine homeostasis. However, neurobehavioral assessment of these mice has not been previously reported. In the current study, Ik-deficient mice were evaluated with detailed neurobehavioral tests. We also describe the spatio-temporal pattern of Ik expression during development in the wild-type mouse and examine the impact of Ik deficiency on striatal neurotransmitter systems. Materials and methods Animals and genotyping Ikaros-null mice were derived as described (Ezzat et al., 2006) from a strain generated by Georgopoulos and colleagues (Wang et al., 1996). Mice were propagated in the original C57BL/6 background. Germline allelic transmission was verified by PCR analysis using tail DNA as described (Wang et al., 1996). Mice were initially housed at the Animal Care Facility at the Ontario Cancer Institute until 1 month of age. They were subsequently transferred to the Centre for Phenogenomics, Toronto, where neurobehavioral testing was performed starting at 2 months of age and was completed at age 3 months. Three batches of mice were tested in order to arrive at sufficient numbers, with a total of 9 homozygous Ik-null mice (Ik−/−; 3 males, 6 females), 12 heterozygotes (Ik+/−; 6 males, 6 females), and 12 wild-type animals (Ik+/+ ; 6 males, 6 females). After the completion of testing, animals were sacrificed and from each animal, one brain hemisphere was fixed in 10% buffered formalin and embedded in paraffin; the other hemisphere was snap-frozen in isopentane. All experiments were carried out in accordance with the rules and regulations of the Animal Care and Use Committee at the University of Toronto. Components of the study that used wild-type mice for the evaluation of normal expression of Ik were performed in the outbred ICR strain (Institute for Cancer Research). This additional strain was chosen to assess the validity of findings across strains. Tissue was harvested at time points E14, E16, E18, as well as P1 through P20, 6 weeks and 3 months, from at least 3 animals per time point. Brains were fixed in 10% neutral buffered formalin and embedded in paraffin. Hemi-brains from postnatal days 1 through 20 were oriented in the coronal plane and combined into a single array block containing all time points. The care of animals was approved by the Institutional Animal Care Facilities at the Ontario Cancer Institute, where animals were housed.

Morphologic and immunohistochemical studies Fetal, neonatal and adult mouse brains were fixed in neutral buffered formalin and embedded in paraffin and sectioned in the coronal plane. Sections were stained with hematoxylin and eosin, and serial sections were used for immunolocalization studies. To examine Ik-null and Ik heterozygote mutant mice for potential neuroanatomical defects, serial sections were taken at 5 μm in the entire rostro-caudal extent (three mice analyzed per genotype group, four age points examined). Every tenth section was stained with hematoxylin and eosin. Immunolocalization of Ik was performed with the 4E9 mouse monoclonal antibody that recognizes the C-terminal tail of Ik, as previously described (Ezzat et al., 2003). The following antibodies were used for the immunohistochemical assessment of the striatal neurochemical architecture: DARPP-32 (Cell Signaling Technologies 2302; dilution 1:100), Glutamic acid decarboxylase/GAD 65 and 67 (Chemicon AB1511; dilution 1:500), Substance P (Chemicon AB1566; dilution 1:3000), Met-enkephalin (Abcam ab22620; dilution 1:500), and Choline acetyl transferase/ChAT (Chemicon AB143; dilution 1:500), Dopamine D1 receptor (Sigma D6692; dilution 1:100), Dopamine D2 receptor (GeneTex GTX71748; dilution 1:100). Elevated plus-maze (anxiety-like behavior) The elevated plus-maze is a commonly used test for measuring anxiety-like behavior and innate fear in rodents (Crawley, 2000; Rogers and Cole, 1994). The maze consists of two open (25 × 5 cm) and two enclosed arms (25 × 5 × 30 cm), arranged such that the two arms of each type are opposite each other and extend from a central platform (5 × 5 cm). The floor and side walls of the maze consist of opaque Plexiglass material. The maze is elevated to a height of 50 cm. Testing was performed in a dimly lit experimental room. Mice were individually introduced to the center, the head facing the open arm. Behavioral parameters were recorded using a digital camera and were analyzed using Observer 50 software (Noldus Information Technology, Netherlands). The percentage of each of the following parameters was measured: 1.) open arm time, enclosed arm time, and central platform time as a percentage of total testing time; 2.) open arm entries, enclosed arm entries, and central platform number of entries as a percentage of total number of entries; 3) total entries; 4) head-dips (exploratory behavior of head and shoulders over the open sides of maze); 5) number of passages from one enclosed arm to another, and 6) risk assessment (posture of body stretched forward and followed by retraction to the original position). The maze was cleaned between sessions using 70% ethanol. Acoustic Startle Response (ASR) and Pre-pulse Inhibition (PPI; motor and autonomic reaction) Testing was performed in four standard startle chambers (Med Associates Inc Startle Reflex System, Georgia, Vermont), as previously described (Lipina et al., 2007). During the test, the animal was confined to a holder, resting on a platform in a

