Pregnancy, Immunity,
Schizophrenia, and Autism
by Paul H. Patterson

10

E N G I N E E R I N G&

S C I E N C E

N O .

3

2006

Opposite: A detail fromThe Temptations of St.
Anthony by Hieronymus
Bosch (d. 1516). The
fantastic—in the strictest
sense of the word—ﬁgures portrayed here are
not unlike some hallucinations reported byschizophrenia sufferers.
Courtesy of the Museu
Nacional de Arte Antiga,

Reprinted from Dierks, et al., Neuron, vol. 22, March 1999, pp. 615–621, © 1999,
with permission from Elsevier.

Lisbon.

Can something as innocuous as the ﬂu cause
schizophrenia? Can a pregnant mom’s sniﬄes
have lifelong consequences for her unborn child?
Does the brain’s own immune system play a role inautism? The answers to these and related questions are indeed surprising, and may suggest new
avenues for treatment or even prevention.
As we learn more about the connections between
the brain and the immune system, we ﬁnd thatthese seemingly independent networks of cells are,in fact, continually talking to each other. As anadult, the activation of your immune system causes
many striking changes in your behavior—increased
sleep, loss of appetite, less social interaction—and,of course, headaches. Conversely, stress in your life
(as perceived by your brain) can inﬂuence immune
function—the brain regulates immune organs,such as the spleen, via the autonomic nervous
system.

Recent evidence shows that this brain-immune
conversation actually starts during the developmentof the embryo, where the state of the mother’s
immune system can alter the growth of cells in the
fetal brain. As we shall see, such alterations canlead to an increased risk of schizophrenia or autism
in the oﬀspring.
First let’s consider schizophrenia, which is a progressive disorder whose initial psychotic symptoms
usually appear in early adulthood. (For a gripping
rendering of how psychotic episodes might appear
to the suﬀerer, see Russell Crowe in A Beautiful
Mind.) People with schizophrenia can be seemingly quite normal part of the time, and then havevery severe problems, which is a huge diﬃculty forthem—people have tended to blame the victim
and wonder why the patient doesn’t get him- or
herself together and behave properly.
In the last decade or two, anatomical andfunctional diﬀerences between schizophrenic andtypical brains have begun to emerge. Magnetic
resonance imaging (MRI) scans of the brains ofidentical twins, one with schizophrenia and one
without, have shown that in 90 percent of thecases the twin with schizophrenia has enlarged
ventricles, which are butterﬂy-shaped, cerebrospinal-ﬂuid-containing voids in the center of thebrain. One explanation for this enlargement isthat the gray matter surrounding the ventricles
might have shrunk, meaning the brain has fewer
or perhaps smaller neurons. Or the neurons might
be more densely packed. An alternative hypothesis
invokes an infection—encephalitis, for instance,
will expand the ventricles. Schizophrenia does not
result from a frank infection of the mature brain,
but there are other indications, which I’ll comeback to, that infections might be involved very
early on.
MRI shows anatomical details, but functional
MRI, which tracks blood ﬂow, shows brain activity. The more blood moving through a particularpart of the brain, the more active it presumably
is. In these renderings of functional MRI scans ofa schizophrenic patient, the head at far left shows,in yellow, that the auditory cortex lit up when aHeschl’s gyrus is thebrain’s main sound-processing center. A realsound lights it up on bothsides of the brain, as seen
at far left in these 3-D renderings. The gyrus lights
up spontaneously during
an auditory hallucination(left), but only on thebrain’s dominant side.

2006

E N G I N E E R I N G&

S C I E N C E

N O .

3

11

Right: There is clearly a genetic predisposition to schizophrenia. This chart shows how your chances of developing the disease increase if you have a close relative with
it—the more genes you share with the affected person,
the higher your susceptibility. Adapted from Schizophrenia

Genesis: The Origins of Madness by Irving I. Gottesman, W.
H. Freeman and Company, New York, 1990.

© Tee and Charles Addams Foundation.

stereophonic sound was played through earphones.
The other head shows the brain activity when thepatient pushed a button to signal that he or she was“hearing voices.” The hallucinations only appear in
the dominant hemisphere, so in this right-handed
patient, only the left hemisphere’s auditory cortex
lit up. It used to be said that the voices in theirheads were imaginary, but since there is activity
in the part of the brain that actually does process
auditory information, they really exist, in a sense.
Schizophrenics are hearing sounds, as far as theirbrains know, and it would be very interesting todiscover what generates this activity spontaneously.
We know that schizophrenia begins in early
development. Statistically, children who will later
develop psychosis are more prone to disciplinary
problems in school, tend to have lower IQs, andare more likely to be beset with emotional and
12

E N G I N E E R I N G&

S C I E N C E

N O .

