Neuroscience and Biobehavioral Reviews, Vol. 22, No. 3, pp. 453–462, 1998 q 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0149-7634/98 $19.00 + 0.00
Pergamon
PII: S0149-7634(97)00053-5
Behavior in Mice with Targeted Disruption of Single Genes RANDY J. NELSON* AND KELLY A. YOUNG Departments of Psychology, Neuroscience, and Population Dynamics, The Johns Hopkins University, Baltimore, MD 21218-2686, USA
NELSON, R. J. AND YOUNG, K. A. Behaviour in mice with targeted disruption of single genes. NEUROSCI BIOBEHAV REV 22(3) 453–462, 1998.—The use of mice with targeted deletion, or knockout, of specific genes provides a relatively new approach to establish the molecular bases of behavior. As with all ablation studies, the interpretation of behavioral data may be limited by the technique. For example, indirect effects of the missing gene may affect behavior, rather than the missing gene per se. Also, because the missing gene might affect many developmental processes throughout ontogeny and because up-regulation or compensatory mechanisms may be activated in knockouts, behavioral data from mice with targeted gene deletions should be interpreted with caution. The development of conditional knockouts, in which a specific gene can be inactivated any time during ontogeny, should allow investigators to avoid these conceptual shortcomings associated with behavioral data from knockouts in the near future. The behavioral alterations reported in knockout mice are reviewed here. Many dramatic changes in complex motivated behaviors including aggression, sexual, ingestive, and parental behaviors, have been reported for knockouts. There have also been many reports of alterations in sensorimotor abilities and spontaneous activity, as well as impairments in balance, coordination, and gait. Impaired learning and memory have also been reported for mice with targeted disruption of specific genes. Taken together, the use of knockouts will provide an important new tool to understand the mechanisms underlying behavior. q 1998 Elsevier Science Ltd. All rights reserved. Knockout
Targeted gene deletion
Learning
Memory
Long Term Potentiation
Aggression
Reproduction
Furthermore, behavioral tests study the effects of the missing gene (and gene product), not the effects of the gene directly. This conceptual shortcoming can be overcome in the same manner as in other types of ablation studies, by collecting converging evidence using a variety of pharmacological, lesion, and genetic manipulations. Finally, correlations among behavioral assessments of knockouts are difficult to make because no standardized behavioral tests are available (14). Because mammalian genome mapping is currently focused on mice (Mus musculus), standardized behavioral testing of mice should be adopted. Against those disadvantages are several important advantages to using knockout mice in behavioral research: 1) disabling a gene is often a very precise and ‘‘clean’’ ablation, 2) the effects of the gene product can be abolished without the side-effects of drugs, and 3) genetic manipulations may be the only way to determine the precise role of many endogenous factors on behavior. The use of newly available inducible knockouts, in which the timing and placement of the targeted gene disruption can be controlled, will likely become an important tool in behavioral research. Importantly, targeted mutation studies do not propose a model of single-gene control of behavior. Our goal here is to review the current state of behavioral research using knockout mice (Table 1). We have limited our review to the behavioral effects of targeted disruption of single genes, and
INTRODUCTION
RECENTLY, A rapprochement has developed between the behavioral sciences and molecular biology. Large segments of the mouse genome have been mapped, and molecular biologists have begun the difficult task of identifying the function of these newly described and sequenced genes. Targeted disruption (i.e. ‘‘knockout’’) of a single gene is an increasingly common genetic engineering technique used to discover gene function (1–4). Molecular biologists have reasoned that the function of a targeted gene can be determined by comparing the phenotype of wild type (WT) and knockout mice. In many cases, the most salient phenotypic change observed in knockout mice is altered behavioral (3–12). Although this genetic technique offers new opportunities to study the mechanisms of behavior, in common with all techniques, there are some potential limitations (11). For example, the products of many genes are essential to normal function, and inactivating the gene may prove lethal or induce gross morphological or physiological abnormalities that can complicate interpretation of discrete behavioral effects. When there are no obvious morphological, physiological, or behavioral changes associated with deactivation of a specific gene, unexpected compensatory or redundancy mechanisms may be engaged that obscure the interpretation of the normal contribution of the gene to behavior (11,13).