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ventilated and sound-attenuating chamber. Background noise was set a 65 dB. Five types of trials were used. Pulse-alone trials (P) consisted of a single white noise burst (120 dB, 40 ms). The pre-pulse + pulse trials (PP69P, PP73P, PP81P) consisted of a pre-pulse (20 ms at 69, 73, or 81 dB, respectively), followed 100 ms after pre-pulse onset by a noise pulse (120 dB, 40 ms). No-stimulus (NS) trials consisted only of background noise. Sessions were structured as follows: 1) 10-min acclimation at background noise level; 2) five P trials; 3) ten blocks, each containing all five trials (P, PP69P, PP73P, PP81P, NS) in pseudorandom order; 4) five P trials. Inter-trial intervals were pseudo-randomly distributed between 12–30 s. The maximum force intensity for each trial was recorded as startle level. The average percent reduction in startle intensity between pulse and pre-pulse + pulse trials at all three pre-pulse levels was defined as the PPI level. Data collection: The percentage PPI induced by each pre-pulse intensity was calculated as (100 − [100·startle amplitude on pre-pulse trial] / [startle amplitude on pulse alone]). Pinch test (catalepsy) Mice were taken from the cage by the tail and placed on the table. Their mobility was restricted by holding the tail. The mouse was then gently pinched with a thumb and forefinger and was quickly lifted up above the table while simultaneously its tail was released. The time of immobility before onset of motor paroxysms (e.g. motor agitation) was recorded in seconds (Fundaro 1998). Porsolt's forced swim test (depression-like behavior) The duration of immobility was measured using the Porsolt's forced swim test (Porsolt et al., 1977; Crawley, 2000). Each mouse was placed individually in a transparent plastic cylinder (24 cm height × 20 cm diameter) that contained water at 25 °C to a depth of 18 cm, and was forced to swim for 5 min. Active swimming, floating and latency (time before the first immobility) were recorded and analyzed using Observer 50 software (Noldus Information Technology, Netherlands). The mouse was determined to be immobile when it floated in an upright position without additional activity other than that necessary for the animal to keep its head above water. Contextual fear conditioning (learning and emotion) The main component of the conditioning chamber was a Perspex arena with a light mounted in the lid (350 × 200 × 193 mm; Technical and Scientific Equipment, Midland, MI). The floor was outfitted with stainless steel bars (4 mm diameter, 5 mm apart). These were connected to a computer, which controlled the duration of the test session and timing, intensity, and duration of the shock. Background noise was set at 52 dB. On day 1, single subjects were allowed to explore the chamber for 180 s before the onset of a discrete conditioned stimulus (CS), which consisted of continuous sound (3600 Hz, 95 dB) lasting 30 s. At the end of this CS period, subjects received three un-signaled foot shocks (duration 2 s, intensity 1 mA) at 60 s