3

2006

social problems. The diﬀerences are too small tobe useful for an early diagnosis, but they’re there.
There’s also a surprising delay in the developmentof motor functions—sitting, standing, walking,and so on.
There’s a genetic component to schizophrenia.
The most important risk factor for predicting
schizophrenia is having a sibling with the disorder.
In the general population, the risk for schizophrenia is approximately 1 percent worldwide. If you
have a schizophrenic cousin or uncle or aunt, therisk is doubled, which is not very signiﬁcant. But
if you have an identical twin with schizophrenia,
the risk is about 50 percent that you will become
schizophrenic as well. But it’s not 100 percent, so
it’s not a classical, dominant genetic disease like
Huntington’s disease, where a single malfunctioning gene gives you the disorder. Rather, people
think there are some six to 12 genes involved, eachof which contributes a small amount of risk. In thelast couple of years, a number of these genes havebeen identiﬁed, including neuregulin, dysbindin,
and one called “Disrupted-in-Schizophrenia,” or
DISC1. Furthermore, each of these genes is wellknown from animal studies to be very important inearly embryonic brain development.
There is also an environmental risk component. Being born in the winter or spring months,
or being born and raised in an urban area bothincrease risk. This is consistent with an infectious
hypothesis—we tend to get sick more often in thewinter and spring, and we’re more likely to sample
other people’s germs if we live in a crowded area.
Another important environmental risk factor ismaternal infection, which will be one of my major
themes. Having a respiratory infection during thesecond trimester of pregnancy increases the risk
for schizophrenia in one’s oﬀspring. In the year
2000, Alan Brown and his colleagues at Columbia
University in New York studied the medical recordsof 12,000 pregnant women who belonged to the
Kaiser HMO in the Oakland area. Brown foundthat there was about a threefold increase in risk if
the woman had a respiratory infection during thesecond trimester, conﬁrming the conclusions ofprevious studies that had not had access to patient
records. The researchers then analyzed frozen
serum samples from those women, and found asimilar, or even larger—up to sevenfold—increased
risk if antiﬂu antibodies were present during theﬁrst half of pregnancy. Moreover, they found a
statistically signiﬁcant association with elevated levels of some members of a group of proteins calledcytokines. Cytokines are produced by the whiteblood cells, and their levels in the blood increase
when we get an infection. A calculation of theso-called attributable risk from this data led to theestimate that about 20 percent of the schizophrenia
cases would not have occurred if ﬂu exposure had
been prevented.
This is a really dramatic piece of information,
particularly given that the researchers had to completely ignore the genetic angle. (Even now, wecannot screen for the susceptibility genes that have
Alarmingly, cases of autism appear to be dramatically on the rise. However,it’s not clear how much of this actually represents an increase in the incidenceof autism, or an increase in the diagnosis of autism rather than, for instance,
mental retardation.

since been identiﬁed.) Thus, the study presumably
included a large number of people who will neverget schizophrenia because they aren’t genetically
predisposed, yet it still found a three-to-sevenfold
risk increase. The actual risk due to maternal infection is therefore likely to be much higher.
Other studies of adult schizophrenic subjects
have found cytokine imbalances and elevated levelsof white cells in the blood. And antipsychotic
drugs such as clozapine, which people take to treat
hallucinations and disordered thoughts, are known
from animal studies to modulate cytokine levels inThis chart shows how the annual number of people diagnosed with autism who
were served by California’s Department of Developmental Services began toskyrocket in the mid-1990s. Courtesy of the DDS.

