The Limbic system is not necessary for fear learning

FEAR CONDITIONING DEVIATES LIMBIC SYSTEM 1

Fear Conditioning Deviates the Limbic System

Kevin E. Hendry

Physiological Psychology, Florida Gulf Coast University


Abstract

Studying the ant Solenopsis invicta as a model for humans, evidence was effectively established,

indicating emotional memory could be obtained without the use of the limbic system. Using a

between group design, two groups were tested before being exposed to a flashing strobe light and

then after exposure to the flashing strobe light. In the experiment group, the flashing strobe light

was paired with shaking for 15 seconds during 12 trials. The control group verified the unpaired

flashing strobe light was a neutral stimulus for the ant. A post test after each trial in the

experimental group verified the shaking produced arousal by an increased level of activity. The

final memory test with the experimental group identified statistical significance in arousal by the

flashing strobe only: Thereby indicating the flashing strobe light became a conditioned stimulus.

This discovery has far reaching implications for further research on emotional memory in

humans.


Fear Conditioning Deviates the Limbic System

It was a matter of unnoticed observation when mowing the lawn, a fire-ant mound was

cut down to almost nothing. After the mower’s blade propelled the ants into the air and onto my

exposed leg did realization hit. A sudden sharp sting emitted from my leg in pulsating fashion,

thanks to the pyrogenic weapon of the fire-ant’s stinger. The next day I returned to the annoying

mound with a weapon of my own: Pesticide. However, only astonishment could describe the

thoughts on how those little hurtful creatures rebuilt their mound overnight. How could an

invertebrate with limited brain size be able to distinguish between enemy and foe, moreover be

capable of reconstructing their dwelling with orchestrated efficacy and perfection? It’s obvious

that humans can because we have a large brain with a frontal lobe for memory, but an ant has no

cerebrum or large brain for memory.

The red fire ant brain and human brain differ greatly from each other. Naturalist, Charles

Darwin concluded in 1859 A.D. that the origin of all species must have a common ancestor, but

the struggle for existence divided all species to what they are today (Darwin, 1975) [Darwin’s

book is significant to this study in theory]. After millions of years, humans and ants are nothing

alike, or are they? Do fire ants and humans learn the same way? Red imported fire ants (RIFA)

and humans are both categorized in the Animalia Kingdom, but differ in the Phylum list, which

evolved approximately 450 million years apart from each other. During that time, differences in

the human brain became more complexed then the more simplistic RIFA’s ganglion brain.

However, it must be noted that the RIFA’s ganglion brain must have had efficient adaptation in

the world’s environment for such little variation of brain change to not have occurred in such a

long time as did the human brain with it’s mass and complexity.


The RIFA (Solenopsis invicta) brain largely consists of three pairs of fused ganglia, a

ventral nerve cord, frontal and olfactory lobes, and pedunculate bodies (Franks et al., 2007).

During the ant’s formation, the ganglia forms an interconnected network located in the ventral

regions of the head, thorax, and abdomen. During the ant’s formation, some pairs of ganglia fuse

in the head area and form a larger ganglion mass, considered the ant’s brain (Franks et al., 2007).

The ventral nerve cord connects the dorsal brain to segmental ganglia in the thorax and abdomen,

completing the central nervous system. The ganglia segments making up the ant’s brain are; the

protocerebrum, deutocerebrum, and the tritocerebrum (Mysore, et al., 2009). The protocerebrum

transports light signals to the tritocerbrum neuropil, which identify’s the sensory input, and then

transmits it to dorsal pedunculate structures of dense nerve fibers (Mayer, 2006). The

invertebrate brain relies on neurotransmitters for functioning, to include; Acetylcholine, 5-

hydroxytryptamine, dopamine, and norepinephrine (Mayer, 2006).

In humans, the basal ganglia is within the cerebral hemisphere of the brain, that acts as

the gate keeper for information flow to other regions of the brain, and control of movement. In

addition, it coordinates mechanisms for attention, action, and cognition (Mestres-Missé et al.,

2012). The basal ganglia includes the caudate nucleus, the putamen, and the globus pallidus

(Imai et al., 2004). According to Imai et al., (2004), the basal ganglia is for controlling

movement, and the limbic system, working with the basal ganglia, is involved in emotion and

learning. The limbic system includes the fornix, olfactory bulb, hypothalamus, amygdala,

hippocampus, mammillary body, and thalamus. The human brain utilizes the same

neurotransmitters as the arthropods, most importantly dopamine (Imai et al., 2004). Dopamine, a

neurotransmitter critical for many aspects of behavior and mood originates in the subsystem of


the basal ganglia called, substantia nigra, then projects throughout the brain by axons in the

mesostriatal pathway to produce emotional feelings (Imai et al., 2004).