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intervals. On day 2, 24 h after the conditioning session, mice were returned to the chamber, and the freezing response was assessed immediately, then every 8 s for 8 min. The freezing response was defined as a lack of any movement except that required for breathing. Assessment of the freezing response occurred in the same conditioning chamber in which the mice received the foot shocks. After each subject was tested, the chamber was cleaned with 70% alcohol. Data analysis and statistics Results were analyzed by t-test, paired t-test, one-way ANOVA, two-way ANOVA followed by post-hoc Student– Newman–Keuls test to identify significant differences. All data are expressed as mean ± SEM In all cases, p b 0.05 was considered statistically significant. Results Ikaros is transiently expressed in striatal medium spiny neurons during development Ik reactivity is first detectable by immunohistochemistry at embryonic day 12 (E12) in very few cells of the striatal primordium. The area enlarges through E14 (Fig. 1A), E16 and E18, at which time only a subset of striatal cells are reactive for Ik (Fig. 1B). Only rare positive nuclei are seen in the subventricular zone. At the interface between SVZ and the developing striatum, there is an abrupt onset of Ik-reactivity (Fig. 1B). Crescent- or ring-shaped Ik-positive structures, 3–4 per nucleus, can be seen in close association with the nuclear membrane. More laterally, the nuclear Ik-pattern becomes weaker and diffuse, and eventually ceases, creating a distinct dorso-ventral and medio-lateral gradient. Around the time of birth and up to postnatal day 2 (P2), distinct geographic clusters of Ik-positive and -negative regions become apparent (Fig. 1C), suggestive of different reactivity in the patch and matrix compartments. After P2, the presence of Ik-reactive cells is restricted to a small rim that lines the ventricular surface (Fig. 1D). Some neuronal nuclear staining can be observed as late as P7, but is no longer visible at P14 (Fig. 1E). Besides the striatum, there is also a weak and diffuse Ik signal briefly around E14 in layers 5 and 6 of the developing cerebral cortex (Fig. 1F). Double-labeling with Ik and the striatal projection marker DARPP-32 (Walaas and Greengard, 1984) confirmed that the Ik-positive cells are the developing medium spiny neurons (not shown). Outside of the CNS, similar dotlike nuclear staining is also observed in the trigeminal ganglion with a time course paralleling that in the striatum (Fig. 1G). Immunohistochemical evaluation of striatal neurochemistry in adult Ik-deficient mice Histologic examination of brain tissue from Ik-deficient mice at the H & E levels revealed no significant neuroanatomical abnormalities. Therefore, an extensive evaluation of the striatal

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Fig. 1. Ik immunoreactivity in the developing mouse brain. (A) At embryonic day 14, there is strong staining in precursors of the striatal patch compartment (5×). Ik is absent in the germinal matrix. Scattered Ik-positive cells are present in other regions of the developing telencephalon. (B) High-power view at E18 reveals an abrupt onset of Ik-reactivity at the interface between SVZ (left) and areas with post-mitotic neurons (right; 40×). There are crescent- or ring-shaped Ik-positive structures, often 3–4 per nucleus in close association with the nuclear membrane. Laterally, the signal becomes diffuse throughout the nucleus, levels off in intensity and eventually ceases. (C) Around the time of birth, distinct geographic clusters of Ik-positive and negative regions become apparent, suggestive of a difference in reactivity in the patch and matrix compartments (10×). (D) At P5, the presence of Ik-reactive cells is restricted to a small rim that lines the ventricular surface (20×). (E) At P14, the neuronal nuclear signal has disappeared while there is continuing nuclear reactivity in microglia (40×). (F) The developing cerebral cortex at E14 also shows a diffuse nuclear Ik signal in layers 5 and 6 (20×). (G) Strong nuclear dot-like reactivity in the trigeminal ganglion at E14 (40×).

