Translating from Animal Models to

Human Schizophrenia: Insights into Pathophysiology, Treatment

and Prevention

Anthony A. Grace, Ph.D.Departments of Neuroscience,

Psychiatry and Psychology

University of Pittsburgh

Disclosures: Johnson & Johnson, Lundbeck, Pfizer, GSK, Merck, Takeda,

Dainippon Sumitomo, Otsuka, Lilly, Roche, Asubio, Akerrmes, Abbott

We have used an animal model based on a disruption of

neuronal development with MAM (methyl azoxymethanol

acetate) that has provided new insights into the complex

circuitry thought to link dopamine system responsivity and

schizophrenia

MAM is a DNA alkylating agent that is given at

gestational day 17, which is a sensitive period

corresponding to the second trimester in humans

MAM developmental model of schizophrenia

1. Anatomical Evidence:- thinning of limbic cortical structures

- increased cell packing density

3. Pharmacological Evidence:- increased response to PCP

- increased locomotion to amphetamine in adult

- no difference in response to amphetamine in prepubertal stage

2. Behavioral Evidence:- impairment in prepulse inhibition of startle

- impairment in reversal learning

-impairments in extradimensional shift

-impairment in latent inhibition

-impairment in social interaction

The increased amphetamine response is consistent with a

role for dopamine in schizophrenia

Moore, Ghajarnia, Jentsch, Geyer, and Grace (2006). Biological Psychiatry 60: 253-264.

Interpretation:

In the MAM model of schizophrenia, there is a hyper-

responsivity of the dopamine system analogous to that

observed in schizophrenia patients.

Current clinical data shows that hyperactivity in the

hippocampus is correlated with the psychotic state of

schizophrenia.

Is the hippocampus hyperactive in the MAM animal as

it is in the schizophrenia patient?

Hippocampal Activity in MAM-treated Rats

Ctrl MAM0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

Avg

FR

(H

z)

Does this also affect DA neuron activity?

Lodge and Grace (2007) Journal of Neuroscience 27: 11424-11430.

Spontaneously active DA neuron

“silent” DA neuron

“Population Activity”

Population

Activity

Firing Rate

Firing Pattern

DA Neuron Activity in MAM-treated Rats

SAL MAM0.0

0.5

1.0

1.5

2.0

2.5 *

Cell

s/T

rack

SAL MAM0.0

2.5

5.0

7.5

10.0

Avg

FR

SAL MAM0

10

20

30

40

50

Avg

% B

urs

t F

irin

gThe increase in the number of DA neurons firing is a likely correlate of

increased F-DOPA uptake in UHR and schizophrenia (Howes, McGuire)

Lodge and Grace (2007) Journal of Neuroscience 27: 11424-11430.

Effects of Hippocampus Inactivation on

DA Neuron Activity

SAL MAM0.0

0.5

1.0

1.5

2.0

2.5 *

Cell

s/T

rack

SAL MAM0.0

2.5

5.0

7.5

10.0

Avg

FR

SAL MAM0

10

20

30

40

50

Avg

% B

urs

t F

irin

gWhy is the hippocampus hyperactive?

Lodge and Grace (2007) Journal of Neuroscience 27: 11424-11430.

Zhang et al, Schiz. Res., 2002

Reduction in density of PV+ GABAergic inhibitory neurons

in schizophrenia

No significant differences in dorsal hippocampus

MAM rats display a regionally selective reduction in PV

interneuron number

PV - Interneuron Cell Counts

MAM

**

mPFC vHipp0

1000

2000

3000

4000Control

cells

/mm

2

Lodge, Behrens & Grace (2009) J. Neuroscience 29:2344-2354

What causes PV interneuron loss in the

Vhipp?

Stress and Psychiatric Disorders:

-Stressful stimuli in schizophrenic patients will exacerbate

the positive psychotic symptoms of this disorder

-Stress may also play a role in the etiology of schizophrenia

(Johnstone, Murray)

-Stress is known to cause a loss of parvalbumin interneurons

in the hippocampus, which are the same neurons lost in

schizophrenia patients and MAM rats

Are MAM rats more stressed prepubertally than controls?

MAM-treated rats show increased anxiety, amygdala

activity, and response to stress prepubertally

Zimmerman, Bellaire, Ewing, and Grace (2013) Neuropsychopharmacology 38: 2131-2139.

