Review

Gabaergic regulation of the neural organization of fear

in the midbrain tectum

Marcus Lira Brandao*, Karina Genaro Borelli, Manoel Jorge Nobre, Julia Maria Santos,

Lucas Albrechet-Souza, Amanda Ribeiro Oliveira, Raquel Chacon Martinez

Laboratorio de Psicobiologia, FFCLRP, University of Sao Paulo, Campus USP, Av. Bandeirantes 3900, 14049-901 Ribeirao Preto, SP, Brazil

Abstract

In midbrain tectum (MT) structures, such as the dorsal periaqueductal gray (dPAG), the superior colliculus (SC) and the inferior colliculus

(IC) GABAergic neurons exert a tonic control on the neural substrates involved in the expression of defensive reactions. In this review, we

summarize behavioral, immunohistochemical (brain Fos distribution) and electrophysiological (auditory evoked potentials) data obtained

with the reduction of GABA transmission by local injections of a GABA receptor blocker (bicuculline, BIC) or a glutamic acid

decarboxylase inhibitor (semicarbazide, SMC) into the MT. Distinct patterns of Fos distribution were obtained following the freezing and

escape reactions induced by MT injections of SMC and BIC, respectively. While only the laterodorsal nucleus of the thalamus was labeled

after SMC-induced freezing, a widespread increase in Fos expression in the brain occurred after BIC-induced escape. Also, injections of

SMC into the IC increased the auditory evoked potentials recorded from this structure. It is suggested that GABAergic mechanisms of MT

are also called into play when sensory gating of the MT is activated during different emotional states.

q 2005 Elsevier Ltd. All rights reserved.

Keywords: Freezing; Escape; Semicarbazide; Bicuculline; GABA; dPAG; Amygdala; SNpr

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1299

2. Tonic inhibition of the neural substrates of defensive behavior by GABAergic neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . 1300

3. Sensory gating of the midbrain tectum in the organization of fear . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1301

4. Regulation from substantia nigra pars reticulata . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1305

5. Regulation by basolateral amygdala . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1305

6. Final comments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1308

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1308

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1308

1. Introduction

Electrical stimulation of the midbrain tectum (MT)—

dorsal periaqueductal gray (dPAG) and deep layers of the

superior colliculus (SC)—in the rat elicits unconditioned

0149-7634/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.

doi:10.1016/j.neubiorev.2005.04.013

* Corresponding author. Fax: C55 16 6331609.

E-mail address: mbrandao@usp.br (M.L. Brandao).

‘fear-like’ behavioral responses, such as alertness, sideways

postures, arching of the back, freezing, fleeing locomotion

and escape leaps (Bittencourt et al., 2004; Brandao et al.,

1982; Graeff et al., 1986; Schenberg et al., 1983). The same

pattern of responses has also been observed with electrical

stimulation of the inferior colliculus (IC) (Brandao et al.,

1988). This defensive behavioral reaction is associated with

sensory changes and autonomic responses, such as increase

in mean blood pressure, heart rate, piloerection, exhophtal-

mus, micturition and defecation. In view of these findings,

the MT has been suggested to play a major role in the neural

Neuroscience and Biobehavioral Reviews 29 (2005) 1299–1311

www.elsevier.com/locate/neubiorev

M.L. Brandao et al. / Neuroscience and Biobehavioral Reviews 29 (2005) 1299–13111300

organization and control of defensive reactions towards

threatening stimuli (for reviews see Brandao et al., 1993,

1999, 2003).

The use of electrical stimulation of brain structures in

these studies frequently leads to problems in the

interpretation of results. Such stimulation frequently

activates fibers of passage, raising the possibility of

stimulation spreading to neighboring structures. In the

case of the IC stimulation, this might include the cuneiform

nucleus. To circumvent this problem researchers have also

examined the effects of microinjecting tiny volumes

(200 nl) of drugs into the MT. The use of this technique

in association with electrophysiological studies has

provided a large body of evidence for the modulatory

influences of an array of neurotransmitters such as GABA,

serotonin, neuropeptides, opioids and excitatory amino

acids (Bittencourt et al., 2004; Brandao et al., 2003; Graeff

et al., 1986). Among them, GABA has been one of the

most studied transmitters regarding its regulatory function

in the defense reaction integrated at the MT level.