neurochemistry was performed by immunohistochemistry on the mice previously used for neurobehavioral studies. There were no significant differences between Ik−/− mice and their wild-type (Ik+/+) or heterozygous (Ik+/−) littermates in markers of striatal projection neurons (DARPP-32, Fig. 2A) and in neurochemistry for GABA (glutamic acid decarboxylase; Fig. 2B), substance P (data not shown) or D1 Dopamine receptors (Fig. 2C). The projections to the external globus pallidus showed unaltered strong reactivity for Met-enkephalin (Fig. 2D). Cholinergic

interneurons (choline acetyl transferase; Fig. 2E) were also unaffected. Elevated plus-maze MANOVA did not detect a significant effect of gender or genotype on most of the tested parameters (all p's N 0.05). Ik+/+ , Ik+/− and Ik−/− mice showed no significant differences in nearly all parameters (Fig. 3A). However, for one component, the

Fig. 2. Striatal neurochemistry as assessed by immunohistochemistry. No differences were seen between wild-type (Ik+/+) and Ik-null (Ik−/−) mice for the following markers (all at 40× magnification): DARPP-32 (A), glutamic acid decarboxylase (B) and D1 dopamine receptors (C) The projections to the external globus pallidus showed unaltered strong reactivity for Met-enkephalin (D). Cholinergic interneurons (E; choline acetyl transferase) were also unaffected.

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Fig. 3. Neurobehavioral phenotyping. (A) Elevated plus-maze. Time spent in open arms (A1), center (A2), closed arms (A3), total entries (A4), passages (A5), risk assessment (A6), number of head-dips (A7) and ends (A8) of male and female Ik−/−, Ik−/+ and Ik+/+ mice are shown, presented as means + SEM (MANOVA). There was no significant effect of gender or genotype on any of the tested parameters. (B) Acoustic Startle Response (ASR): lower ASR in male and female Ik−/− compared to Ik+/+ or Ik−/− mice within each gender when measured by 20 ms startle stimuli at 69, 73, or 81 dB, respectively (MANOVA). (C) Pre-pulse Inhibition of the startle response: no effect of gender or genotype on PPI (ANOVA; 69, 73 and 81 dB). (D) Porsolt's forced swim test (FST): major effect of genotype on floating time in Ik+/+, Ik+/− and Ik−/− mice. (E) Pinch test (catalepsy): Ik−/− mice showed a markedly increased duration of immobility when compared to Ik+/+ or Ik+/− animals. (F) Contextual Fear Conditioning: no effect of gender or genotype on any of the behavioral parameters assessed (⁎statistically significant difference).

number of head-dips, there was a trend towards significance for an effect of genotype [F (2,26) = 382, p = 0.058]. The number of head-dips was significantly lower in Ik+/− when compared to Ik+/+ animals but was not different in Ik−/− mice.

is consistent with decreased “behavioral despair”. The performance of heterozygotes (Ik+/−) did not differ from those of wildtype mice (Ik+/+; p N 0.05). Pinch test (catatonic-like state)

Acoustic Startle Response (ASR) and Pre-pulse Inhibition (PPI) MANOVA detected a significant effect of genotype on the ASR [F (2, 24) = 47, p b 0.05] and gender effect [F (1, 24) = 57, p b 0.05] (Fig. 3B). Post-hoc comparison revealed a lower ASR in both male and female Ik−/− compared to Ik+/+ or Ik+/− mice within each gender. ASR was lower in female Ik+/+ and Ik+/− animals compared to males in these genotypes (both p's b 0.05). Fig. 3C demonstrates PPI at three pre-pulse intensities and startle amplitude in Ik+/+, Ik+/− and Ik−/− Ikaros mice. ANOVA revealed a main effect of gender on pre-pulse intensities (69, 73 and 81 dB) [F (2, 48) = 33, p b 0.05], but there was no effect of gender on PPI [F (1, 24) = 15, p N 0.05] or genotype [F (2, 24) = 06, p N 0.05]. Porsolt's forced swim test (FST) Mice were also analyzed in the FST, which is widely used as a screening test for antidepressants (13, 17). MANOVA detected a major effect of genotype on floating time in the FST [F (2, 22) = 91, p b 0.001], but there was no effect of gender [F (2, 22) = 04, p N 0.05]. Fig. 3D shows the mean of floating time in Ik+/+,Ik+/− and Ik−/− Ikaros mice. Ik−/− mice spent significantly less time in immobility than their Ik+/+ littermates ( p b 0.001). This phenotype