2006

the blood. So these drugs might not only be acting
in the brain, but on some aspect of the immune
system to achieve their eﬀectiveness. I think thisis a very interesting observation, but it hasn’t mademuch of an impression on the research community
yet, so the possibility hasn’t really been investigatedcarefully.
A recent, very impressive paper by William
Eaton and colleagues at Johns Hopkins University
Medical School analyzed the remarkably comprehensive records of Denmark’s health system, which
tracks every Dane from the cradle to the grave.
The investigators accessed the ﬁles on all 7,704
people who were diagnosed with schizophrenia
between 1981 and 1998, including the details of
every hospital visit those people ever made in their
entire lives. It turns out that people who developed
any of nine diﬀerent autoimmune disorders—diseases in which the body’s immune system begins
attacking one’s own cells—had a 45-percent
increase in risk for developing schizophrenia.
So there is a link between the immune systemand schizophrenia, but we don’t know what it is.
We know that a genetic predisposition to autoimmune disease exists—are the genes responsible
for this predisposition somehow linked to theones predisposing to schizophrenia? Or is there
something about having an autoimmune disorder,
such as the creation of antibodies against certain
molecules, which increases risk for schizophrenia?
Now let’s turn to autism, which was originallydescribed by Leo Kanner at Johns Hopkins in 1943
as a type of schizophrenia. We don’t think that way
anymore, but there are some interesting similarities—particularly in the withdrawal of patientsfrom the world around them. The hallmarksof autism are, of course, deﬁcient social skills—
patients don’t read other people’s emotions well or
respond to them appropriately—and the lack ofdevelopment of language. Heartbreakingly, about
30 percent of patients actually experience a regression in these areas that starts at about age three.
Unlike schizophrenics, however, autistic children
frequently display odd, repetitive gestures—banging their heads against the wall, or a ﬂapping
motion with the hands that is a classic symptom
often used by teachers as a possible indication thata problem may exist. And autistics tend to ﬁxate
on objects and rituals. A patient might spend
hours playing with a piece of string, for example, or
eating her dinner in just the right way. There’s also
fear of new situations or objects, and oftentimes
considerable problems with sensory stimuli—
extreme sensitivity to noises, for example. Alarmingly, cases of autism appear to be dramatically on
the rise. However, it’s not clear how much of thisactually represents an increase in the incidence ofautism, or an increase in the diagnosis of autism
rather than, for instance, mental retardation.
Like schizophrenia, there’s a strong genetic component to autism—the single biggest risk factor
is having a sibling with it. Autism is also a multiE N G I N E E R I N G&

S C I E N C E

N O .

3

13

organs all develop in thefetus according to a very
strict timetable, and thetypes of birth defects
seen in thalidomide babies
correlate very precisely
to when the mother-tobe took the drug. Somethalidomide babies arealso autistic, revealing awindow of vulnerability
in early brain development. Autism data from
K. Strömland et al. in

Developmental Medicine
and Child Neurology, April
1994; graphic after Patricia
Rodier, Scientiﬁc American,
February 2000.

14

E N G I N E E R I N G

genetic disorder, with six to 10 genes involved, andagain, the genes that have been identiﬁed thus far(neuroligins 3 and 4, En-2, and Hox-a1) are very
important in embryonic brain development. Furthermore, there are environmental risk factors for
autism. Valproic acid, which is used to treat epilepsy, causes a dramatic increase in the risk of autism
when taken by women before they know they’repregnant. This drug is still commonly prescribed,
but people are beginning to get concerned about its
use by pregnant women.
We have a valuable insight into the fetus’s periodof vulnerability, thanks to the thalidomide tragedy.
Those of you who are old enough will remember
the use of thalidomide as an anti-morning-sickness
drug in the 1960s. Severe birth defects resulted,
as did an increased incidence of autism. But what
is key here is that the kind of physical abnormality one got—missing ears, stunted arms, stunted
legs—was found to depend on how far along the
pregnancy was. In other words, the child’s deformity told us exactly when, sometimes to withina day or two, the mother took the drug. The
window of risk for autism proved to be days 20
through 23 after conception—a very early stage
in neural development. At this time, the neuraltube is just closing, and the ﬁrst neurons are justbeing born. A similar window of risk is found withvalproic acid, and with an ulcer-preventing drug
called misoprostol. We don’t know the cause orcauses of autism in most cases, but this window ofvulnerability is clearly a very important clue to how
the brain is altered in this disorder.
Again, as in schizophrenia, there’s a maternalinfection risk factor. A review of the literature byAndrea and Roland Ciaranello at Stanford concluded that “the principal nongenetic cause of autism isprenatal viral infection.” This was based primarily
on studies of an epidemic of rubella, or German
measles, in New England in 1964. On the order of
10 percent of the children born to infected mothers
exhibited symptoms of autism, which is really an&

S C I E N C E

N O .

