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Learning with Half a Brain

David D. Lent,1 Marianna Pinter,2 Nicholas J. Strausfeld1,3

1Arizona Research Laboratories Division of Neurobiology, The University of Arizona, Tucson, Arizona

2 Department of Molecular and Cellular Biology, University of Arizona, Tucson, Arizona

3 Center for Insect Science, University of Arizona, Tucson, Arizona

Received 15 August 2006; revised 16 October 2006; accepted 12 December 2006

ABSTRACT: Since the 1970s, human subjects thathave undergone corpus callosotomy have provided impor-

tant insights into neural mechanisms of perception, mem-

ory, and cognition. The ability to test the function of each

hemisphere independently of the other offers unique

advantages for investigating systems that are thought to

underlie cognition. However, such approaches have been

limited to mammals. Here we describe comparable experi-

ments on an insect brain to demonstrate learning-associ-

ated changes within one brain hemisphere. After train-

ing one half of their bisected brains, cockroaches learn

to extend the antenna supplying that brain hemisphere

towards an illuminated diode after this has been paired

with an odor stimulus. The antenna supplying the nave

hemisphere shows no response. Cockroaches retain this

ability for up to 24 h, during which, shortly after train-

ing, the mushroom body of the trained hemisphere alone

undergoes specific post-translational alterations of micro-

glomerular synaptic complexes in its calyces. ' 2007 Wiley

Periodicals, Inc. Develop Neurobiol 67: 740751, 2007

Keywords: insect; memory; mushroom bodies; PCamK;

cockroach

INTRODUCTION

Research into mechanisms underlying learning and

memory usually relies on comparisons between

trained and nave subjects. However, assessing the

results of tests between individuals is complicated by

the fact that it is almost impossible to ensure that they

have shared the same life experience before being

tested. Even such genomic model systems as the fruit

fly are unable to provide true control material. Indi-

viduals are tested against other individuals withoutknowing if these individuals have actively learned

and memorized environmental events before being

tested for molecular changes that might have

occurred as a consequence of memory acquisition.

Here we use an insect model system that overcomes

this by using the same individual for the test and con-

trol. The experiments described here were performed

on Periplaneta americana, the brain of which is not

significantly different from that of Drosophila but is

far more robust, allowing defined surgical interven-

tion. The two supraesophageal hemispheres of the

brain can be entirely separated while still eliciting

unilateral motor actions from either hemisphere. Theuse of the cockroach as a model for learning and

memory is evidenced by studies demonstrating both

Pavlovian-type associative memory as well as mam-

malian-like place memory (Mizunami et al., 1998;

Watanabe et al., 2003; Kwon et al., 2004; Lent and

Kwon, 2004; Pinter et al., 2005). In part, such studies

have been enabled by making use of specific behav-

ioral repertoires and relying on the brains resilience

to chronic recording and lesioning (Mizunami et al.,

Correspondence to: D.D. Lent ([email protected]).Contract grant sponsor: National Science Foundation; contract

grant number: NSF IBN0411958.

' 2007 Wiley Periodicals, Inc.Published online 21 February 2007 in Wiley InterScience (www.interscience.wiley.com).DOI 10.1002/dneu.20374

740


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1998). The cockroach has also been used to demon-

strate learning-dependent gene expression (Pinter

et al., 2005).

Here we use a modification of an established para-

digm in which directional movements of the cockroach

antennae (the antennal projection response: APR (Lent

and Kwon, 2004) provide information about the progressand establishment of a memory trace (Lent and Kwon,

2004). Unilateral conditioning of the APR is demon-

strated by a learned response localized to one half of the

split brain whereas the antenna serving the other half,

when tested with the same conditioned stimulus, remains

entirely unresponsive. This allows for one brain hemi-

sphere to serve as the test subject and the other half as

the control. Microscopic learning-associated alterations

of the mushroom bodies, which are the assumed learning

and memory centers of the insect brain (Heisenberg,

2003; Davis, 2005), have been explored using an anti-

body specific to autophosphorylated neuronal calmodu-

lin kinase (PCamK), a protein associated with memory

acquisition in mice and Drosophila (Miller and Ken-

nedy, 1986; Silva et al., 1992; Griffith et al., 1993).

When compared with the nave half, the trained brain

half of the mushroom bodys calyx reveals an increase

in the size of PCamK-positive active domains within the

microglomeruli and in the neurites that supply Kenyon

cell dendrites that form synaptic complexes at this level

(Yasuyama et al., 2002).