In this paper, we review a series of studies aimed at

investigating the role of GABA in sensory gating processes

in the MT during different emotional states produced either

by electrical or local injections of GABA blockers into

the MT, with special emphasis on dPAG and IC. Possible

changes in sensory processes have been neglected as a

possible source of the defensive behavior reaction that is

elicitable via MT stimulation (Brandao et al., 2003; Huston

et al., 1990). However, the MT processes aversive sensory

inputs and transduces them into behavioral and vegetative

nervous system reactions. For example, the IC-induced

defensive behavior is accompanied by changes in auditory-

evoked potentials in this structure, indicative of a

modification of sensory input channels (Brandao et al.,

2001). Thus, the behavioral patterns of the defense reaction

elicited at the MT level are unlikely to be the result of a

localized output process. As in the case of hypothalamic

aggression (Bandler, 1982a,b; Bandler and Flynn, 1971,

1972; Flynn et al., 1971; MacDonne and Flynn, 1966) and

the central activation of the perioral biting reflex, they are

probably linked to changes in sensorimotor gating processes

(Huston et al., 1980; Welzl et al., 1984).

While it is well established that there are four columns in

the central gray, as identified anatomically, their functional

role is still a subject of great debate (Bandler and Carrive,

1988; Bandler and Shipley, 1994; Carrive, 1993). It seems

very likely, however, that different pools of neurons in the

midbrain central gray are responsible for the elaboration of

distinct aspects of the defensive behavior. Dorsolateral and

dorsomedial columns have been associated with freezing,

escape, hypertension, tachycardia, and serotonin-dependent

analgesia, the lateral column with attack and the

ventrolateral column with quiescence, fear conditioned

freezing, recuperative-like behaviors, hypotension, brady-

cardia and opioid-dependent analgesia (Brandao et al.,

2003; Bittencourt et al., 2004; Canteras and Goto, 1999;

De Oca et al., 1998; Walker and Carrive, 2003; Vianna and

Brandao, 2003).

2. Tonic inhibition of the neural substrates of defensive

behavior by GABAergic neurons

GABA exists in appreciable density in the MT, and

GABAergic inhibition controls the firing rate of neurons in

this region (LeBeau et al., 1996, 2001; Roberts and Ribak,

1987; Thompson et al., 1985). As aversive states are

produced by GABA-A blockers and inhibited by GABA-A

agonists locally injected into the dPAG and the IC, it has

been suggested that these structures contain a tonically

active GABAergic network that regulates these states

through GABA-A receptors (Audi and Graeff, 1984;

Behbehani et al., 1990; Brandao et al., 1982, 1986, 1988,

1999; Coimbra and Brandao, 1993; DiScala et al., 1983;

Sandner et al., 1981; Schenberg et al., 1983). Consistent

behavioral evidence has also been provided for an anti-

aversive action of benzodiazepines in the MT. Indeed, local

injections of benzodiazepines into the MT depress the

defensive behavior induced by stimulation of this region

(Audi and Graeff, 1984; Brandao et al., 1982; Melo et al.,

1992; Pandossio and Brandao, 1999).

In this review, we will concentrate on the distinct

sensorimotor effects following the injections of bicuculline

(BIC) and semicarbazide (SMC) into the MT. BIC is a post-

synaptic GABA receptor antagonist, while SMC causes a

reduction in GABA levels due to its inhibition of glutamic

acid decarboxylase (GAD), the enzyme responsible for the

GABA synthesis (Brandao et al., 1986; Killam and Bain,

1957). Injections of SMC or BIC into the MT produce

defensive behavior, which mimics the effects of its electrical

stimulation (Brandao et al., 1982, 1986, 1988; DiScala and

Sandner, 1989). However, while BIC causes a full-blown

behavioral activation with escape responses predominating,

the defensive reaction caused by SMC has a slow onset and

freezing behavior predominates. Freezing and escape are

negatively correlated, suggesting a competition between

these fear-related motor systems. The distinct defensive

responses induced by these drugs could be due to different

degrees of GABA inhibition, as BIC (being a receptor

antagonist) would cause an immediate GABA inhibition

whereas the SMC, by reducing its synthesis, would cause

less intense antagonism.