3

2006

Vargas, et al. Annals of Neurology, vol. 57, no. 1, pp. 67–81, 2005, © 2004 American
Neurological Association. Reprinted with permission of John Wiley & Sons, Inc.

Fingers, toes, limbs, andastronomical increase in risk. Of course, rubella is
not common anymore, because we get vaccinatedagainst it, but the point is that maternal infection
can increase the risk for autism. Other infections
have also been implicated—a paper just came out
last week linking genital herpes infection with anincreased risk for autism.
Without going into the details, the rates of autoimmune diseases and allergies are higher in families
with autism, particularly in the mother. There arealso reports of immune dysfunctions in the bloodof autistic individuals. These various connections
to the immune system are, of course, reminiscentof schizophrenia.
There is also very striking evidence of immune
dysregulation in the brain itself. Just last year, agroup led by Carlos Pardo at Johns Hopkins found
what they’re calling a “neural inﬂammation” inpostmortem examination of brains of patients with
autism who died between the ages of eight and 44
years. But these people weren’t infected—they diedof such things as drowning or heart attacks. The
study found that the microglial cells, which act as
the brain’s own immune system, were activated.
The study also found amazing increases of certain
cytokines in the brain, and of others in the cerebrospinal ﬂuid. This is a landmark paper, in my opinion. It presents the ﬁrst evidence that there’s anongoing, permanent immune-system activation in
the brains of autistic people. It’s a subclinical state,because there’s no overt infection. But it’s there.
To try to untangle how the immune system isintertwined with the development of these diseases,
we turned to an animal model. Animals are vital to

In this cross section of the cerebellum of an autistic
patient, the microglial cells have been activated, as shownby their absorption of a red dye that binds to an immunesystem protein called HLA-DR.

medical progress. If you think a gene is importantin a particular disease, you can introduce that gene
into a mouse, and note whether it gets something
like the human disease. You can also test bacteria, viruses, and environmental toxins. You canstudy pathogenesis—how the stages of the diseaseprogress, and how it spreads from tissue to tissue—in animals much much easier than you couldin humans. And you can, of course, test treatments. By law, you have to test drugs on animals
ﬁrst. It’s also how we work out the details of new
surgical procedures and explore the potential of
new therapies, such as those involving stem cells.
Without the animal studies that preceded them,such common but highly complex procedures asbone marrow, kidney, and heart transplants would
not be available today.
That’s all well and good, but what about animalmodels of mental illness? How do you psychoanalyze a mouse? How can you tell if it’s hallucinating? (I think we can, but that’s a topic for a future
Watson lecture.) And how do we even model a disease like autism, which is supposed to be uniquely
human? How can you measure an autistic mouse’s
impaired language skills when—sorry, Walt—theyaren’t capable of speech in the ﬁrst place? Or atleast not speech that we can understand—they docommunicate via alarm and distress calls, and thereis even some speculation that they can recognize
other mice by their voices. But that, again, isanother story.
Fortunately, that isn’t what we really do withanimal models. We don’t mimic the whole diseasein any model—we mimic features of the disease.
This might be the kinds of neurons that die. It
Top: Elevated cytokine

ing mouse psychology.

might be some change in the electrical propertiesof the neurons, or some molecular change suchas the cytokine levels. Or it could be the tremorsand shuﬄing gait of Parkinson’s disease. In fact,
the mouse models of Alzheimer’s and Huntington’sdiseases that are in routine use in labs around theworld do not display some of the diseases’ key
features. The neurons that typically die in humanpatients somehow survive, for example. So a model doesn’t have to be perfect to be extremely useful,even when testing potential human therapies.
Our laboratory is exploring a model of maternal
infection. We give a pregnant mouse the ﬂu by
touching a pipette containing a solution of thehuman inﬂuenza virus to her nose, which she theninhales. The mousegets lethargic, stops
grooming herself,hunches in the cornerof the cage, and ina few days recovers
and behaves normallyagain. In due time
she gives birth, and westudy the pups, both as
infants and adults. We
watch their behavior,and then examine theirpathology—what theirbrains look like.
What types ofmouse behavior might
be relevant for schizophrenia or autism?
People often use what’s
called an open ﬁeld
test to study anxiety
under mildly stressful
conditions. The mouseis placed in an enclosure with a camera
overhead and grid
Vargas, et al. Annals of Neurology, vol. 57, no. 1, pp. 67–81, 2005, © 2004 American Neurological Association.
Reprinted with permission of John Wiley & Sons, Inc.

levels were found in three

A handy guide to decipher-different parts of thebrains of autistic patients.
The ﬂat gray rectangle atthe bottom of the graphshows the corresponding
levels in typical brains.
Bottom: Cytokine levels in
the cerebrospinal ﬂuid ofautistic (purple) and unaffected (yellow) subjects.