MATERIALS AND METHODS

Animals

Experiments were conducted on adult male P. americana

from a laboratory colony. Animals were maintained on

IAMS cat food and water and kept as described by Lent and

Kwon (2004). Animals with any external damage (e.g.

missing antennal segments or legs) were discarded.

Split-Brain Preparation

In preparation for surgery, animals were anesthetized by

cooling to 48C. Cockroaches remained anesthetized

throughout surgery by fixing them to a cold plate, whichconsisted of a plastic dish with an ice-filled center. To split

the brain as far as its esophageal foramen, a single antero-

posterior incision was made from above into the middle of

the head capsule, to a depth of 2 mm and between 11.5

mm long. The incision was then sealed with a low melting-

point wax and the animal was isolated in a small chamber

with access only to water. It was allowed 48 h to recover.

Following recovery animals were monitored for general

health and behavioral abnormalities. Animals that demon-

strated forward locomotion and normal (but not coordi-

nated) antennal movements were used for experiments.

These animals comprised the split-brain group.

Control surgeries, which consisted of an incision

through the cuticle without contacting the brain, were done

in parallel with split-brain surgeries. Animals that under-

went control surgery comprised the intact brain group used

in behavioral experiments. As with split-brain animals only

those that demonstrated normal motor activity were used

for learning and memory experiments. Results were eval-

uated after postmortem histology demonstrated that a brain

had been bisected as far as the esophageal foramen. Brains

that had suffered asymmetric lesions were not included in

the analysis.

Training Protocol

Restricted Training. In this procedure only one half of the

head receives sensory information during training. Follow-

ing recovery from surgery animals were anesthetized with

CO2, restrained in a small plastic tube, and placed in the

middle of a circular arena (Fig. 1). The wall of the arenahad a single green LED in the left hemifield and a single

green LED (CS) in the right hemifield both positioned 58

from midline of the roach. A food odor source (US) was

released through a syringe immediately adjacent to each of

the green LEDs. The protocol (Fig. 1) consisted of six pre-

training trials of the conditioning stimulus (green light cue

of 2 s duration) presented three times to each side of the

roach to test the preconditioned response. Following pre-

training, the eye and antenna on one side, (side randomly

selected), were respectively occluded with black wax and a

narrow plastic tube which was then sealed at both ends so

as to totally isolate the olfactory receptor array. The

occluded side belonged to the \nave" half. The unoccluded

eye and antennae other belonged to the \trained" half. Fol-lowing pretraining, the animal was presented with five

training trials to the trained half. The light cue and odor

were onset simultaneously at each trial: the light was pre-

sented for 2 s and the odor was presented for 1 s. Six test

trials were performed randomly, three to the trained half

and three to the nave half, in which only the CS was pre-

sented. Inter-trial intervals (ITIs) were 1 min. Cockroaches

that elicited APRs towards the CS following training were

considered trained.

Nonrestricted Training. After recovery from surgery, ani-

mals were trained using the same stimulus protocol but

without sensory input blocked to the designated nave half.

This group of animals was used to analyze structural differ-

ences between nave versus trained brain hemispheres. The

protocol provided sensory input to each brain half, the

trained half receiving paired stimulation and the nave half

receiving unpaired stimulation of the eye and antenna.

Immunoblot

Sodium dodecylsulfate polyacrylamide gel electrophoresis

(SDS-PAGE) was performed according to Laemmli (1970)

Learning with Half a Brain 741

Developmental Neurobiology. DOI 10.1002/dneu


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in 4% stacking and 7.5% separating gel. Brains of adult

male animals were dissected in phosphate buffered saline,

pH 7.4 (PBS), transferred into Laemmli loading buffer and

homogenized. Five percent of one brain corresponding to

10 lg protein was loaded per lane. Protein concentration

was determined according to Bradford (1976) using Protein

Assay Dye Reagent (BioRad). Blotting was performed as

described by Towbin et al. (1979) except for the use of

0.05% Tween-20 in Tris buffered saline throughout the

washing steps. Electrotransfer was checked by staining with

0.1% Ponceau S in 5% acetic acid. Blocking solution

(Roche) in maleic acid buffer was used to quench non-spe-

cific binding. Anti-PCamK (sc-12886-R, Santa Cruz Bio-

tech) primary antibody was added to the blocking solutionat 2000 times dilution. Horseradish peroxidase conjugated

secondary antibody combined with SuperSignal West

Femto substrate (Pierce) was used to visualize immunoreac-

tivity. For preabsorption experiment anti-PCamK antibody

was diluted 200 times in PBS and a five-fold excess (by

weight) of the blocking (sc-12886 P) or substrate peptide

(sc-24562) was added. After incubation for 2 h at 208C, the

mixture was diluted 10 times in blocking solution buffered

with maleic acid and the standard immunodetection proto-

col was performed.