These same defensive responses may also be produced

by drugs acting at glutamate receptors, as recently reported

in a Fos study from this laboratory. Glutamate injected into

the dPAG caused a selective activation of the laterodorsal

nucleus of the thalamus and other structures involved in the

sensory processing of aversive information, such as the

superior and inferior colliculi. NMDA, similarly injected,

produced a distribution of Fos in the brain that was quite

different from glutamate. NMDA caused widespread

activation throughout the forebrain but only in structures

M.L. Brandao et al. / Neuroscience and Biobehavioral Reviews 29 (2005) 1299–1311 1301

involved in the motor output of defensive behavior in the

brainstem. Therefore, the freezing resulting from the

activation of non-NMDA receptors appears to be related

to the acquisition of aversive information, whereas that

resulting from the activation of NMDA receptors could

serve as a preparatory response for flight (Ferreira-Netto

et al., 2005).

The characteristics of the defense reaction elicited by MT

stimulation will depend on the particular structure under

study so that the neural substrates of fear might subserve

different aspects of the defensive reaction. The main

defensive responses produced by chemical stimulation of

the SC, dPAG and IC are turnings, uncoordinated and

undirected escape behavior, and a more oriented and

coordinated escape, respectively (for a review see Brandao

et al., 1999).

The specific contribution of sensory changes in MT

structures for the production of defensive behavior is less

clear. As we will see, it is likely that GABAergic

mechanisms are involved in the gating of distinct sensory

information of aversive nature, depending on the midbrain

structure which is activated: tactile/nociceptive in the dPAG

or auditory in the inferior colliculus (Brandao et al., 1999;

Schmitt et al., 1986). As to the SC, it remains unresolved

whether this structure is merely sensorimotor or whether it

also possesses attentional (Stein et al., 1975; Drager and

Hubel, 1976) or motivational functions (Dean et al., 1989;

Redgrave et al., 1981). The SC is able to process sensory

integration of visual, auditory and somesthesic information,

aversive or not, so as to produce appropriate orienting

reflexes of eyes, head and trunk via tecto-reticulo-spinal,

tecto-pontine and tecto-cuneiform pathways (Dean et al.,

1989; Mitchell et al., 1988; Redgrave et al., 1981). Recent

studies have provided evidence for the role of these neurons

in the processing of visual information necessary for

attentive reflexes and orienting behavior (turnings), which

are relevant for the expression of defensive behavior

(Bittencourt et al., 2004; Beleboni et al., 2004).

3. Sensory gating of the midbrain tectum

in the organization of fear

Several studies have suggested that GABAergic mech-

anisms in the dPAG may be involved in the gating of

sensory information towards the neural substrate of

defensive response (Schmitt et al., 1985; DePaulis and

Vernes, 1986; Bagri et al., 1989; Nobre et al., 2004).

For example, unilateral microinjections of GABA antagon-

ists in the dPAG produced a hyporeactivity to tactile stimuli

applied to the side of the body ipsilateral to the injected side,

and caused hyperreactivity to stimuli applied to the

contralateral body side (Schmitt et al., 1985). This

hyperreactivity resulted in withdrawal reaction or even

jumping. However, these forms of defensive behaviors were

not accompanied by lunge-and-biting attacks, suggesting

that they represent submissive behavior rather than defense

reactions, in the way the latter has been defined previously

as fleeing and freezing, and also attacks directed at the face

or protruding parts of the body of the opponent (Adams,

1979). Fig. 1 shows the defensive behaviors and its

asymmetrical elicitation in rats injected unilaterally with

bicuculline into the dPAG.

Conversely, unilateral injections of GABA agonists into

the dPAG produced a contralateral hypoactivity with an

ipsilateral hyperreactivity (DiScala et al., 1983). Similarly,

using a social interaction situation, it was shown that

microinjections of GABA antagonists into the dPAG would

increase defensive reactions, such as withdrawal and

jumping to tactile stimulation applied to the contralateral

flank, while the perioral bite reflex was reduced (DePaulis

and Vernes, 1986). These data suggest that GABAergic

mechanisms may intervene, either in the gating of sensory

information towards the PAG substrate involved in the

expression of the defense reaction or in the selection of an

appropriate behavioral output in response to given sensory

information (Adams, 1979; Schmitt et al., 1986).