* Corresponding author: Tel.: +1 410 5168407; Fax: +1 410 5166205; E-mail:
[email protected]
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NELSON AND YOUNG TABLE 1 THE BEHAVIORAL EFFECTS OF SPECIFIC TARGETED GENE DELETION
Gene
Protein/target
Behavior
Reference
AT 2
Angiotensin II type receptor
(15) (16)
Atm
Atm-ataxia telangiectasia mutated
Attenuated exploratory behavior, increased fear, hypoalgesia Impaired drinking response to H 2O deprivation; spontaneous movement reduction Defective balance; abnormal gait; reduced rearing and horizontal activity Defective coordination, movement, and balance
BDNF-NT 4
neurotrophin and brain derived neurotrophic factor Brn-3a Brn-3a c-fos proto-oncogene c fos aCaMKII CREBaD CREM D2 D3 DAT ER
FMR1 fosB fyn GFAP GLP1 GluRd2 hexa hexb hexab HO-2 5-HT 1B Igf1 InsP3 MAOA mGluR1 mGluR2 MOR b2 nAChR Ncam NPY Neurotropin-3 receptor gene trkC NGFI-A (Egr-1)
Defective suckling; uncoordinated limb and trunk movements Impaired spatial and non-spatial learning Increased latency to male mounting behavior; reduced number of mounts acalcium-calmodulin kinase II ‘‘Jumpy’’, nervous behavior; impaired LTP and spatial learning Reduced fear; elevated aggression cAMP-responsive element-binding protein Reduced morphine abstinence (CREB) a and D isoforms CREM Normal mating behavior despite infertility Dopamine D 2-receptor Reduction in locomotor activity; gait and balance impairment; abnormal posture D 3 dopamine receptor Increased locomotor and rearing behavior; normal gait, reflexes and coordination Dopamine transporter Hyperlocomotion; indifference to cocaine and amphetamine Estrogen receptor Absence of female mating receptivity Reduced aggressive behavior Females display reduced ability to elicit ejaculation; males produce fewer ejaculations Fmr1 protein Hyperactivity; macroorchidism; spatial learning deficits Immediate early gene fosB Deficient ability to nurture young Fyn tyrosine kinase Spatial learning deficit; LTP deficits Suckling behavior deficit Increased fearfulness; enhanced seizure susceptibity GFAP-glial fibrillary acid protein Normal behavior Glucagon-like peptide 1 Normal feeding behavior Glutamate receptor d2 subunit Impaired motor coordination and balance; altered gait; reduced horizontal activity Lysomal b-hexosaminidase a subunit Normal behavior Lysomal b-hexosaminidase b subunit Defective coordination and motor balance; reduced feeding behavior Lysomal b-hexosaminidase a, b subunits Severely defective coordination and motor balance; cessation of feeding behavior Cerebral isoform of hemeoxygenase Normal LTP; normal behavior 5-HT 1B serotonin receptor Elevated alcohol consumption Elevated aggression; reduced fear Igf1 Males fail to display mating behavior 1-inositol 1,4,5-triphosphate receptor Ataxia; impaired balance; propensity for seizures Monamine oxidase A Elevated adult aggression; persistence in mating; pups display increased fear; abnormal posture and balance; trembling Metabotropic glutamate receptor 1 Mild ataxia; moderate deficit in associative learning; decreased LTP Metabotropic glutamate receptor Impaired LTD m-opiod receptor Reduced morphine analgesia Neuronal nicotinic acetylcholine receptor Improved performance on passive avoidance, associative memory tasks N-CAM Spatial learning deficit Neuropeptide Y Increased propensity for seizures; normal feeding behavior Neurotropin-3 receptor Abnormal movements
NMDA e1 nNOS NT-3
NGFI-A immediate early transcription factor NMDA e1 subunit Neuronal nitric oxide Neurotropin-3
OT P p 6H Pax5 Pkcc
Oxytocin P p 6H BASP transcription factor PKCg-gamma isoform of protein kinase
X-linked Plp
Proteolipid proteins PLP and DM20
PR
PR progesterone receptor-PR a and PR b
(17) (18) (19) (20) (21) (22,23) (9) (24) (25) (26) (27) (28) (29) (30) (31,32) (92) (34) (33) (35) (36,37) (38,39) (40) (41) (42) (43) (43) (45) (46) (12) (47) (48) (49) (7,8) (50) (51) (52) (53) (54) (55)
Normal behavior
(56)
Reduced spatial learning and hippocampal LTP; ‘‘jumpy’’ Increased aggressive behavior; inappropriate sexual persistence Reduced activity; athetotic gait; abnormal limb postures; no evidence of food intake Reduced aggression; normal maternal behavior Reduced motor coordination; tremors Nervous jerky gait Abnormal clasping behavior and reflexes Mild deficits in spatial and contextual learning; reduced LTP Reduced behavioral reactions to ethanol Severe defects of neuromotor coordination; reduced spontaneous activity Females fail to exhibit lordosis response
(57) (58) (59) (60) (61) (62) (63) (5,6) (64) (65) (98)
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455 TABLE 1 CONTINUED
Prn-p RyR-3 Thy-1 TN Tyrosine hydroxlase (TH)