Immunohistochemistry

Split-brain animals were decapitated 30 min after training.

The heads were placed into fixative (2% paraformaldehyde

dissolved in PBS) and incubated overnight at 68C. Brains

were dissected in phosphate buffered saline, pH 7.4 (PBS),

embedded in gelatin/ovalbumin and postfixed in 10% neu-

tral buffered formalin solution overnight at 68C. Frontal

sections of gelatin embedded brains were sectioned at 45

lm with a Leica vibratome. Immunostaining was per-

formed on free-floating sections at room temperature. Sec-

tions were equilibrated with 0.5% Triton X-100 dissolved

in PBS (PBSTx), blocked with 5% normal goat serum

(NGS) in PBSTx, and incubated simultaneously with the

primary antibodies added to the blocking solution for at

least 24 h. The primary antibodies were rabbit anti-PCamK

and mouse anti-tubulin (MAB3408, Chemicon Int.)

employed at 1:500 and 1:4000 dilutions, respectively. Sec-

tions were washed with PBSTx and incubated for approxi-

mately 16 h with the secondary antibodies; Alexa Fluor 488

F(ab0) fragment of goat anti-rabbit IgG (Molecular Probes),

and Cy5-conjugated goat anti-mouse IgG (Jackson) diluted

in PBSTx containing 1% NGS to 1:400 and 1:500, respec-

tively. After washing with PBSTx and rinsing with PBS,

Figure 1 The experimental protocol. Upper left: diagram of the training arena with the cock-

roach placed at its center with the two sets of spatially coincident cues (odor source and green

diode) labeled A and 1, each offset 58 from the midline. Upper right: Training protocol. The para-

digm employs two randomized sets of three pretraining trials, five training trials, and two random-

ized sets of three testing trials. Lower left: micrograph of a surgically split brain after fixation and

removal from the head capsule. Scale bar 1 mm.

742 Lent et al.



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sections were mounted on slides and coverslipped under

Elvanol (Rodriguez and Deinhardt, 1960). Images were

acquired with a Zeiss Pascal confocal microscope.

Measurements of Microglomeruli andKenyon Cell Fiber Bundles inSplit-Brain Cockroaches

Given that the split extends through the supraesophageal

ganglion the two halves of any one brain are linked

throughout treatment by its subesophageal ganglion. This

allows the tissue from each brain half to be fixed, embed-

ded, and sectioned at the same level and, therefore, imaging

of tissue belonging to the nave or trained hemispheres to

be under the exact same conditions of excitation and image

capture. Confocal images were reconstructed from three se-

rial optical sections each with a total depth of 4 lm. Flat-

tened stacks were processed using Adobe Photoshop. For

microglomeruli analysis, images of the calyx were collected

anteriorly above the apex of the vertical lobe. Merged

images of PCamK and tubulin immunoreactivity were usedto measure differences of microglomerular dimensions at

equivalent locations of the calyces of each brain hemi-

sphere. The maximum edge-to-edge widths of identified

microglomeruli were measured, as were center-to-center

distances between microglomeruli. Between 105 and 180

microglomeruli were measured from 2 to 3 areas from the

lateral and medial calyces in each brain half. For calculat-

ing PCamK immunoreactivity in bundles of Kenyon cell

neurites, images were collected from calyces at a level ante-

rior to the apex of the vertical lobes and from the calyces at

a level that is immediately posterior to the mushroom body

pedunculi. Sections of these parts of the calyx allowed

imaging of neuropil and clusters of neuronal cell bodies

within the calyx cup. The number of neurite bundles immu-nostained with a-PCamK were calculated as a percentage

of all fibers stained with a-tubulin.

Statistical Analysis

For behavioral data, nonparametric statistics were used as

described by Lent and Kwon (2004). Friedman ANOVA

(ANOVA v2) was used to compare differences between

repeated measures within a group. The Sign test (Z) was

used compare values within the repeated measures within a

group. The MannWhitney U test (U) was used to test dif-

ferences between two groups. Values shown represent

responses\

0"

or\

1"

in percentages; therefore, standarddeviations are not depicted. Confocal images of microglo-

meruli and neurite fiber bundle were measured and scored in

a blind test by an observer without knowledge of the identity

(nave or experimental) of the images being measured or of

the behavioral data. Differences were analyzed using two-

tailed Students t-test. Corrections for small sample size

were made when calculating the Tvalues of the fiber differ-

ences. Using a correction for n 10 the 95% confidence

interval is set as Xavg6 2.262r/N1/2. Differences were con-

sidered statistically significant using a p value of 0.05.