Although the RIFA and human brain are much different in their complexity and structure,

it can be concluded that the human brain is a much more complex version of the arthropod brain.

For example, the arthropod compound eyes transmit signals through the optic nerves to the

protocerebrum, which is a pair of fused ganglion, and in turn process the signal and transmit the

information to the tritocerebrum. In human, the eye itself contains the ganglion that receives the

signals, then transmits those signal through it’s axons, the optic nerve, and gets channeled to duel

locations: Most to the thalamus, and the rest to the visual cortex (Scott et al., 2006). In

arthropods the tritocerebrum acts in a more simplistic way as the thalamus in humans. The

ganglion system alone does not imply memory or learning capacity, but it indicates a duel

functioning of motor control and information transmission.

One study illustrate how the ganglion brain contains the capacity for learning. Lu et al.,

(2011) indicated the dorsal positioned pedunculate structures and olfactory lobes have been

frequently compared with the human’s cerebrum and olfactory lobes, and to regard them as an

organ of intelligence. The pedunculate structure is mushroom shaped bodies of nerve fibers,

with a stem of nerve fibers connected to the protocerebrum (See figure 1). Cammaerts (2004)

demonstrated classical conditioning to successfully train Myrmica sabuleti ants using green

triangles as the conditioned stimulus (CS) and liquid sugared food as the unconditioned stimulus

(UCS). Using 12 successive presentations, statistical significance (p < 0.01) was found when the

CS was presented a short time before, just before, or together with the UCS, but failed if the

UCS was presented 15 minutes after removal of the CS, or was presented before the CS

(Cammaerts, 2004).


In the 1960s, chimps exhibited the ability to use tools (Goodall, 1964), then in the 1970s

Morrill (1972) revealed ants use tools as well. The ants would drop leaf pieces or sand into a

pool of food to absorb it like a sponge and then return it to the nest (Morrill, 1972). They also

used tools by means of dropping small stones or sand into the holes of competitor ant mounds to

prevent their exit in order for the stone dropping ant to be first to forage for food in the area

(Pierce, 1986). In that study, ants also drop sand or small stones into the holes of ground nesting

bees, which would precipitate an attack by a massing of ants that killed the bee. It would be

probable that the stone dropping ant laced the stone with pheromone before dropping it into the

hole as a guide for others to navigate. It could be argued that such mental complex processes

would require attention, planning, and anticipation.

Another arthropod verified the ability to anticipate their route for food before actually

committing to it: The jumping spider. Tarsitano et al., (1997) conducted a study with the

jumping spider (genesis Portia), and discovered that the motionless spider visually followed a

pathway from a food source, maintaining attention to various parts of the of the path, which

supports preplanning and cognition. In a similar study using ants, Franks et al., (2007) found a

significance in latent learning that was irrelevant during the time it was learned, but later used.

Dopamine was previously noted as a neurotransmitter in ants (Mayer, 2006), and

dopamine is an active component in the reward-driven learning of mammalians, vertebrates,

reptilians, and invertebrates (Tobiansky et al.2013). Classical conditioning using positive CS

can be accounted for by the presence of dopamine. Norepinephrine, another previously noted

neurotransmitter (Mayer, 2006) in ants, is an active component in fear for humans (Marieke et

al., 2011). Increases in noradrenaline (Latin form of norepinephrine: a Greek word) during or

imminently after a stressful event strengthened the formation of associative fear memory


(Lorenzetti et al., 2011). It is hypothesized that because RIFA utilizes norepinephrine as a

neurotransmitter, although lacking a limbic system, they are capable of fear conditioned learning.

It is thought that the RIFAs tritocerebrum acts as a more primitive and limited version of the

human limbic system’s amygdala and hippocampus. In turn, the tritocerebrum transmits the fear

signals to the dorsal pedunculate structure of dense nerve fibers where it is stored as memory.