PrPc or prion Ryanodine receptor type 3 Thy-1 neuronal glycoprotein Tenascin Dopamine (DA)
Normal spatial learning Increased locomotor activity; ‘‘restless’’ Normal spatial learning Hyperlocomotion; swimming ability reduced Hypoactive adipsic; aphagic
therefore exclude behavioral phenotypes of natural mutants and transgenics. PRODUCTION OF KNOCKOUT MICE
In order to inactivate, or knock out, a gene, molecular biologists rearrange the nucleotide sequence that encodes for the gene under investigation (71,72). Because the chromosomes of most organisms used in behavioral studies are paired, there are two copies of each gene that must be inactivated. The creation of a mouse with a targeted disruption (i.e. knockout) of a specific gene is an arduous task that combines several low probability events. The gene in question must be identified, targeted, and marked precisely. This has been accomplished for an astounding number of murine genes during the past several years (53,72). Next, mouse embryonic stem cells are harvested and cultured. A mutated form of the gene is created, and introduced into the cultured embryonic stem (ES) cells either by microinjection or electroporation transfection (4). Homologous recombination can then incorporate a very small number of the altered genes into the DNA of the ES cells (1,73). The mutated ES cells are inserted into otherwise normal mouse embryos (blastocysts), then implanted into surrogate mothers (2,72,74). Within the developing mice, all of the descendant cells from the mutated stem cells will have the altered gene; the descendants of the original blastocyst cells will have normal genes. The resultant mouse, possessing copies of both mutant and normal, wild-type (WT) cells is called a chimera. If the mutated stem cells are incorporated into the germ lines (the cells destined to become the sperm and ova), then some of the gametes will contain the mutant gene. Chimeric mice are then bred with WT mice; some of the offspring produced will be heterozygous for the mutation; i.e. possess one copy of the mutant gene. If the heterozygous mice are interbred, then approximately one fourth of their offspring will be homozygous for the mutation. These homozygous mice can be interbred to produce pure-lines of mice with the gene in question knocked out (75,73). The product for which the gene typically encodes will be missing from the progeny (73). Behavioral performance is typically compared among WT (þ/þ), heterozygous (þ/¹), and homozygous (¹/¹) mice in which the gene product is produced normally, usually produced at reduced levels, or completely missing, respectively (11), though this assumption must be tested directly. One limitation to the interpretation of behavioral data from knockout mice is that the targeted gene is missing throughout ontogeny. Thus, any behavioral deficits may be due to the missing gene product per se or due to the missing gene product during key developmental processes throughout ontogeny. Furthermore, it is possible that
(110) (67) (68) (69) (70)
compensatory or redundancy mechanisms might be activated when a gene is missing. For example, in mice lacking the gene for the neuronal isoform of nitric oxide synthase (nNOS¹/¹), there is a 20% increase in the expression of the endothelial isoform of nitric oxide synthase (13). A compensatory mechanism may spare behavioral function, and cloud interpretation of the normal contribution of the gene to behavior. The availability of ‘‘inducible’’ or ‘‘conditional’’ knockouts, in which a specific gene can be inactivated at any point during development, or only inactivated in tissue-specific cells, should provide an important tool to bypass this problem of ontogenetic interactions (11). These conditional knockouts are currently being developed for a variety of genes (e.g. (10,76,77)). Region- or cell-specific promoters are combined with genes that can be activated at any time by specific events induced by the investigators (e.g. exposure to tetracycline, ecdysone, or interferon) (10,76,77,78). These substances serve as activators that terminate expression of a gene by binding to a promoter transgenically attached to the gene. Restricted gene activation can also be accomplished with a Cre–lox bacteriophage site-directed recombination method in which DNA recombination sites flank targeted genes, and these sites bind to the site-specific recombinase Cre (10). Ecdysone can also be used to inactivate a gene during any point of pregnancy because it easily crosses the placenta–blood barrier (77). The wide-spread availability of inducible knockouts should prove extremely useful in behavioral studies because they will provide a method