2006

E N G I N E E R I N G&

S C I E N C E

N O .

3

15

The path followed by acontrol mouse exploring
an unfamiliar place. The
asterisk marks where themouse was placed in thebox, and the red circles
show where the mousestood up on its hind legs
for a better whiff of its
surroundings.

By contrast, a mousewhose mother got the ﬂu
tends to stay hunkered
down in the corner where
it was placed.

16

E N G I N E E R I N Glines on the ﬂoor so we can track where the animal
moves. A normal mouse usually spends a lot oftime creeping along the edges of the box at ﬁrst,
because it’s afraid that it’s dangerous to go out into
the middle—which, obviously, it might be. But
it will eventually inspect most of the box, pausing
frequently in the process to rear up on its hind legs
and sniﬀ the air. Our normal mice, which we call
control mice, are born to “sham-infected” mothers
who were given a sterile saline solution without thevirus. These mice do exactly the same thing—they
are timid at ﬁrst, but they’re soon traipsing all overthe box.
Below is an example of an adult mouse who wasborn to a ﬂu-infected mother, and you can tell
immediately from the fecal pellets that it hasn’t
moved beyond its corner very much at all. We
would interpret thisas excessively fearful
behavior, given themildly stressful natureof the situation, andwe can quantitate it bysimply measuring theamount of time spentin the center squares ofthe box. This mouseenters the center many
fewer times, and it rears
and sniﬀs much lessoften as well.
The so-called
novel-object test is also
relevant. Remember,
autistic children areoften afraid of unfamiliar things. So when weput something strange
and new in the ﬁeld,
say a coﬀee cup, thecontrol mouse carefully investigates it,&

S C I E N C E

N O .

3

2006

touching and sniﬃng it from all sides, whereas ourmouse born to an infected mother is very reluctant
to go anywhere near it. In fact, this mouse turns
its head away and acts as if the object isn’t there.
We measure the time lapse before the mouse ﬁrst
touches the object, which we call the latency toﬁrst contact, and we count how often contacts are
made. Again, the diﬀerences are dramatic. The“autistic” mouse waits much longer, and touches
the object far fewer times.
We also do simple social interaction tests. We
put two mice who don’t know each other in thebox, and ask how long it takes them to makephysical contact, and how often they do so. And
not surprisingly, pairs of mice born to infectedmothers make contact less than half as often andhave more than four times the latency. So clearly
they’re not socializing properly. Grad student Steve
Smith is now following up on that observation by
Adapted from Shi, et al., The Journal of Neuroscience, vol. 23, no. 1, pp. 297–302, January
1, 2003. © 2002 Society for Neuroscience. Reprinted with permission of Wiley-Liss,
Inc., a subsidiary of John Wiley & Sons, Inc.

Mice whose mothers were given the ﬂu virus ventured into
the great empty middle of the box much less often (left)
and spent much less time there overall (center). They also
reared up to sniff less often (right).

At least rodents don’t run
up bar tabs—biology staff
member Limin Shi puts apair of mice in a threeroom box designed to test
their social skills.

Top: A normal mouse inspects an unfamiliar object withavid curiosity.
Bottom: An “autistic” mouse ignores it, seeming to act on
the theory that if it can’t see the object, the object doesn’t
exist.