Sal MAM

Firin

g R

ate

(H

z)

0.0

0.1

0.2

0.3

0.4

0.5

0.6 ***

If stress plays a role in the

developmental etiology of

schizophrenia, this would suggest

that an early intervention in

susceptible individuals may help to

prevent the transition to psychosis

This was tested in the MAM model by

administering the anti-anxiety agent

diazepam peripubertally and testing the

animals as adults

A Dose of Diazepam was used that Normalizes Anxiety in

Peripubertal MAM Rats

BLA spontaneous firing with acute

diazepam administration (PD35-42)

• Elevated plus maze with acute diazepam administration (PD38-42)

MAM:Veh MAM:DZ

Tim

e S

pe

nt

in

Op

en

:Op

en

+C

lose

d A

rms

0.0

0.1

0.2

0.3

0.4

0.5

0.6

MAM:Veh MAM:DZ

En

trie

s in

to

Op

en

:Op

en

+C

lose

d A

rms

0.0

0.1

0.2

0.3

0.4

0.5

0.6

**

**

Sal MAM MAM:DZ

Firin

g R

ate

(H

z)

0.0

0.1

0.2

0.3

0.4

0.5

0.6 *** ***

Du and Grace (2013) Neuropsychopharmacology 38:1881-1888.

Du and Grace (2016) Neuropsychopharmacology doi: 10.1038/npp.2016.42. [Epub ahead of print]

Sal MAM MAM:DZ

PV

In

tern

eu

ron

Pop

ula

tio

n E

stim

ate

0

1000

2000

3000

4000

5000

6000

Sal MAM MAM:DZ

PV

In

tern

eu

ron

s /

mm

3

0

1000

2000

3000

4000

5000 *

***

**

*

***

***Peripubertal Administration of Diazepam Partially Prevents

PV Neuron Loss and DA neuron activation in the Adult

Du and Grace (2016) Neuropsychopharmacology doi: 10.1038/npp.2016.42. [Epub ahead of print]

Puberty: 35-36 DO

Inject dams: E17

MAM or Saline

x

Inject males: 31-40 DO

Diazepam (5mg/kg) or Saline

Record population activity in VTA

2+ months old

x

Design:

0

0.5

1

1.5

2

2.5

Sal:Veh Sal:DZ MAM:Veh MAM:DZ

Cell

s/ T

rack

Converging evidence from clinical and basic science

studies suggest that the inability to regulate stress early in

life can lead to pathological changes in the hippocampus

that may underlie the emergence of schizophrenia in the

adult.

This suggests that major stress by itself during this critical

peripubertal period could lead to parvalbumin loss and

induce a psychosis-like state

The fact that MAM can be circumvented suggests that

genetic predisposition likely doesn’t CAUSE schizophrenia,

but instead facilitates the impact of environmental factors on

disease susceptibility.

PD31 PD40 PD65

Behavioral

tests

Stress

exposure

Behavioral tests:

- EPM (PD65)

- NOR (PD66: habituation;PD67: acquisition

and retention trials)

- Locomotor response to Amph (PD68-PD69)

VTA recordings

PD69 PD77

Stress exposure (groups):

- Naïve

- Restraint stress (PD31, PD32, and PD40 – 1h)

- Footshock (PD31-40 – 25 shocks, 1.0mA/2s)

- Footshock + Restraint stress

Stress exposure during adolescence

Only FS+RS induced higher amphetamine-induced locomotion

Naiv

eR

SFS

RS+FS

0

2 0 0 0

4 0 0 0

6 0 0 0

8 0 0 0

1 0 0 0 0

B a s e lin e

Lo

co

mo

to

r a

ctiv

ity

(c

m)

Naiv

eR

SFS

RS+FS

A fte r a m p h

*

510

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

0

3 0 0

6 0 0

9 0 0

1 2 0 0

1 5 0 0

1 8 0 0

Lo

co

mo

to

r a

ctiv

ity

(c

m)

T im e (m in )

R S

N aive

510

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

0

3 0 0

6 0 0

9 0 0

1 2 0 0

1 5 0 0

1 8 0 0

Lo

co

mo

to

r a

ctiv

ity

(c

m)

T im e (m in )

N aive

F S

510

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

0

3 0 0

6 0 0

9 0 0

1 2 0 0

1 5 0 0

1 8 0 0

Lo

co

mo

to

r a

ctiv

ity

(c

m)

T im e (m in )

R S + F S

N aive

Gomes and Grace (2016) doi 10.1093/schbul/sbw156.