of studying genetic influences on behavior in the absence of ontogeny issues that currently obscure interpretation of knockout mouse behavior. Behavioral reports of mice with targeted disruption of a single gene appear to fall into one of two categories. In the first type, specific outcomes are predicted based on previous work. For example, based on ovariectomy and hormone replacement studies, estrogens have been determined to be important in mediating female mating behavior (79). If the gene encoding the estrogen receptor is eliminated, then the signal transduction pathway for estrogen effects on behavior is presumably disrupted and mating behavior should not occur. Serendipitous findings comprise the second type of behavioral studies with knockout animals. Typically, altered behaviors of knockout mice are often sufficiently obvious or unusual that they catch the attention of animal care personnel, who then notify the investigators. Currently, therefore, dramatic behaviors including increased aggression, altered maternal care, seizures, impaired motor coordination and sensory abilities are commonly reported for knockout mice (e.g. (9,12,13,17,80)). Although it has been noted that, ‘‘...it is difficult...to recognize minor neurological abnormalities in mice’’ ((33), p. 25), presumably additional behavioral changes, both dramatic and subtle, of knockout mice await
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discovery by behavioral biologists. Thousands of genetic knockouts are being created, but few are examined for specific behavioral changes. Importantly, many knockouts have been examined in behavioral tests, but display no behavioral impairments (e.g. (39,45,56)). The behavioral phenotypes of mice with targeted disruptions of specific genes tested to date are described in the following sections.
SENSORIMOTOR BEHAVIOR AND SPONTANEOUS ACTIVITY
The most commonly observed behavioral change in knockout mice is impairment of sensorimotor or reflexive activities. Many investigators simply report ‘‘abnormal movements’’ or ‘‘changes in behavior’’ in their description of a knockout phenotype (e.g. neurotropin receptor gene trkC¹/¹ mice (55), adenylyl cyclase type 1D (81)); however, precise behavioral descriptions have also been noted. For example, deficiencies in balance and coordination occur in mice lacking the gene for the glutamate receptor d 2 subunit (GluRd 2) (41,82). From postnatal Day 12, the GluRd 2¹/¹ mice were reported to walk with a tottering, scrambling gait, and lacked the ability to maintain balance after rearing (41,82). Mice with targeted deletion of the gene for brain-derived neurotrophic factor and neurotrophin-4 (BDNF-NT4) (18), or the gene for 1-inositol 1,4,5-triphosphate receptor (InsP 3) also failed to display wellcoordinated behavior; the InsP 3¹/¹ mice display ataxia with loss of balance during walking or standing (48). In contrast, sensorimotor skills have been reported to be normal in many knockout mice including nNOS¹/¹, HO2¹/¹, FGFAP¹/¹ (glial fibrillary acidic protein), 6FAP¹/¹, and fosB¹/¹ mice (e.g. (34,39,45,58,80)). Assessment of the sensorimotor abilities of knockouts can be complicated by extrinsic factors. For example, behavioral performance of nNOS¹/¹ mice changes throughout the day. Although motor coordination and balance were equivalent between nNOS¹/¹ and WT mice when tested during their inactive period (lights on) (58), behavioral tests during the dark phase of the daily photoperiod revealed significant impairments in balance and coordination (104). The behavioral impairments were negatively correlated with a daily fluctuation of nNOS staining in the cerebellum. The extent to which other behavioral phenotypes are affected by the time of testing remains unspecified. Reduction in spontaneous activity and ‘‘hypolocomotion’’ as a result of targeted deletion have been reported in several different knockout mice. Functional deletion of the dopamine D 2 receptor results in reduction and impairment of locomotion (26), while the neurotropin-3 (NT-3) deletion produces mice with both reduced activity and abnormal posturing (59). Mice lacking two integral myelin proteins, proteolipid protein (PLP) and DM2O display significantly reduced activity with profound neuromotor coordination abnormalities (65). Atypical posturing and reflex reactions are also seen in a B cell specific transcription factor (Pax5) null mutants, that display abnormal clasping behavior (63). Uncoordinated limb and trunk movements and atypical posturing are observed in neonatal knockouts of Brn-3a, a member of the POU DNAbinding domain family (19). The neurological dysfunctions of mice lacking the Atm gene mimic the symptoms of the recessive human