RESULTS

Classical Conditioning with RestrictedSensory Input in Intact andSplit-Brain Cockroaches

The antennal projection response (APR) of restrainedcockroaches were conditioned and measured during

pretraining, training, and testing in cockroaches with

intact brains (control surgery group; n 51) and split

brains (split surgery group; n 48). In both groups

input was restricted to either the left or right side

[Figs. 1 and 2(a)]. Restricted input was used to ensure

that the behavioral observations and comparisons

made between intact and split brains were due to sen-

sory input being associated unilaterally and not con-

founded by interactions of bilateral sensory input.

Both groups of animals performed APRs in less than

10% of the pretraining trials, with no significant dif-

ference between the two groups (n 51, 48; U 1197.00, p 0.85). There was also no significant

difference between the two groups during training, in

which the odor stimulus was coupled with the green

light cue (n 51, 48; U 3363.50, p 0.9027).

However, in both groups after training, the side of the

animal exposed to paired stimulation by the CS and

US exhibited vigorous APRs that were significantly

different from pretraining (intact brain, n 51, Z

5.2223, p 1.7669e-7; split brain, n 48, Z

5.0289, p 4.932e-7). Both groups exhibited

greater than 60% APRs to the green LED presented

alone 5 min after training and were not significantly

different from each other (n 51, 48; U 1201.50,

p 0.8748). This indicated that split-brain cock-

roaches actively sample the olfactory milieu as do

animals that are intact and that, at least with regard to

antennal motor actions, surgery does not adversely

affect behavior. Testing the nave half of intact and

split-brain animals yielded different results. Present-

ing the conditioned stimulus to the nave side of the

split brain after training elicited APRs that were not

significantly different from pretraining (n 51, Z

0.5000, p 0.6171). However, at 5 min, APRs

of the nave half of animals with intact brains were

significantly different from both pretraining (n

51,Z 2.7735, p 0.005546) and from the APRs eli-

cited from the trained half (n 51, Z 3.590662, p

0.00033). Using a 6 min testing window 5 min af-

ter training and randomizing the stimulus between

the trained and nave sides of the brain of intact ani-

mals provided some insight into these results. Com-

parison of APRs of the nave half at different times

during the test (irrespective of whether the nave half

was tested before or after the trained half) revealed




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that if APRs were tested in the latter half of the 6 min

testing window they were significantly increased

compared with pretraining (n 26, Z 2.5981, p

0.009375), but if testing occurred during the early

stages of the testing window APRs were not signifi-

cantly different from pretraining (n 25, Z

1.2247, p 0.2207). This suggests that informa-tion acquired by the trained half of the brain is trans-

ferred or made accessible to the nave brain half nei-

ther during nor immediately after training, but after a

delay of minutes.

To further investigate the dynamics of this apparent

memory transfer, APRs of the trained and nave half of

intact brains were tested over a period of 24 h

[Fig. 2(b)]. APRs of the trained half at 5 min, 1 h, 3 h,

6 h, and 12 h after training were significantly differentfrom pretraining (n 33, ANOVA v2 23.5946, p

0.0026) but not different from one another (n 33,

ANOVA v2 2.3158, p 0.6779). As described,

APRs from the nave half at 5 min after training were

significantly different from APRs before training and

different from the trained half at 5 min. However, APRs

of the nave half at 1, 3, 6, and 12 h were not signifi-

cantly different from each other (n 33, ANOVA v2

2.8868, p 0.40942) or different from the trained half

(n 33, ANOVA v2 3.4656, p 0.83885) at the

same time intervals. While the trained half received

both the US and CS during training, and thereafter had

access to the learned association, these results show

that access by the nave half to the learned association

was delayed. Once the memory was transferred, the

Figure 2

Figure 2 Comparisons of APRs of intact (non-split) and

split-brain cockroaches. (a) No significant difference is

seen between the APRs of intact (n 51) and split-brain (n

48) cockroaches during pretraining, training, and testing

of the trained half. However, there is a significant differ-

ence in APRs of the two groups between testing of the na-

ive half and pretraining. There is no difference between the

pretraining and the testing of the nave half of the split-brain (n 48). There is a significant difference between

