Expression of IA-2, IA-2β and neurotransmitters in brain of WT and DKO mice
IA-2 and IA-2ß are widely distributed in cells throughout the brain. By immunofluorescence, both of these proteins are found in the cerebral cortex, hippocampus, thalamus and hypothalamus with somewhat wider expression of IA-2ß as compared to IA-2 in the cerebral cortex and hippocampus (see Supplementary Fig.1
). In contrast to WT mice, DKO mice do not express either of these proteins as determined by immunofluorescence (see Supplementary Fig.1
) or Western blot ().
Decreased neurotransmitters in brain of DKO mice
To see whether the deletion of IA-2/IA-2ß had any effect on neurotransmitters, brain homogenates were analyzed by HPLC (). At 12 weeks after birth, the level of norepinephrine, dopamine and serotonin in DKO as compared to WT mice was decreased by 23.5%, 50.5%, and 37.0%, respectively. At 30 weeks, the decrease was 19.6%, 34.2%, and 60.9%, respectively. Thus, the deletion of IA-2/IA-2ß has a substantial effect on the level of a number of neurotransmitters in the brain.
Physical examination of DKO mice
Physical examination (Crawley, 2000
) of the DKO mice revealed normal appearing animals with no gross abnormality: body position and gait were normal as was walking speed (). Heart rate, respirations and body weight similarly were normal. In a battery of neurological reflex tests including righting and corneal reflexes no significant differences were found between the WT and DKO mice, except the DKO appeared somewhat less active than the WT mice.
Behavioral changes in open field (A-E) and height-fear (F) tests in DKO mice
Anxiety-like behavior in DKO mice
To see if the deletion of IA-2/IA-2ß had any effect on behavior, WT and DKO mice were evaluated in an open field test. Because this test places an animal in a novel environment imposing a conflict between an innate desire to explore versus safety, it is used as a surrogate for anxiety-like behavior. The DKO mice showed a highly significant increase in the time required for them to begin exploratory activity () at both 12 and 30 weeks of age as compared to the WT mice. In addition the frequency of rearing (standing on two feet to look at the surroundings) (), total distance traveled () and frequency of visits to central area of cage () were all significantly reduced.
Further evaluation of anxiety-like behavior was obtained with the height-fear test, which measures the length of time (latency) to step down from increasingly high platforms on which the mouse is placed (). At 1.5 and 3.0 cm there was no significant difference between the DKO and WT mice, but at 6.0 and 7.5 cm there was a marked increase in the latency of descent, requiring two to three times as long for the DKO as the WT mice to step down.
Conditioned Learning in DKO mice
To evaluate the effect of the knockout of IA-2 and IA-2β, the conditioned taste aversion (CTA) test was used. During the training period DKO and WT consumed the same volume of water () and both showed an equally strong preference for saccharine-flavored water () indicating that taste and ability to consume fluid was not impaired in the DKO mice. In the conditioning phase, mice were given saccharine-flavored water, followed by an intraperitoneal injection of LiCl which causes abdominal discomfort. At different times thereafter the mice were given a choice of saccharine-flavored or plain water. As seen in , one day after exposure to the conditioning stimulus, less than 5% of the water consumed by the WT mice was saccharine-flavored as compared to 22.7% for the DKO. At 7 and 14 days after the conditioning stimulus 7.5% and 19.4%, respectively, of the water consumed by the WT mice was saccharine-flavored as compared to 72.4% and 89.9%, respectively, for the DKO mice indicating highly accelerated extinction of conditioned learning.
Conditioned learning in DKO mice as evaluated by taste aversion, olfactory-place preference and rotarod-latency to fall
In the olfactory conditioning test an odorant (lemon or peppermint) was placed in a dish in the conditioning cage with a sugar cube. After conditioning (a total of 8 trials), animals were placed in the central compartment of a 3 compartment interconnected cage in which the conditioning odorant (but without a sugar cube) was placed in one of the side-compartments and the non-conditioning odorant (also without a sugar cube) was placed in the other side-compartment. The amount of time that the mouse spent exploring each compartment was determined. As seen in , whereas the WT mouse spent nearly 4 times as long in the compartment with the odorant previously associated with the sugar cube, the DKO mouse spent an equal amount of time in the two side-compartments demonstrating a lack of place-preference learning for the conditioning stimulus.
Motor learning was evaluated by the rotarod test which measures the length of time that a mouse can remain on a rotating drum and its capacity to learn motor coordination upon repeated trials (). The latency to fall for DKO mice on the first trial was 27 ± 5 seconds, whereas for WT mice it was a 105 ± 12 seconds indicating a possible coordination problem. However, upon repeated trials, the DKO mice learned how to maintain their balance and the latency to fall was increased to approximately 232 ± 17 seconds nearly reaching the latency of fall (288 ± 4 seconds) observed in the WT mice. These findings argue that the DKO mice may suffer from some impairment of coordination, but maintain much of their motor learning ability.
Spontaneous and induced seizures and sudden death in DKO mice
Up to about 12 weeks of age handling the DKO mice rarely induced seizures. However, with increasing age, handling-induced seizures increased from about 10% at 13 to 30 weeks of age to nearly 60% at 50 to 80 weeks of age (). The seizures were characterized by facial twitching and myoclonic jerks followed shortly thereafter by generalized clonic-tonic seizures and loss of posture. Most of the animals recovered within several minutes and resumed a standing position.