NELSON AND YOUNG
neurologic disorder, ataxia telangiectasia (17). Atm¹/¹ mice have significantly shorter latencies to fall off a moving rota-rod than WT mice. These mice also display reduced horizontal activity and significantly less rearing than WT mice. Locomotor gait was also affected in the Atm¹/¹ mice; mutant mice have shorter stride lengths with inconsistent ‘‘stepping patterns’’ (17). This lack of coordination is present from the onset of locomotor behavior. However, not all motor dysfunctions are evident at an early age. In mice lacking the b subunit for the lysosomal enzyme b-hexosaminidase (hexb), motor function remains normal until 3 months of age when both coordination and balance begin to progressively deteriorate (43). Mutations in hex a and b subunit genes lead to Tay Sachs and Sandoff diseases in humans. Onset of gait abnormalities also occurs at 3 months; horizontal movement is complicated with hindlimb spasms that progress into a complete lack of hind-limb mobility by 5 months of age. At this time feeding behavior also ceases. While these behavioral abnormalities are not evident in Hexa¹/¹ mice (42,43), the reduction of balance, motor coordination, and horizontal activity observed in the Hexb¹/¹ mice is intensified when both Hexb and Hexa genes are deleted (44). These double knockout mice are significantly more impaired than either single hex gene knockout. Some targeted gene deletions can also result in severely hyperactive mice. For instance, NMDAe receptor channel, and a calcium–calmodulin kinase II receptor knockouts are classified as ‘‘jumpy’’ and ‘‘nervous’’ (9,57). Recent reports indicate that the ryanodine receptor type 3 (RyR3) null mutants also display ‘‘restless’’ behavior (67). The hyperactive behavior of the dopamine transporter (DAT) knockouts was reported to be ‘‘indistinguishable’’ from WT mice on psychostimulatory drugs (28). D 3 dopamine receptor knockouts displayed normal gait and coordinated behaviors, but showed 57% more locomotor activity and 93% more rearings than WT mice in the open field test of anxiety (27). Mice missing the tailess gene also exhibit distinctive elevation of activity levels (Simpson, E.; Young, K.A.; Nelson, R.J., unpublished data). Deletion of the gene involved in fragile X mental retardation, Fmr1, results in increased exploratory behavior and motor activities (33).
AGGRESSIVE BEHAVIOR
Production of knockouts has often resulted in mice that are extremely aggressive; these knockouts have been important in understanding the physiological mechanisms underlying aggressive behaviors. Reduced brain levels of serotonin have been implicated in aggression (84). Not surprisingly, mice with deleted genes that affect serotonin are more aggressive than WT mice. For example, mice with targeted disruption of genes for a-calcium–calmodulin kinase II (9), serotonin receptor 5-HT 1b (12), and monamine oxidase A (MAOA) (49) display increased aggressive behavior as compared to WT animals. Increased aggression has also been serendipitously discovered in tailess ¹/¹ mice (85), and the gene encoding nNOS (58,106). However, increased aggressiveness is not a general behavioral phenotype of mice with a targeted disruption of any gene (cf (86,106)); mice with targeted disruption of the heme oxygenase-2 gene display no alterations in aggressive
DISRUPTION OF SINGLE GENES
behavior (80), and animals with targeted disruption of the gene for estrogen receptors or oxytocin actually display markedly reduced aggressiveness (30,60,87). The elevation in aggression can be dramatic. For example, when examined in an intruder–resident test of aggression, nNOS¹/¹ mice engaged in 3–4 times more aggressive encounters than WT mice (58). Nearly 90% of the aggressive encounters were initiated by the nNOS¹/¹ animals. Similar results were obtained in dyadic or group encounters in neutral arenas. In all test situations, male nNOS¹/¹ mice rarely displayed submissive behaviors. Importantly, elevated aggression does not appear to reflect nonspecific effects of the targeted disruption of the nNOS gene. Treatment of male mice with specific pharmacological nNOS inhibitors (7-nitroindazol) also increased aggression (88). This impulsive aggression is also observed in adult MAOA¹/¹ mice that exhibited elevated offensive aggression compared to WT mice in a resident–intruder test (49). Biting behavior in MAOA knockout pups was also noted, as a propensity to bite the experimenter was evident when the mice were a few weeks of age (49). Although elevated testosterone is often associated with increased aggressive behavior (58), there were no differences between