*

Naive RS FS FS+RS

FS+RS also increased the number of spontaneously active DA cells in the VTA, but did not change the firing rate and burst activity

Medial Central Lateral

VTA subregions

….with greatest effect in the lateral VTA

*

Gomes and Grace (2016) doi 10.1093/schbul/sbw156.

PD65 PD75 PD81

FS+RS

PD88

Stress exposure during adulthood

VTA recordings(1-2 weeks after stress)

Gomes and Grace (2016) doi 10.1093/schbul/sbw156.

This decrease in VTA activity

in the medial VTA is consistent

with models of depression

STRESS(vulnerable periods)

↑ Oxidative stress PV loss in the vHIP

↑ vHIP activity

Psychosis

↑ BLA activity

↑ VTA activity

Stress leads to PV neuron loss in the hippocampus

mPFC

Our previous work shows that the mPFC can potently inhibit stress responses in the BLA

We tested whether inactivation of the plPFC would exacerbate the deleterious effects of stress

mPFC dysfunction would increase

vulnerability to stress

PD31 PD40 PD65

Behavioral tests

Stress exposure

Behavioral tests:- EPM (PD65)- NOR (PD66: habituation; PD67: acquisition and retention trials)- Locomotor response to AMPH (PD68-PD69)

VTA recordings

PD69 PD77

Stress exposure:- Naïve- Footshock (PD31-40 – 25 shocks, 1.0mA/2s)

PD25

Lesion of the mPFC

Experiment 3: mPFC lesion increases vulnerability to stress?

mPFC dysfunction increases vulnerability to stressmPFC lesions alone increase anxiety

in EP

With mPFC lesion, FS alone increased amphetamine locomotion

Gomes and Grace (2016) doi 10.1093/schbul/sbw156.

If we can identify susceptible individuals and intervene early

enough, we may be able to prevent the deleterious effects

of stress and the transition to schizophrenia.

Therefore the deleterious impact of stress, combined with

factors predisposing an individual to the impact of stress,

may underlie susceptibility to schizophrenia

The increased response to stress may be mediated by a

failure of the mPFC to limit the impact of stress exposure

This is critically dependent on the timing of the stress:

-exposure during adolescence → PV loss and

schizophrenia

-exposure during adulthood → PV protected and

depression

Ali Charara Dan Lodge Collaborators:

Mark Bellaire Witek Lipski Benjamin S. Bunney, Yale

Pauline Belujon Michael Mana Margarita Behrens, UCSD

Brandon Bizup Jared Moreines James Cook, UWM

David Bortz Holly Moore Lorenz Deserno, Charite Univ.

Deanne Buffalari Eric Nisenbaum Andreas Heinz, Charite Univ.

Chun-hui Chang Patricio O’Donnell Philip McGuire, Kings Coll.

Pierangelo Cifelli Shao-Pii Onn Urs Meyer, ETH Zurich

Cynthia Correll Mary Patton Gemma Modinos, Kings Coll.

Yijuan Du Vince McGinty Celeste Napier, Rush

Samuel Ewing Michele Pucak Michael Pogue-Geile, Univ. Pitt

Stan Floresco Meera Ramsooksing Susan Sesack, Univ. Pitt

Krysta Fox Millie Rincon-Cortes Linda Rinaman, Univ. Pitt

Mehdi Ghajarnia Heather Rose Mark Ungless, Imperial Coll.

Kathryn Gill J. Amiel Rosenkranz Dean Wong, Hopkins

Felipe Gomes Thibault Sesia

Yukiori Goto Ian Smith

David Harden Susie Sonnenschein

Jeffrey Hollerman Judy Thompson

Hank Jedema Chris Todd

David Jentsch Ornella Valenti

Antonieta Lavin Anthony West

Steve Laviolette Eric Zimmerman

Xiyu Zhu

Acknowledgements

Funding: NIMH