Method

Subjects

The experiment was conducted on RIFA collected from two colonies in Cape Coral,

Florida during March 2013. They were contained in a one gallon plastic jug, and closed with a

plastic lid with small air-holes. The container had about 2 inches of soil from the ant’s original

colony. They were maintained in an outdoor environment and under constant shade to maintain

external validity. They were fed small chunks of bacon as a protein source, along with humming

bird nectar diluted 10 times in water as sucrose. Sixty RIFA subjects were randomly removed

from the colony container and randomly separated into two groups. For unknown reasons,

sixteen of the subjects in the experimental group died and all data for those samples were not

included in the study. Twelve of the subjects in the control group died and their data were

removed from the study. In addition, two more subjects in the control group were randomly

removed from the control group to maintain an even distribution between the two groups:

Leaving fourteen samples per group.

Materials

The sixty RIFAs were placed into individual 100×15 polystyrene petri dishes. Included

in each petri dish was a piece of bacon and the cutoff end of a cotton q-tip soaked in water and

humming bird nectar. The petri dish lid was sealed onto it’s base using a glue gun with all


purpose 101 mm ╳ 6.8 mm glue sticks. A center hole was drilled into the top of the dish, only

wide enough for a 5 ml syringe with blunt fill needle (18G ╳ 1 1/2, 1.2 mm ╳ 40 mm) to enter,

then the hole was covered with a drop of glue from the glue gun to prevent escape. Every two

days the syringe was inserted into the top of the glued hole and three drops of water-sucrose

solution was added, then the hole was resealed with glue. The dishes were aligned side by side

on a table over top of square graph paper. An Intertec 3187095 flashing strobe light was utilized

for paired conditioning. A stopwatch timer application on an I-Phone was used to maintain

consistency in time intervals.

Data Collection

During evening hours between 6pm and 11pm during the month of April in Southwest

Florida, each RIFA subject was transported one at a time in it’s petri dish about 70 feet to another

table outside, and onto graph squared paper. A two minutes interval was provided between

movement to the table and experiment initiation to allow the RIFA to calm down from the

transport. For the manipulation group, a 5 second pretest measured how many squares the head

of the RIFA entered to measure its rate of excitability. Then, for 15 seconds, the strobe light

flashed at 10 flashes per second while the dish was shaken violently back and forth. Afterwards,

the strobe light and shaking stopped to allow for a 5 second post test, which counted how many

squares the RIFA’s head entered to determine it’s excitability. Five sessions were conducted in

the manipulation group. The ambient outside light was 1.27Fc emitting from a single source 7.2

feet about the petri dish. The flashing strobe light was 5.11Fc and 6 inches above the petri dish.

The control group was conducted in the same manner, except there was no shaking. The

flashing strobe light was not paired with any fear stimulus, remaining neutral. A pre and post

test was conducted to validate the flashing light’s neutralness. Five sessions were conducted in


the control group. After the training sessions were completed, after a twenty-four hour period, a

final test was conducted on the manipulation and control group to determine if fear conditioning

was established. To determine differences among means, data were analyzed by analysis of

variance (ANOVA).

Results

To determine the effect of the flashing strobe light as a neutral stimulus, an ANOVA was

conducted using SPSS. The analysis showed no significant difference between the control group

means from the first trial to the means of the twelfth trial (M = 1.42 vs. 1.75, F = 21.64, p < .88).

It was concluded that the non paired flashing strobe light in the control group had no effect on

the RIFA.

An ANOVA was conducted to test whether the flashing strobe light became a

conditioned fear stimulus in the conditioned trial verses the first training trial (M = 6.54 vs. 1.37,

F = 28.77, p < .029). The analysis showed that RIFA learned to fear the strobe light after twelve

trials of pairing the strobe light to shaking. Thus, rejecting the null hypothesis. The limbic

system is not a requirement for learning. For RIFA, the dorsal pedunculate structures of the

ganglia brain serves as the mecca region for stored memory.