2006

using a box divided into three rooms. We put our
test mouse in the middle room, and then we put
an unfamiliar mouse in one of the side rooms. We
leave the room on the opposite side empty in sometests, and in others we put a cage mate of our test
mouse in there. Then we sit back and watch where
our test mouse goes. Normal mice like novelty,
and almost always go to the strange mouse, evenwhen there is a familiar mouse in the other room.
Preliminary results with our “autistic” mice, however, show that they prefer to remain in the central
chamber regardless of who else is in the box withthem.
Another pertinent test is the startle response,
which is a lot like sneaking up behind someone
with an inﬂated paper bag and popping it. We put
the mouse in a tube inside a soundproof box, andunderneath that tube is a motion sensor. There’s
a speaker in the box, and when a loud sound isplayed, the mouse is startled, and we measure howhigh it jumps. But if we precede the loud soundwith a softer sound that doesn’t startle the mouse—called a prepulse—it doesn’t jump so much. Thisis called prepulse inhibition, or PPI, and when
the same type of test is done in people, a striking
deﬁcit is observed in schizophrenic and autistic
subjects. In other words, they get startled just as
much regardless of whether they got a prepulse ornot. The loud noise surprises them every time. We
think this relates to the attention-deﬁcit issues. On
the next page is a plot of the amount of the mice’s
PPI versus prepulse intensity. As we increase theprepulse intensity, we get a bigger inhibition acrossthe board, but our “autistic” mice have a PPI deﬁcit
at every intensity.
E N G I N E E R I N G&

S C I E N C E

N O .

3

17

Right: The mouse-startlingmachine. The mouse sits
in its comfy burrow—aplastic tube, seen end-on
in these pictures, that inturn sits on a platform
with a motion sensor (theblack unit connected tothe black cable) on its
underside.
Far right: Regardless of
how loud the prepulse was

Adapted from Shi, et al., The Journal of Neuroscience, vol. 23, no. 1, pp. 297–302, January
1, 2003. © 2002 Society for Neuroscience. Reprinted with permission of Wiley-Liss,
Inc., a subsidiary of John Wiley & Sons, Inc.

(the numbers are in decibels), the “autistic” mouse
was always more startled—that is, had a lower
prepulse inhibition (PPI)
than a control mouse.

The PPI is thought to be a measure of sensory-motor gating—the connection between theﬁltering of incoming sensory information and thecreation of motor outputs to the muscles—which
is likely to be related to attention deﬁcits anddistractibility. In fact, a PPI deﬁcit is also foundin attention deﬁcient disorder. Importantly, thoseantipsychotic drugs mentioned earlier can restore
the PPI in schizophrenic subjects, whereas psychomimetic drugs—hallucinatory drugs—disrupt PPI.
We have shown the same thing to be true in ourmice.
We presume that these behavioral abnormalities
are based in brain pathology—changes in the nerve
cells, or in their connections. In fact, postmortem
examinations of at least some brains of schizophrenia patients have shown nerve cells that are not intheir appropriate locations. So recently, biology
staﬀ member Limin Shi, postdoc Natalia Malkova,
and Steve Smith have been looking at fetal brain
development in the mice. For this analysis, thepregnant mice are given the ﬂu at mid-pregnancy,
day 9.5 of gestation, which corresponds to theperiod of very early brain development in humans.
In other words, it’s similar to the thalidomide

The human (right) andmouse (far right) brains,
not to scale.

18

E N G I N E E R I N G&

S C I E N C E

N O .

3

2006

window of autism vulnerability. However, because
fetal mice develop so fast, the illness also extends
through the period corresponding to that secondtrimester stage in humans when maternal infections lead to an increased risk of schizophrenia.
Five days into the infection, a dye that marks newly
formed neurons is injected into the mice, and theygive birth six days after that. At right is the brainof a normal pup. The green neurons have taken
up the dye, and most of them have migrated out
to what neuroanatomists call layers 2 and 3 of thecerebral cortex. This is similar to how a normal
newborn human brain would look, too. But thislayer is barely present in the pups from infectedmothers. Something has gone very wrong, because
the green cells have wandered oﬀ all over insteadof forming the normal, tightly packed layers. We
plan to repeat the experiment but let the pups growto adulthood to see if this disorganization persists,
and whether it looks similar to what was found in
those few human schizophrenia examples.
Another human pathology occurs in the cerebellum. The cerebellum has lobes, called lobules,
which look like a cauliﬂower in cross section, andcontain neurons called Purkinje cells that are pres-A 20-month-old childparticipates in Pierce andCourchesne’s current setof exploration studies.
The tape grid on the ﬂoor
helps the researchers map
the child’s movements.
Photo courtesy of Karen
Pierce.