nNOS¹/¹ and WT mice in blood testosterone concentrations either before or after agonistic encounters (58). However, recent data on castrated nNOS¹/¹ males suggest that testosterone is necessary, but not sufficient to promote increased aggression (89). Importantly, inappropriate aggressiveness has never been observed among MAOA¹/¹ and nNOS¹/¹ female mice; however, aggressive behavior was not examined in females knockouts in the context of maternal aggression during which WT females are highly aggressive toward an intruder. Targeted disruption of the tailess gene causes dramatic increases in aggression in both sexes (85), although males (and females) have serum testosterone concentrations in the normal sex-specific range. Tailess ¹/¹ females engaged in 96% more aggression than WT females in resident and grouped aggression tests. Tailess ¹/¹ females were more aggressive than WT males. The tailess ¹/¹ females also displayed unprovoked offensive aggression in both resident and grouped aggression tests toward other females or males; again, inappropriate aggressiveness was not examined in female Zfa or other knockouts during maternal care when WT females display a natural increase in aggressiveness during nest defense.
PARENTAL BEHAVIOR
Mice lacking the fosB gene display poor maternal behavior (34). The lack of maternal behavior among the fosB knockouts does not correspond to a lack of fertility or the ability to lactate. FosB¹/¹ mothers have similar endocrine profiles to WT dams (34), but fail to display nest-building, cleaning/retrieving pups, nursing or protective crouching postures (34). These maternal behaviors are retained by female oxytocin (OT) knockout mice (OT¹/¹) mice, that fail to eject milk in response to suckling (60,103,112). These observations potentially open up a new area of inquiry into the mechanisms underlying hormonal mediation of maternal behavior. Behavioral studies of knockout mice may also evoke questions about established hormonal
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relationships. For example, the display of overtly normal maternal behavior in oxytocin knockout mice suggests that oxytocin may not be necessary for maternal behavior in mice (60,87). There may be subtle effects of missing genes on maternal behavior. Indeed, most knockout mice studied to date remain with their knockout mothers. The extent to which the altered behavior of knockout mice reflects atypical maternal behavior remains unspecified. Cross-fostering studies comparing the behavior of knockout mice reared with WT dams to knockout mice reared with knockout mothers will be critical in the future to untangle the role of extrinsic and intrinsic factors on behavior further.
LEARNING AND MEMORY
Knockout mice have been particularly useful in studies of long-term potentiation (LTP), long-term depression (LTD), spatial learning and memory, phenomena in which the molecular bases underlying the behavior have been wellestablished (3,4,90,91). The strategy in using knockouts in studies of learning and memory has been to confirm and extend what is known about the molecular mechanisms. Knockouts also provide a unique opportunity to study the molecular bases of these phenomena, and their relation to learning and memory. Mice with targeted disruption of the gene encoding acalcium calmodulin kinase II or the g isoform of protein kinase C display altered hippocampal long-term potentiation and impaired spatial learning (5–8,22,23,91). Similar deficits are also noted in NMDA e1 receptor channel null mutants (57), type 1 adenylyl cyclase mutants (81), and fyn tyrosine kinase ¹/¹ mice (92). Reduction of LTP and spatial learning in the fyn¹/¹ mice corresponds to altered hippocampal neuroanatomy; these mice display abnormal cell layers in the CA3 and dentate gyrus (36,92). Contextspecific associative learning and LTP are severely impaired in mice lacking the metabotropic glutamate receptor 1 (mGluR1¹/¹); however, no abnormalities in LTD or short-term potentiation were noted in mGluR1¹/¹ mice (7,8). Impaired spatial and nonspatial learning in immediate early gene c-fos¹/¹ mice were only observed during complex tasks; c-fos¹/¹ mice were able to complete simple discrimination tasks (20). Increased latency to learn any spatial task is observed in Fmr1¹/¹ mice that model human Fragile X mental retardation, but this observation may reflect hyperactivity in this knockout rather than deficits in learning or memory (33). Targeted deletion of a common neuronal protein, Thy-1, does not affect LTP or spatial learning (68). LTP is a stable and enduring increase in the magnitude of neuronal responses after afferent cells to the region have been stimulated with bursts of electrical stimulation. LTD, in contrast, is a stable and enduring decrease in the magnitude of neuronal responses after afferent cells to the region have been stimulated with bursts of electrical stimulation of relatively low frequency. Because afferent neural activity affects firing patterns in neurons ‘‘upstream’’, the existence of retrograde messengers was proposed (90). Two candidate molecules for the role of retrograde messengers are nitric oxide (NO) and carbon oxide (CO) (93–95,105,109). Treatment with pharmacological NOS inhibitors suppressed LTP and cerebellar LTD, but nNOS¹/¹ mice display normal