Ikaros Ik−/− mice displayed a pronounced catatonic response in the Pinch test compared to Ik+/+ animals, as shown in Fig. 3E. MANOVA detected a highly significant effect of genotype on the duration of immobility [F (2, 26) = 41,4, p b 0.001]. Post-hoc analysis identified a significantly higher immobility in Ik−/− mice time detected in the Pinch test ( p b 0.001). There was no effect of gender [F (2, 26) = 11, p N 0.05]. Further analysis revealed a significant negative correlation between immobility in the Pinch test and FST for Ik−/− Ikaros mice (r = −06, p b 0.001). Contextual Fear conditioning There was no effect of gender or genotype on any of the behavioral parameters assessed in the Fear Conditioning component (all p values N 0.05), as shown in Fig. 3F for Ik+/+, Ik+/− and Ik−/− mice. Discussion Ikaros transcription factors were previously found to be expressed in the developing striatum (Georgopoulos et al., 1992). A putative Ikaros binding site (5′-GGGA-3′) in the promotor of the mouse and rat enkephalin gene was identified

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(Dobi et al., 1997). A subsequent study then focused on the relevance of Ik to the enkephalinergic system (Agoston et al., 2007) and concluded that Ik is co-expressed with enkephalin mRNA and that Ik may act as a positive regulator of enkephalinergic specification in the developing striatum. While enkephalin is one of the striatal co-transmitters, expressed by a subset of MSNs, there are many others, including GABA, substance P and dopamine D1 and D2 receptors (reviewed in: Holt et al., 1997). In the postnatal striatum, there is a precise regional distribution of neurons and neurotransmitters across distinct compartments termed the patch and matrix (Graybiel and Ragsdale, 1978). The presence of Ik in precursor cells of both compartments suggests that it may play a role as a general maturation factor for medium spiny neurons. At any time point, only a subset of nuclei are immunoreactive for Ik, so it is possible that most, if not all MSNs pass through a brief stage of Ik expression. In our study, when these markers were evaluated by immunohistochemistry, they showed similar patterns in Ik +/+ and Ik −/− mice, consistent with recent observations in this mouse model (Agoston et al., 2007). The nuclear pattern of Ik-reactivity described here in developing MSNs is reminiscent of similar prior observations in lymphocytes (Avitahl et al., 1999), where different patterns were linked to stages of activation and of the cell cycle. We show that Ik is almost completely absent in the proliferating cells of the subventricular zone but is abruptly induced during neuronal maturation (Fig. 1B). Toroidal and crescent-shaped nuclear Ik-positive structures (Fig. 1B) resemble the pattern seen in activated T and B cells (Brown et al., 1997). More laterally, the nuclear Ik-pattern becomes diffuse, a pattern which in lymphocytes is associated with the resting state. As shown in Fig. 1B, immunohistochemistry did not detect Ik in the proliferative population of the SVZ. Rather, an abrupt onset of Ik expression occurs at the interface of SVZ and maturing regions. This is consistent with a role whereby Ik may act in the transition from committed neuronal precursor to post-mitotic neuron. This could result in altered connectivity or other changes that would impact neuronal function and account for behavioral alterations. Behavioral phenotyping of Ik−/− mice showed no effect of Ik deficiency on parameters of learning and emotion or on anxietylike behavior (Fig. 3A and F). However, in the Porsolt's forced swim test (FST), Ik−/− mice had markedly reduced immobility times (i.e., decreased floating) when compared to Ik+/+ or Ik+/− animals (Fig. 3D). The FST creates a situation of inescapable stress, causing mice to display variable times of immobility. An increase in immobility (i.e. “floating”) is considered a “depression-like” phenotype and the FST is therefore widely used in the evaluation of antidepressant drugs (Crawley, 2000; Porsolt et al., 1977). The phenotype of Ik−/− is suggestive of an antidepressant-like response, with a degree of immobility reduction that is comparable to a pharmacologic antidepressant response (Hill and Gorzalka, 2005) or sleep deprivation (LopezRodriguez et al., 2004). Startle is a stereotypical reflex to a sudden and unexpected stimulus that results in rapid, involuntary contraction of the