pretraining and the testing of the nave half of the intact

brain (n 51) indicating access of the information by the

nave half (see Results). (b) APRs of the trained half and

nave half in intact cockroaches for 24 h. The APRs of the

trained half at 5 min, 1 h, 3 h, 6 h, and 12 h are not signifi-

cantly different (n 33). The APRs of the nave half at 1,

3, 6, and 12 h are not significantly different from each other

and not different from the trained half at the same time

intervals (n 33). The APRs of both the trained half and

nave half at 24 h are not significantly different from pre-

training (n 33). This suggests that memory is transferred

from the trained to the nave half after 5 min and that after

24 h the memory decays. (c) APRs of the trained half andnave half in split-brain cockroaches for 24 h. The APRs of

the trained half at 5 min, 1 h, 3 h, 6 h, and 12 h are not sig-

nificantly different (n 33). The APRs of the nave half at

5 min, 1 h, 3 h, 6 h, 12 h, and 24 h are not significantly dif-

ferent from pretraining, suggesting no learning (n 33).

The APRs of both the trained half and nave half at 24 h are

not significantly different (n 33). This suggest that mem-

ory is not transferred from the trained to nave half after 5

min due to splitting of the brain and that after 24 h the

memory decays in split-brains as it does in non-split.

744 Lent et al.



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Figure 3 Two examples (ag & hl) of differences of PCamK-labeled microglomeruli in the

calyces of the mushroom bodies of trained versus nave brain halves of split-brain P. americana 30

min after the last training trial. Left: optical sections from the na ve brain half (a,c,e) are comparedwith equivalent locations in the lateral calyx in the trained brain half (b,d,f). Tubulin-immunoreac-

tivity extends between microglomeruli that are revealed by an antibody against PCamK. g. The

range of PCamK immunoreactive microglomerular dimensions is compared. Microglomerular

PCamK domain/size distribution shifts towards higher values in the trained compared with the na-

ve brain half. Right, h, i: two optical stacks (depth 12 lm) showing structural differences of micro-

glomeruli between trained and nave brains (enlarged in j, k). Maximum diameters are compared in

panel l. Scale bar for panels a-f and j, k 20 lm.




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Figure 4

746 Lent et al.



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cockroach responds equally well to the conditioning

stimulus presented to either the left or right visual field.

The learned response was maintained for 12 h, after

which there was a steady decay of the response until at

24 h there was no significant difference of APRs eli-

cited by either side from pretraining APRs (trained half,

n 33, Z 1.5119, p 0.1306; nave half, n 33,Z 17678, p 0.0771).

APRs of the trained and nave sides of split-brain

cockroaches were also examined for a period of 24 h

to determine whether any transfer might occur via

pathways that linked one side of the brain to the other

through subesophageal pathways such as a poststo-

modeal commissure linking the tritocerebra (Gundel

and Penzlin, 1995) [Fig. 2(c)]. APRs from the trained

half measured at 5 min, 1 h, 3 h, 6 h, and 12 h were

significantly different from pretraining (n 33,

ANOVA v2 32.4324, p 0.00001), but not each

other (n 33, ANOVA v2 3.7949, p 0.4345).

APRs from the nave half at 5 min, 1 h, 3 h, 6 h, 12 h,

and 24 h were not significantly different from pre-

training (n 33, ANOVA v2 0.8696, p 0.9724).

This indicates that the nave half remained nave

throughout the 24 h period after training. However,

after 24 h, APRs of the trained half were not signifi-

cantly different from pretraining (n 33, Z

0.0000, p 1.0000) suggesting that a decay of reten-

tion, as indicated by APRs, occurs between 12 and 24

h when the trained brain hemisphere was disassoci-

ated from the untrained one.

Classical Conditioning withNon-Restricted Sensory Input inSplit-Brain Cockroaches

In a third experiment, split-brain cockroaches were

trained under the same protocol previously described,

but the eye and antenna on both left and right side were

exposed to sensory stimulation. This non-restricted pro-

tocol was necessary to control for possible alterations

of structures or molecular changes that might be

incurred by sensory exposure alone rather than learned

sensory associations. Prior to utilizing the split-brain

animals for analysis of morphological change it wasnecessary to compare the behavioral responses and

observe if providing bilateral sensory input impacted

the previously described learning. Comparisons of

APRs elicited from the trained and the nave half fol-

lowing training from split-brain animals conditioned in

this non-restricted sensory condition were not signifi-

cantly different from split-brain animals that were

trained with sensory information restricted (n 48, 48,

trained half, U 961.50, p 0.2516; n 48, 48, nave

half, U 519.00, p 0.5134). This experiment pro-

vided paired olfactory and visual cues that were associ-

ated in the trained half and unpaired olfactory and vis-

ual cues that were not associated in the nave half;

unlike that demonstrated in split-brains that have been

trained under sensory restricted conditions, which only

received paired olfactory and visual cues. Training

under the described non-restricted sensory condition

could be used for effectively controlling observed dif-

ferences due to sensory stimulation. Animals trained

under these conditions were used for the analysis of

PCamK positive dimensions of microglomeruli and the

Kenyon cell neurites.