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LTP and LTD (96). Furthermore, mice lacking the gene encoding heme oxygenase-2 (HO-2), which produces CO, also display normal LTP (45). Obviously, conflicting data between drug and knockout research require additional studies to be conducted. Consequently, the physiological roles of NO and CO in LTP, LTD, and learning remain unknown. ANXIETY AND FEAR
Increased fearfulness, usually defined by increased number of boli expressed or amount of time spent in the closed arms of an elevated ‘‘plus’’ maze, is also a commonly observed behavioral phenotype of knock-out mice. Both angiotensin receptor II (AT 2) (15) and Fyn tyrosine kinase (37) null mutants demonstrate increased fearfulness as exhibited by a lack of exploration of open arms in an elevated plus maze. Additionally, Fyn¹/¹ mice avoid bright chambers and show a stronger learned fear response in a passive avoidance task than Fynþ/¹ or WT animals. Mice with targeted disruption of genes for a-calcium– calmodulin kinase II display decreased measures of anxiety and fear responsiveness (i.e. reduced freezing response after foot-shock and increased time spent in the open arms of the elevated plus maze) as compared to WT mice; this decreased fearfulness may contribute to elevated levels of aggressiveness (9). Importantly, the attenuated freezing response of a-CaMKII¹/¹ mice is not associated with reduced pain threshold; the mutant mice display elevated pain responsiveness as compared to WT mice (9). Increased aggression levels are also associated with reduced fear in mice with targeted deletion of the serotonin 1b receptor (12). SEXUAL BEHAVIOR
Altered sexual behavior of knockouts is often noted as researchers attempt to establish a breeding colony of mice. For example, when nNOS¹/¹ and WT males were paired with anestrous females, there initially was no difference in the amount of attempted sexual behaviors (58). However, mounting behavior diminished rapidly among WT mice, but continued at high rates among nNOS¹/¹ animals (58). There were no obvious disruptions in mating behavior among the female nNOS¹/¹ mice, and no apparent sensorimotor deficits among either sex of the mutant mice. Male nNOS¹/¹ mice exhibited fewer penile intromissions and ejaculations than WT males when paired with estrous females (13), but male nNOS¹/¹ males displayed elevated sexual motivation when paired with nonestrous females (58). Female nNOS¹/¹ mice display normal estrous behavior (97). In contrast, males with the insulin-like growth factor-1 (Igf1) gene deleted fail to display mating behavior (47). There was a trend toward longer latency to mount, decreased percentage of males mounting, and decreased number of mounts with or without intromissions in male knockouts of the immediate early gene, c-fos (21). These c-fos¹/¹ males also displayed significant reduction in mounting rates, despite no general lack of motivation or sexual arousal (21). Female mice lacking the progesterone (P 4¹/¹) receptor gene fail to exhibit lordosis (98). Absence of receptive behavior is also reported for female estrogen
receptor knockout (ERKO) mice (29,31,99). Although female ERKO mice elicit an equal number of mounts from males as female WT mice, no ERKO females allowed males to mate to ejaculation, regardless of hormone priming (31). Importantly, hormonally-primed, ovariectomized ERKO females were equally attractive to males as similarly treated WT females (i.e. males spent equivalent time in proximity to both genotypes). Male ERa¹/¹ mice also are deficient in mating behavior; the number of ejaculations are significantly reduced. However, male ERa¹/¹ mice display equivalent numbers of mounts and intromissions as WT mice and they appear to be feminized in mating behavior (32,104). Differences in sexual motivation were also reported for the ERa¹/¹ mice; WT mice spent significantly more time with tethered female stimulus animals than in a neutral area compared to ERa¹/¹ males (32). Mating behavior was normal in male and female cAMP response element modulator (CREM) ¹/¹ mice (25,107). The CREM gene is important for spermatogenesis and these males are infertile (25). SEIZURES