skeletal musculature (Koch, 1999). Startle amplitude is affected by a variety of factors, such as noradrenergic tone (Astrachan and Davis, 1981; Kehne and Davis, 1985; Mishima et al., 2004) or the serotonergic system (Dulawa et al., 1991; Dirks et al., 2001). Decreased startle activity has also been observed with striatal hypodopaminergic activity (Chen et al., 2000; Dassesse et al., 2001). The endocrine system can also modulate acoustic startle response. It has been consistently observed that, in rats, centrally administered CRH increases startle amplitude (Swerdlow et al., 1986; Liang et al., 1992; Jones et al., 1998). Loss of Ikaros in vivo results in contraction of the pituitary corticomelanotroph population and reduced circulating ACTH levels (Ezzat et al., 2005). Therefore, the reduction in startle amplitude seen in our study could be linked to glucocorticoid insufficiency. The characteristically low body weight of Ik−/− mice (Ezzat et al., 2006) may have contributed to the difference in startle reactivity. Impaired hearing can be excluded as an explanation because Ikaros-null mice show no deficit in prepulse inhibition and respond well even to very soft startle tones. Experimental pinch-induced catalepsy is similar to the behavior elicited in rodents when attacked by a predator, and is affected by different pathways. In the striatum, there is a reciprocal interaction between adenosine A2A receptors and dopamine D2 receptors on GABA release (Dayne-Mayfield et al., 1996). Catalepsy is induced by adenosine A2A agonists (Ferré et al., 1991) and by dopamine antagonists (Sanberg, 1980). If these major systems are indeed affected in the Ik−/− mouse, the phenotype is consistent with reduced activity of D2 transmission or of increased A2A activity. A variety of other systems, such as the serotonergic (Bantick et al., 2005; Kondaurova et al., 2006) and cholinergic pathways (Castello et al., 1992; Dains et al., 1996) also have an effect on catalepsy, but these are not among the primary neurotransmitters of medium spiny neurons. Lack of Ik in development may affect multiple systems simultaneously. Whether the impact of Ik loss on neurobehavioral functions is linked to dysregulation in the enkephalinergic system remains to be determined. The findings in our study do not support that assumption. Indeed, the functional changes observed here in Ik−/− mice differ significantly from those observed in ENKdeficient strains. For example, Ik−/− mice did not have altered emotional responses, in contrast to preproenkephalin knockout mice (König et al., 1996; Ragnauth et al., 2001). Preproenkephalin-deficient mice display higher levels of anxiety and startle response (Bilkei-Gorzo et al., 2004), in sharp contrast to the blunted startle response seen in Ik−/− mice. Endogenous enkephalins exert strong antidepressant effects and, therefore, the performance of Ik−/− mice in the FST is also not consistent with enkephalin deficiency. Lastly, activation of opioid receptors has been shown to promote behavioral arrest and catalepsy, and, therefore, the marked increase in catalepsy in the Ik−/− mice would be unexpected in a hypo-enkephalinergic state. Nonetheless, additional behavioral studies could focus on enkephalins, which mediate reward-and motivation-related behavior via enkephalinergic MSNs (Kelley et al., 2005). Deficits in the enkephalinergic system have been shown to be associated with a selective reward deficit (Hayward et al., 2002)