CamK Autophosphorylation is Elevatedin the Mushroom Body Calyces ofTrained Brain Halves

A body of evidence has suggested that mushroom

bodies support learning and memory either at the

level of their afferent supply in the calyces, or within

Figure 4 Comparison of PCamK immunoreactivity in Kenyon cell neurites. (ad) Comparable

regions of the lateral calyces in nave and trained hemispheres labeled with anti-PCamK (panels a, b)

and anti-tubulin (panels c, d) of the same animal reveal differences in the number of Kenyon cell neu-

rites that show double labeling (examples arrowed in panels e, f). This increase in PCamK immunor-

eactivity is further manifested by the thickness of the layer (bracketed in e, f) of Kenyon cell neurites

projecting over the inner surface of the calyx bowl. Images represent 4 lm thickness. Scale bar 20 lm.(g) Percentages of the PCamK-labeled fiber bundles, co-labeled in the tubulin-immunoreactive bundles

were calculated from both the nave and trained brain halves of five split-brain trained animals. Fiber

counts were performed in a blind test using composite images of PCamK and tubulin staining. Counts

were made from frontal images of the medial (n 10), lateral (n 9), anterior (n 10) and posterior

walls (n 9) of both the medial and lateral calyces. Statistics are corrected to small sample size. Error

bars Standard deviation. h. Immunoblot characterization of the specificity of anti-PCamK. Anti-

PCamK recognizes one discrete band of approximately 50 kD of roach brain homogenate (left col-

umn). The blocking peptide with phosphorylated Thr286 inhibits staining when anti-PCamK is preab-

sorbed with it (second column: block). The substrate peptide with a non-phosphorylated Thr286 has

no effect on staining when anti-PCamK is preabsorbed with it (right column: subs).




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the lobes, or at both levels (Hammer and Menzel,

1998; Zars et al., 2000; Heisenberg, 2003). We have

investigated calycal organization, comparing this

neuropil in the trained brain half versus the nave

brain half of split-brain animals. The focus on the

calyces is due to their distinctive composition of

measurable synaptic complexes called microglomer-uli (Yasuyama et al., 2002; Frambach et al., 2004)

and because changes in the lobes, if they occur,

would be difficult to discern among the approxi-

mately 200000 parallel fibers. Microglomeruli com-

prise synaptic complexes that are formed between the

boutons of presynaptic terminals from the ipsilateral

antennal lobes, ipsilateral GABAergic interneurons

from the protocerebrum and dendritic spines of Ken-

yon cells, the axons of which supply the mushroom

body lobes (Farris and Sinakevitch, 2003). These syn-

aptic complexes underlie divergence from calycal

afferents to intrinsic pathways within the lobes.

Changes of the calyces between the trained and nave

halves of split-brain animals were analyzed by label-

ing the brain with an antibody raised against anti-

PCamK, which is specific to the autophosphorylated

amino acid P286Thr that renders human CamKIIa

constitutively active (Miller and Kennedy, 1986). Im-

munohistochemical staining was performed on brain

tissue collected 30 min after training (see Fig. 2). The

size of the PCamK immunoreactive microglomeruli

and spacing in the trained versus nave calyces of

split-brain cockroaches were analyzed using double

labeling with anti-PCamK and anti-tubulin antibodies

(Fig. 3).

PCamK immunoreactive microglomeruli are bor-

dered by tubulin-stained neuronal processes [Fig.

3(e,f)]. Comparison of trained and nave halves of

split-brain cockroaches (n 5) showed significant

alterations in the sizes of autophosphorylated micro-

glomerular domains in the trained half. Measure-

ments of the maximum widths of the domains (n

105180) showed increases of widths by 4.85,

7.85, 8.47, 12.3, and 16.2%, respectively, in the

trained halves compared with the nave halves. These

differences were significant in all animals measured

(t 4.374430, 5.468498, 8.357868, 10.412, 13.43387;

p

2.1722e-5, 2.045e-7, 1.7304e-14, 8.971e-18,6.6199e-29). Plots of the normal distributions of the

PCamK immunoreactive microglomerular domains in

the trained and nave half of a single cockroach where

there was a 16.2% increase in size is shown in Figure

3(g) and the corresponding labeled sections shown in

Figure 3(af). As a control, measurements of the

number of glomeruli and the center-to-center distance

between glomeruli were also compared. No signifi-

cant differences were found between trained and

nave halves (n 25, t 1.4285, p 0.1660; n 25,

t 0.1432, p 0.8897).