Neurons from an earlystage of brain development have been labeledwith a ﬂuorescent green
dye. These neurons form
clearly visible layers in ahealthy newborn mousebrain (top), but when themother was infected in
midpregnancy (bottom),the neurons are scatteredalmost at random.

ent in all mammalian species. Some 90 percent
of postmortem autism samples show a substantial
reduction in the number of Purkinje cells in lobules
VI and VII. In some cases there are even misplaced
Purkinje cells. And MRI studies of living autistic
subjects reveal that lobules VI and VII are underdeveloped.
There is a fascinating correlation between
abnormalities in lobules VI and VII and children’s
exploratory behavior. In 2001, Karen Pierce andEric Courchesne at UC San Diego did a study
where they put a child (aged three to eight) in aroom with a lot of brightly colored boxes and otherintriguing objects, and counted how many of themthe child played with in eight minutes. The control kids, on average, explored about 10 of the 14
items. But the autistic children tended to ﬁxate ona few objects to the exclusion of all others—in one
extreme example, the child got no further than thevery ﬁrst item it encountered. All of these children
had previously had MRI scans as part of anotherstudy, and a dramatic correlation popped out—thesmaller the autistic child’s lobules VI and VII, thefewer objects the child showed interest in.
Because our “autistic” mice were similarly
immune to the allure of an unknown object,
we wanted to see if they had the same cerebellar
abnormality. Treating the cerebellum with a dyethat just stains Purkinje cells reveals a consistentdiﬀerence in these mice, as you will see on the next
page. In addition, we occasionally see what we
think are misplaced Purkinje cells. The cell bodies
are supposed to line up in a neat row along theboundary between the red and the black zones, andnot dawdle in the dark interior. We think that thismisplacement must have occurred in embryonicdevelopment.
Now let’s consider the mechanism for how thisworks, which is where the animal model comes invery handy indeed. Does the virus actually infectthe fetal brain itself, or is it working indirectly
through the mother’s immune system? We think
2006

From Karen Pierce and Eric Courchesne, Biological Psychiatry, vol. 49, pp. 655–664,
2001. © 2001. With permission from the Society of Biological Psychiatry.

An MRI scan of a control child’s cerebellum (left) and an
autistic child’s (right), with lobules VI and VII shaded red.

E N G I N E E R I N G&

S C I E N C E

N O .

3

19

Control and “autistic”
mouse cerebellums stainedwith a dye that binds toPurkinje cells. The bottom
two images are close-ups
of lobule VII. The bright
red globs are the Purkinje
cell bodies, and the darkvoids are actually chockfull with other types ofcells. The right-hand
image—an extreme
example from an adultmouse born to an infectedmother—shows almost no
Purkinje cells.

Errant Purkinje cells(white arrowheads) in the
middle of lobule VI.

20

E N G I N E E R I N G

it’s the latter, because we can’t ﬁnd the virus in theoﬀspring, either in the embryonic brain or at birth.
That’s not surprising because, after all, inﬂuenza is
primarily a respiratory virus. It hardly ever gets outof the lungs, throat, and nose and into the rest of
the body. When it does, you have viremia, which
is a very serious disease.
Furthermore, we can evoke an immune responsein the mother without using a virus, simply by
injecting her with a piece of double-stranded RNA.
Mammals don’t make double-stranded RNA but
many viruses do, so the immune system knowsthat when it sees double-stranded RNA, it needs toswing into action. It starts secreting cytokines andin general mounting a vigorous antiviral response,even though there’s no infection. Tellingly, theoﬀspring of mothers whose immune systems havebeen artiﬁcially activated in this way display thesame PPI deﬁcit that we saw before. So we don’t
need the virus; activation of the maternal immune
system is suﬃcient to alter the behavior of theoﬀspring.
A second example of “autistic” behavior broughton by maternal immune activation was discoveredby Natalia Malkova. Anecdotal evidence suggeststhat autistic human infants may be less bondedwith their mothers. When Natalia removes themother mouse from the family cage, it normallyinduces considerable crying in the control pups,
although since mouse pups vocalize at ultrasonicfrequencies, we have to use a special microphone
to hear them. So Natalia counted how often thepups cried in three minutes, and the mice born toa double-stranded-RNA-exposed mother cried lessthan pups born to a normal mother.
&S C I E N C E

N O .

3

2006

We think that maternal immune activation alters
brain circuits. Besides that dramatic abnormal
layering Limin ﬁnds in the mouse cortex, and a lossof Purkinje cells that’s been seen in the human cerebellum, there’s that permanent, subclinical, alteredimmune state in the autistic brain—those increased
cytokine levels. Are those cytokines an irrelevant,residual footprint—a fossil, if you will—of someearlier event, like a maternal infection? Or are
they actually interacting with the brain in an
ongoing fashion, with consequences visible in thepatients’ behavior? I favor the latter hypothesis.