Increase in spontaneous and induced seizures and shorten lifespan in DKO mice
To see if the DKO mice were more susceptible to drug-induced seizures, 9 to 12 week old mice were injected with pentylenetetrazol (PTZ), a GABA receptor antagonist, before the onset of handling-induced seizures. DKO mice showed an increased susceptibility to PTZ-induced seizures as demonstrated by a shorter latency to first fall (225 seconds) as compared to the WT mice (525 seconds) (). Similarly, the DKO mice were more susceptible to kainic acid (KA) as shown by the severity of seizures based on a 1 to 5 scale (Otani et al., 2006
) (). Approximately 80% of the DKO mice died within 2 hours after 20 mg/kg KA injection, whereas all the WT mice survived. Moreover, injections of KA, which is particularly toxic to neurons in the hippocampus, produced more severe neuronal degeneration in the DKO than in the WT mice as characterized by the loss of neurons and pyknotic nuclei (data not shown).
The KO of IA-2/IA-2ß also resulted in a significantly shorter life span. Up to 40 weeks of age none of the WT mice died, whereas approximately 50 % of the DKO mice died during that time period (). The deaths were sudden. Animals appearing perfectly normal at the end of the day were found dead the next morning. Although death following spontaneous seizures was not observed, seizures appear to be a likely explanation for the sudden deaths.
IA-2ß colocalized with synaptic vesicles
IA-2 and IA-2ß are transmembrane proteins of DCV. There is very little information about the relationship of these two proteins to SV. The fact that deletion of IA-2/IA-2ß resulted in a decrease in the levels of norepinephrine, dopamine and serotonin () and also resulted in behavioral, learning and neurological abnormalities suggested that some of these changes might be related to alterations in SV. To see if IA-2 or IA-2ß colocalized with SV, brain homogenates were subjected to sucrose gradient fractionation. shows a different distribution for IA-2 and IA-2ß. Both of these proteins were found in the fractions associated with the DCV marker, secretogranin II, whereas IA-2ß, but not IA-2, was found in fractions associated with the SV marker, synaptophysin. A similar distribution was found upon sucrose gradient fractionation of PC12 cells (data not shown).
Distribution of IA-2 and IA-2β in SV and DCV
Subcellular fractions enriched for SV also showed that IA-2ß colocalizes with SV (see Supplementary Fig.2
). Fractions sequentially enriched in SV (e.g. SG-V) contained primarily IA-2ß and little or only trace amounts of IA-2. Further evidence for the presence of IA-2ß in synaptic vesicles comes from biotinylating proteins on the surface of synaptosomes and from separating intracellular non-biotinylated proteins from biotinylated surface proteins. By Western blot, with antibodies to the luminal domain of IA-2ß and to SNAP-25 (), both IA-2ß and SNAP-25 were found both on the surface of the synaptosomes (biotinylated) and within synaptosomes (i.e., cytosolic, non-biotinylated). That the surface expression of IA-2ß was not the result of contamination with cytosolic proteins was demonstrated by the absence of HSP60 in the biotinylated surface fraction. Moreover, IA-2 was not found on the surface of the synaptosomes and only trace amounts were found within the cytosolic fraction of the synaptosomes. These finding suggest that when SV fuse with the surface of the synaptosome, the luminal domain of IA-2ß becomes exposed. In other experiments, primary neuronal cultures from the hippocampus were immunostained with antibodies to IA-2ß and synapsin I, a marker for SV (). Both antibodies produced a speckled pattern that colocalized when the images were merged.
Functional analysis of synaptosomes from DKO mice
No difference was found in the uptake of radiolabelled dopamine, GABA or glutamate () by synaptosomes prepared from DKO as compared to WT mice. Moreover, no difference was found in the release of dopamine, GABA or glutamate under basal conditions when the synaptosomes of the DKO mice were compared to those of the WT mice (). However, upon stimulation with K+ there was a significant decrease ranging from 20.3 %, to 26.7% in the fractional release of all three neurotransmitters from the synaptosomes of the DKO mice. Electron microscopic studies on tissue secretions from the hippocampus revealed normal appearing synapses (not shown), but approximately a 60% decrease in the number of SV ().
Decreased secretion of neurotransmitters from synaptosomes of DKO mice
Effect of neuropharmacologic agents on behavior
To determine whether neuropharmacologic agents could reverse some of the behavioral abnormalities found in the DKO mice, the tail suspension test (TST) was used. In this test rodents become immobile in aversive situations from which they cannot escape. As seen in , the DKO mice treated with saline remained immobile considerably longer (225 seconds) than the WT mice also treated with saline (150 seconds). Many anti-depression drugs are known to reduce immobility time in the TST test (Cryan et al., 2004
). Treatment of WT mice with fluoxetine, bupropion or pargyline, as expected, markedly reduced immobility from 150 seconds (saline control) to between 25 and 50 seconds. Treatment of DKO mice with fluoxetine and bupropion, but not pargyline, reduced immobility from 225 seconds (saline control) to about 125 seconds. This is within the time range of the untreated WT saline controls, but does not approach the reduction in immobility seen when fluoxetine and bupropion were used to treat WT mice.