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and substance abuse in mice (Banks et al., 2004; BeadlesBohling and Wiren, 2005). In conclusion, the findings in this study extend the role of Ikaros, a hematologic transcription factor, to a regulator of maturation and differentiation of striatal medium spiny neurons and a determinant of depression. Future molecular and neurophysiologic phenotyping will be required to reveal the mechanisms upstream and downstream of Ik to elucidate where exactly Ik fits into the landscape of signaling events that control the structural and functional development of the striatum. Potential human applications of this information will improve the understanding of malformative and neurodegenerative diseases of the striatum and depression and may contribute to future cellular therapies. Acknowledgments This work was supported by the Canadian Institutes of Health Research (grant MT-14404 to S. E. and S. L. A.) and by the Toronto Medical Laboratories. We thank K. Georgopoulos for generously providing the Ikaros-null mice and Ikaros antibody. The technical assistance of K. So is also gratefully acknowledged. We also thank J. Becz and R. Lopez from the Ontario Cancer Institute for the animal care and advice. References Agoston, D.V., Szemes, M., Dobi, A., Palkovits, M., Georgopoulos, K., Gyorgy, A., Ring, M.A., 2007. Ikaros is expressed in developing striatal neurons and involved in enkephalinergic differentiation. J. Neurochem. 102, 1805–1816. Astrachan, D.I., Davis, M., 1981. Spinal modulation of the acoustic startle response: the role of norepinephrine, serotonin and dopamine. Brain Res. 206, 223–228. Avitahl, N., Winandy, S., Friedrich, C., Jones, B., Ge, Y., Georgopoulos, K., 1999. Ikaros sets thresholds for T cell activation and regulates chromosome propagation. Immunity 10, 333–343. Banks, W.A., Kumar, V.B., Morley, J.E., 2004. Influence of ethanol dependence and methionine enkephalin antisense on serum endomorphin-1 and methionine enkephalin levels. Alcohol., Clin. Exp. Res. 28, 792–796. Bantick, R.A., De Vries, M.H., Grasby, P.M., 2005. The effect of a 5-HT1A receptor agonist on striatal dopamine release. Synapse 57, 67–75. Beadles-Bohling, A.S., Wiren, K.M., 2005. Alteration of kappa-opioid receptor system expression in distinct brain regions of a genetic model of enhanced ethanol withdrawal severity. Brain Res. 1046, 77–89. Bilkei-Gorzo, A., Racz, I., Michel, K., Zimmer, A., Klingmüller, D., Zimmer, A., 2004. A Behavioral phenotype of pre-proenkephalin-deficient mice on diverse congenic backgrounds. Psychopharmacology (Berl). 176, 343–352. Brown, K.E., Guest, S.S., Smale, S.T., Hahm, K., Merkenschlager, M., Fisher, A.G., 1997. Association of transcriptionally silent genes with Ikaros complexes at centromeric heterochromatin. Cell 91, 845–854. Castello, M.E., Bolioli, B., Dajas, F., 1992. Catalepsy induced by striatal acetylcholinesterase inhibition with fasciculin in rats. Pharmacol. Biochem. Behav. 41, 547–550. Chen, J.F., Beilstein, M., Xu, Y.H., Turner, T.J., Moratalla, R., Standaert, D.G., Aloyo, V.J., Fink, J.S., Schwarzschild, M.A., 2000. Selective attenuation of psychostimulant-induced behavioral responses in mice lacking A(2A) adenosine receptors. Neuroscience 97, 195–204. Crawley, J.N., 2000. What's Wrong With My Mouse? Behavioral Phenotyping of Transgenic and Knockout Mice. John Wiley and Sons, Ltd., Chichester, UK. Dains, K., Hitzemann, B., Hitzemann, R., 1996. Genetics, neuroleptic response and the organization of cholinergic neurons in the mouse striatum. J. Pharmacol. Exp. Ther. 279, 1430–1438.

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