Mouse pups born to uninfected mothers (yellow bar)cried about 60 times in three minutes, or once every
three seconds, when separated from Mom. So did control
pups (purple) whose mothers had inhaled a sterile saline
solution. Pups whose mothers’ immune systems had been
activated, either by a virus (lavender) or a piece of doublestranded RNA (green), cried much less often.

Vargas, et al. Annals of Neurology, vol. 57, no. 1, pp. 67–81, 2005, © 2004 American
Neurological Association. Reprinted with permission of John Wiley & Sons, Inc.

Top: Purkinje cells (purple-blue dots) in a normal
human cerebellum.
Bottom: Nine of the 10
autistic brains analyzed
showed patchy Purkinje
cell loss.

In some clinical trials where cancer patients were
given cytokines in the hopes that these molecules
would attack their tumors, dramatic diﬀerencesin behavior and mood became apparent—up to
severe depression, in the worst cases. And otherresearchers have found that high levels of cytokines
in animals can alter learning and memory.
If this hypothesis is true, what would happen if
we changed the brain’s immune state? Antipsychotic drugs are known to suppress the immune
system. Is that relevant to psychotic behavior? We
are very interested in this possibility. In fact, Carlos Pardo of Johns Hopkins and I are organizing ameeting with the Cure Autism Now and Autism
Speaks foundations to examine the possibilityof immune intervention in autism. People takeanti-inﬂammatory drugs such as aspirin to modulate their immune response all the time—is this astrategy worth exploring in this context?
We are just starting to explore the interactions
between the immune system and the developing
brain. Cytokines aren’t the only possible conduit
from a mother’s infection to the fetus’s developing
brain—there are other changes brought about bycorticosteroids, which are released following an
infection or sickness, that also have eﬀects on thefetus. And don’t forget the genetic component—on what are those genes acting to increase thesusceptibility? They might aﬀect fetal brain development directly, or they might aﬀect the brain’s
susceptibility to such other factors as cytokines,or the response of the placenta to the mother’s
immune activation, or they might even be acting in
the mother, to aﬀect her response to infection. We
should be able to sort these possibilities out eventually, using this animal model.
Finally, I want to ask a question that’s come up
in the literature in the last few years—should wereally be promoting universal maternal vaccination?
The ﬂu vaccine has been recommended routinely
to pregnant women in the United States since1957. The oﬃcial policy of the Centers for Disease
Control states that “administration of vaccines towomen seeking prenatal care is an opportunity
for preventative intervention that should not be
wasted.” Now you might say, “Well, of course,
you don’t want to get the ﬂu if you’re pregnant!”
But remember that double-stranded RNA experiment—we activated the immune system, and it
caused all these downstream eﬀects on the fetus.
And what does a vaccination do? It activates theimmune system. That’s the point of vaccination.
In practice, not all pregnant women receive ﬂu
shots, and I think that universal vaccination ofpregnant women could get us into a whole new
set of problems. I’m hoping, therefore, that a way
will be found to intervene somehow and repair thedamage or reregulate the immune system. This
mouse model is an excellent place to start.

Postdoc Natalia
Malkova andfriend.

Paul Patterson, the Biaggini Professor of Biological Sciences at Caltech and a research professor ofneurobiological surgery at the Keck School of Medicine
at USC, got his BA in biology at Grinnell College
in Iowa in 1965, and his PhD from Johns Hopkins
in 1970. He was a professor of neurobiology at theHarvard Medical School before coming to Caltech in
1983, following in the footsteps of his uncle, the lateProfessor of Geochemistry Clair Patterson. This article
was adapted by Douglas Smith from a Watson lecture
given May 17, 2005, at which Patterson was introduced by Caltech trustee Ted Jenkins (BS ’65, MS’66), who has a schizophrenic son, and who with hiswife, Ginger, underwrote the cost of the mice for the
beginning of this work. Other support came from thelate Ruben Mettler (BS ’44, MS ’47, PhD ’49), theCure Autism Now and Autism Speaks foundations,
the Stanley Medical Research Institute, the McKnight
Foundation, and the National Institute of Mental
Health.

Mouse limos.
PICTURE CREDITS: 17, 18, 21—Bob Paz; 14, 18 — Doug
Cummings

2006

E N G I N E E R I N G&

S C I E N C E

N O .

3

21