Differences were also resolved in immunolabeling

of bundles of Kenyon cell neurites leading from the

cell body layer above the calyx to dendrites supplying

the calyces [Fig. 4(ag)]. An increase of PCamK

immunoreactivity, which is here interpreted to reflectmobilization of PCamK, was revealed in calyces of

the trained brain half [Fig. 4(b)] compared with the

nave half [Fig. 4(a)]. An anti-tubulin antibody was

employed to reveal bundles of Kenyon cell neurites

that were not stained for PCamK [Fig. 4(c,d)]. Per-

centages of PCamK-labeled fibers calculated from

equivalent areas of the lateral calyx of the nave and

trained brain half [Fig. 4(g)] showed a greater than

50% increase in labeling in the calyx of the trained

half. These increases were observed in equivalent lat-

eral areas of the medial (n 10, t 8.795, p

0.0000103) and the lateral (n 9, t 13.093, p

0.0000011) calyces and at equivalent anterior (n

10, t 8.893, p 0.00000941) and posterior (n

9, t 9.112, p 0.0000169) areas [Fig. 4(g)].

Immunoblot characterization of anti-PCamK shows

that it recognized only one *50 kD band in roach

brain homogenate ([Fig. 4(h)], first panel). Immuno-

reactivity was quenched if the antibody was preincu-

bated with an excess of the antigen blocking peptide

containing P286Thr, which corresponds to autophos-

phorylated CamKIIa of human origin ([Fig. 4(h)],

second panel). Immunoreactivity was unaffected

when anti-PCamK was preincubated with an excess

of the unphosphorylated substrate peptide containing

286Thr corresponding to nonphosphorylated CamKIIa

of human origin ([Fig. 4(h)], third panel).

DISCUSSION

The robust nature of the cockroach allows chronic

surgical intervention without diminishing antennal

motor action by the restrained animal. Split-brain

preparations can therefore be used to compare behav-

ioral and structural differences within circumscribed

brain regions after training only one half of the brain.

In the present experiments, in which half of a surgi-cally split-brain P. americana was conditioned to as-

sociate a light cue with an odor source, the learned

association, which is restricted to the half that

received paired visual and olfactory information,

decays within 24 h. This compares to intact animals

tested with the same association paradigm in which

memory was still present at 72 h (Lent and Kwon,

2004). Interestingly, animals with an intact brain

also showed reduced memory at 24 h when the pre-

748 Lent et al.



10/12

sentation of the CS and US is restricted to only the

trained side by sheathing the contralateral antenna

and covering the contralateral eye (Fig. 2). These

findings suggests that in intact animals the dynamics

of long term memory involve more than simple asso-

ciation of the conditioned and unconditioned stimu-

lus by one side of the brain but require unimpededsampling of the sensory surroundings by the opposite

side.

Interestingly, following unilateral conditioning,

cockroaches with intact brains demonstrated that the

visual-olfactory memory trace is either localized in

both halves or has been made accessible to both

halves after a delay of several minutes. This suggests

that the memory trace develops rapidly, possibly

within the mushroom body, in the trained half of the

brain and is then transferred, or is made accessible to,

the mushroom body on the contralateral side. These

results in the cockroach provide for a comparison of

memory transfer dynamics in the honey bee. Analysis

of the olfactory memory trace in honey bees, using

proboscis extension response (PER) conditioning, has

shown that there is also a transfer of learned informa-

tion between sides, but after a significantly longer

delay. It was only when honey bees were tested with

longer retention times of 3 or 24 h that bilateral mem-

ory could be demonstrated (Sandoz and Menzel,

2001). Sandoz and Menzel (2001) suggest that a

short-term unilateral olfactory memory trace may be

established in the antennal lobe whereas the mush-

room bodies are involved in the consolidation pro-

cess. In Drosophila, experiments have supported this

proposition in that the initial phases of olfactory

memory are represented, to some extent, by changes

in the representation of odors by the projection neu-

rons (Yu et al., 2004). There are, however, critical

differences between the conditioning paradigms of

the cockroach and honey bee that may account for

the observed differences in memory transfer dynam-

ics. The sensory modalities that are involved in the

conditioning procedures are likely to be reflected by

which areas of the brain are involved in particular

memory processes. PER conditioning involves the

integration of gustatory and olfactory information.