Knockouts demonstrating abnormal propensities for seizing behavior have also been studied. Fyn-tyrosine kinase¹/ ¹ and neuropeptide Y knockout mice display enhanced seizure susceptibility (37,54). P0 gene knockouts (P0 is a glycoprotein found predominantly in Schwann cells) exhibit tremors that are associated with motor incoordination (61). Mice that lack type 1 inositol 1,4,5-triphosphate receptor suffer neonatal ataxia and tonic or tonic-clonic seizures that resemble ‘‘epileptic’’ seizures (48). PAIN RESPONSES
The mechanisms of pain perception and responses have been recently examined using mice with targeted disruption of specific genes. Loss of morphine analgesia was reported in mice lacking the m-opioid receptor gene (MOR), although no other behavioral abnormalities were observed among MOR¹/¹ mice (51). Acute analgesic responses to morphine were not affected in mice heterozygous for the cAMP response element binding protein (CREB) a and D isoforms; however, reduced morphine abstinence and attenuated withdrawal symptoms were reported (24). FEEDING AND DRINKING
Alterations in both feeding and drinking behavior have been observed as a result of targeted gene deletion. Inactivation of the tyrosine hydroxalase (TH) gene results in dopamine-deficient mice that are adipsic and aphagic (70). Neurotropin three receptor (NT-3) ¹/¹ mice fail to ingest food and die within 24 h of birth (59), as do the Brn-3a¹/¹ mice that lack suckling behavior and perish without evidence of milk ingestion (19). As noted above, Hexa¹/¹ and Hexb¹/¹ mice stop feeding behavior at approximately three months of age (43,44). Reduced spontaneous ‘‘behavior’’ accompanies impaired drinking responses to water deprivation in angiotensin II type receptor (AT2) deficient mice (16). However, feeding behavior was not affected in mice that lacked glucagon-like peptide 1, a
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primary regulator of satiety and blood glucose (40). Mice lacking the heat generating uncoupling protein (UCP) in brown adipose tissue have no changes in ingestive behavior, though they resist obesity (100). The inability to synthesize noradrenaline and adrenaline due to targeted deletion of the gene for b-hydroxylase (dbh) results in increased food intake corresponding to a higher metabolic rate (101). DRUG INTERACTIONS
Alcohol and other drug testing paradigms have recently employed knockout technology to elucidate genetic and cellular bases for drug and alcohol interactions, as well as addictive behaviors. With dopamine (D1) receptor knockouts, the administration of both amphetamines and cocaine fails to produce the neural and behavioral effects stereotypical of these drugs (102). As mentioned above, loss of morphine analgesia was reported in MOR¹/¹ mice (51). Acute analgesic responses to morphine were not affected in mice heterozygous for the CREB a and D isoforms; however, reduced morphine abstinence and attenuated withdrawal symptoms were reported (24). The use of knockout mice in psychopharmacological research will likely yield important new information. SUMMARY
The use of animals with targeted disruption of genes
provides a novel method of understanding the genetic bases of behavior. Importantly, the use of knockouts does not imply acceptance that complex behaviors are controlled by a single gene. Rather, knockouts provide information about the mechanisms underlying behavior. Knockouts have already provided important insights into behavioral mechanisms despite the well-known limitations (reviewed by (75,11)). Additional progress likely awaits the development of conditional knockouts. In the meantime, the results of behavioral studies of knockout mice should be confirmed with other methods, including pharmacological methods. Progress in behavioral studies of knockouts will also profit from the development of standardized tests to assess the effects of targeted disruption of specific genes (e.g. (14)). Taken together, the use of genetic manipulations will likely become an important tool for behavioral biologists to study the mechanisms underlying behavior.
ACKNOWLEDGEMENTS
Partial support during the preparation of this manuscript was provided by USPHS grant MH 57535 (formerly HD 22201). We thank Drs. Ted Dawson, A. Courtney DeVries, and Gregory Ball, as well as Gregory Demas, Sabra Klein, and Lance Kriegsfeld for helpful comments. We are also grateful to Violette Renard and Sue Yang for bibliographic assistance.
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