Discussion

The implication that the limbic system is not required for memory creates many more

questions than answers. This study does not determine the magnitude of learning capability in

the dorsal pedunculate structures of the ganglia brain. This study does not investigate the length

of time memory is stored in the RIFA. Acknowledging the neurotransmitter’s presence without

the limbic system present, yet memory is still possible, illustrates that plasticity in the central

nervous system and the brain. Many more studies are needed with various forms of invertebrates


to determine if this phenomenon is restricted to the RIFA or to general life itself. Implications to

these findings have plausible beneficial outreach to stem cell research with neurons for patients

with damaged limbic systems. This study also suggests future studies on alternatives to the

limbic system.

References

Cammaerts, M. C. (n.d.). Classical conditioning, temporal learning and spatial learning in the ant

myrmica sabuleti. (2004). Biológia (Bratislava), 59, 243-246.

Darwin, C. (1975). The origin of species. (6th ed., p. xvi). Franklin Center, Pennsylvania: The

Franklin Library.

Franks, N. R., Hooper, J., Dornhaus, A., Aukett, P. J., Hayward, A. L., & Berghoff, S. (n.d.).

Reconnaissance and latent learning in ants. (2007). Proceedings of the Royal Society:

Biological Sciences, 274, 1505-1509.

Imai, K., Yamaguchi, K., Watanabe, M., Kainuma, E., Hikake, N., Saitoh, S., . . . Kimura, R.

(2007). Crucial role of thalami and basal ganglia in emotional memory and cognition:

Association with the recognition of niigata ken chuetsu earthquake 2004. Psychogeriatrics,

7(2), 58-63.

Lorenzetti, F. D., Baxter, D. A., & Byrne, J. H. (2011). Classical conditioning analog enhanced

acetylcholine responses but reduced excitability of an identified neuron. The Journal of

Neuroscience, 31(41), 14789-14793.

Lu, H., & Pietrantonio, P. V. (2011). Immunolocalization of the short neuropeptide F receptor in

queen brains and ovaries of the red imported fire ant (solenopsis invicta buren). BMC

Neuroscience, 12 Mayer, J. (n.d.).


Morrill, W. L. (n.d.). Tool using behavior of pogonomyrmex radius (hymenoptera: Formicidae).

(1972). The Florida Entomologist, 55, 59-60.

Mysore, K., Subramanian, K. A., Sarasij, R. C., Suresh, A., Shyamala, B. V., VijayRaghavan,

K., & Rodrigues, V. (2009). Caste and sex specific olfactory glomerular organization and

brain architecture in two sympatric ant species camponotus sericeus and camponotus

compressus (fabricius, 1798). Arthropod Structure & Development, 38(6), 485-497.

Pierce, J. D. J. (n.d.). A review of tool use in insects. (1986). The Florida Entomologist, 69, 95-

104.

Scott, D. J., Heitzeg, M. M., Koeppe, R. A., Stohler, C. S., & Zubieta, J. (2006). Variations in the

human pain stress experience mediated by ventral and dorsal basal ganglia dopamine

activity. The Journal of Neuroscience, 26(42), 10789-10795.

Tarsitano, M. S., & Jackson, R. R. (n.d.). Araneophagic jumping spiders discriminate between

detour routes that do and do not lead to prey. (1997). Animal Behaviour, 53, 257-266.


Tobiansky, D. J., Roma, P. G., Hattori, T., Will, R. G., Nutsch, V. L., & Dominguez, J. M.

(2013). The medial preoptic area modulates cocaine-induced activity in female rats.

Behavioral

Neuroscience, 127(2), 293-302.

Figures


Figure 1. Head of RIFA drawn under magnification with the brain, eyes, and

ocelli viewed as transparent objects. oc, Median ocellus; pb, pedunculate bodies;

og, optic ganglion; on, optic nerve; ol, olfactory lobe; an, antennary nerve.


Figure 2. Mean difference values for learning test conducted after twelve trials of fear

condition training. Control group received non-paired flashing strobe light only,

while the manipulation group received flashing strobe light paired with shaking.

During learning test, both groups received non-paired flashing strobe light only.

Standard errors are represented in the figure by the error bars attached to each

column. The means are for a 5 seconds duration before the strobe light and then 5

seconds after the strobe light. The strobe light was on for a 15 seconds duration

for each trial in both groups.

Control Group Manipulation Group0

3

6

9

12

15

1.42 1.371.75

6.54

Before strobe light After strobe light

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The Limbic system is not necessary for fear learning