The convergence of these modalities in the deutocere-brum, at a level prior to their representation relayed

to mushroom bodies, may result in delayed involve-

ment or recruitment of the mushroom bodies in the

formation of the memory trace because antennal

lobes themselves are capable of supporting short-

term memory. Conditioning of the APR involves the

integration of converging visual and olfactory infor-

mation. Because such convergence is know to occur

at the level of the mushroom bodies (Li and Straus-

feld, 1997) an immediate involvement of the mush-

room bodies might be expected to form short-term

memory and support the longer memory trace. Thus,

like that demonstrated from honey bees, bilateral

transfer of a memory trace in the cockroach is time-

dependent, but on a much faster scale than in the hy-

menopteran system with the modalities employed.The present experiments demonstrate that the phe-

nomenon of bilateral transfer is probably not uniform

across different modalities but appears also to be mo-

dality dependent.

The present split-brain preparation has the singular

advantage in that it allows a single animal to be both

the control and test subject. The split-brain paradigm

eliminates individual variation, thereby offering an

unprecedented advantage for molecular studies of

learning and memory (Pinter et al., 2005). The pres-

ent results reveal the sites at which there are

increased levels of autophosphorylated CamK 30 min

after training as a consequence of sensory association.

It has been shown in mice that the CamK signaling

pathway is critical for both explicit and implicit

memory (Mayford et al., 1996). How the increase in

PCamK relates specifically to associative memory

and general memory processes in the cockroach is

unknown. Here the observed increases in PCamK

have been used as a correlative marker of sensory

association and not a direct marker of learning or

memory formation. Differences within the calyces

specifically relate to synaptic complexes, called

microglomeruli, that consist of Kenyon cell dendritic

spines clustered around, and postsynaptic to, termi-

nals of afferent neurons (Yasuyama et al., 2002; Far-

ris and Sinakevitch, 2003; Frambach et al., 2004).

Such changes suggest either upregulation or translo-

cation of PCamKII (Lai et al., 1987; Gaertner et al.,

2004) to synaptic sites (Shen and Meyer, 1999), a

phenomenon also reported from studies of mamma-

lian hippocampus (Gleason et al., 2003). The present

results also show a corresponding increase in consti-

tutively activated CamK in neurite bundles of Ken-

yon cells in the trained brain half during the early

phases of memory. Previous in vivo studies describe

CamK translocation to postsynaptic densities after

Ca2

/calmodulin activation and after activation-induced autophosphorylation stabilize its synaptic

association (Gleason et al., 2003). We propose that

during sensory association, and persisting for some

minutes thereafter, CamK is constitutively activated

within Kenyon cell neurites, and within dendrites and

their postsynaptic spines, resulting in increased size

of active domains within the microglomeruli.

Transient learning-induced changes of CamKII

have been demonstrated in the hippocampus of rats




11/12

following inhibitory avoidance training. Following

one trial learning there was an increase of between

2575% of both total and calcium-independent activ-

ities of CamKII in the hippocampus of rats 0 min af-

ter training, but there was no observable difference

after 120 min. Additionally, an increase of 24% in the

in vitro phosphorylation of both the alpha and betasubunits of CamKII was observed in the synaptoso-

mal membranes of trained rats 30 min after training,

but not 120 min after training (Cammarota et al.,

1998). Comparisons of these significant increases

observed in the hippocampus to those of the mush-

room bodies within minutes of training demonstrate

that those increases quantified in the mushroom

bodies, while quite large, are actually on the conserv-

ative side compared with those identified in the hip-

pocampus. These changes in the mushroom bodies

are also assumed to be transient and not to lead to

persistent structural alterations of the microglomeruli.

Comparable increases are not seen in sampled indi-

viduals that are confronted on a daily basis by sensory

associations. Microglomerulus size and number how-

ever, are subject to other environmental and develop-

mental events, such as rearing conditions and changes

of behavioral repertoire (Farris et al., 2001; Groh

et al., 2006), as in the transition from nurse to forager

honey bee. The presence of f-actin within Kenyon

cell spines of microglomeruli (Frambach et al., 2004)

is a further indication that, as demonstrated during

learning by the hippocampus (Fukazawa et al., 2003),

specific structures within the insect mushroom bodies

reveal learning-related plasticity.

DDL was supported under a National Science Founda-

tion Graduate Research Fellowship Award. The authors

thank Norman T. Davis for advice in immunohistochemis-

try, Elizabeth Riordan for behavioral tests, Chhitij Bashyal

for performing the blind measurements, and Dr. Katalin

Gothard for valuable discussion.

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