Mapping techniques, including electrophysiological

recordings, autoradiographic labeling with 2-deoxyglucose,

and Fos immunohistochemistry have revealed arrangements

of isofrequency contours within the central nucleus of the

inferior colliculus (Clerici and Coleman, 1986; Coleman

et al., 1982; Huang and Fex, 1986; Merchan et al., 1994;

Pierson and Snyder-Keller, 1994; Saint Marie et al., 1999;

Schreiner and Langner, 1997). Generally, neurons, which are

tuned to high-frequency stimuli are clustered in band-like

arrays located in ventral IC while those responding best to

low-frequency pure tones are found in more rostral locations.

However, the biological relevance of sensory processing has

also to do with the behavioral meaning of the information

provided. What an individual animal senses may change with

the behavioral program, developmental stage, hormonal

influences and individual expectations. With this in mind,

our studies have attempted to assess the processing of

acoustic information, which is concomitant with the display

of defensive behavior.

A rapid means to assess the state of fear or anxiety of an

animal consists of evaluating the startle reaction (Campeau

and Davis, 1995; Davis et al., 1994). In the startle reflex test,

the animal is placed in a stabilimeter inside an acoustically

attenuated chamber, and the amplitude of the startle

response to loud sounds is measured (Davis et al., 1994).

Moderate, but not high, levels of fear enhance the amplitude

of the startle response (Davis and Astrachan, 1978; Walker

et al., 1997; Santos et al., 2005). While separate evidence

has been provided for the involvement of GABA-mediated

mechanisms in startle and acoustically evoked potentials

(AEP) (Bagri et al., 1989; Brandao et al., 2001; Fendt,

1999), studies combining sensory processing and motor

responses are lacking in this field of research. Thus, taking

advantage of the properties of BIC and SMC in producing

distinct defensive behavior, we were also interested in

Fig. 1. A time course of the behavioral effects produced by microinjections of bicuculline methiodide (BIC—35 ng/0.2 ml) and semicarbazide (SCBZ—6 mg/0.

2 ml) into the dPAG. B. Incidence of the behavioral responses (from top to bottom: contact or bite, head orientation, withdrawal responses and jumps) over time

observed on application of a tactile stimulus ipsilaterally (open symbols) or contralaterally (close symbols) to the injection site, before and after a

microinjection of bicuculline methiodide into the right dPAG. The data are reported as the % incidence of response to tactile stimulus applied to the animal’s

body every minute over 30 min (see Schmitt et al., 1985, for details).

M.L. Brandao et al. / Neuroscience and Biobehavioral Reviews 29 (2005) 1299–13111302

measuring the sensory changes (AEP) concomitant to

freezing induced by SMC.

The processing of sensory information was evaluated by

the AEP recorded from electrodes implanted bilaterally in

the central nucleus of the IC in freely moving rats. The

method used made it possible to record freezing behavior,

startle amplitude and the AEPs (from both sides of the

brain), simultaneously. Microinjections of SMC into the IC,

at doses that induced freezing behavior, caused a clear

enhancement of the AEPs recorded from electrodes

implanted in the central nucleus of this structure with a

concomitant decrease in the startle reflex (Fig. 2).

These effects of SMC on the AEP and startle response

support the notion of the existence of sensory and motor

components for the defense reaction organized at the MT

level. AEPs evoked by sounds have been previously

recorded from the IC of the rat, under drugs and aversive

conditions (Bagri et al., 1989; Brandao et al., 2001; Sandner

et al., 2002). These studies have shown that AEPs are also

increased by local injections of BIC into the IC.

Interestingly, light used as conditioned stimulus (light-CS)

or ultrasound signals presented at the frequency of 22 kHz,

which have been considered to emulate the alarm calls of

nearby predators, caused significant increases in the

AEPs recorded from electrodes implanted into the IC

(Brandao et al., 2001). These effects are similar to those

caused by injections of SMC into this structure, also at doses

that produce freezing (Nobre et al., 2004). This same

chemical stimulation of the IC did not produce any

vocalization at the frequency range studied (16–30 kHz)

(Nobre and Brandao, 2004).

These findings support the contention that IC is a

structure involved in sensory processes relating to aversion.

Neurons of the IC only fired during external acoustic

stimulation or during stimulation of the dPAG, but never

before vocalization onset as occur with the dPAG, one of the

most important vocalmotor structures in the midbrain

(Pieper and Jurgens, 2003). It has been suggested that

these changes in AEP amplitude may be considered to

reflect synaptic modifications within the IC, rather than

upstream of it (Szczepaniak and Moller, 1995). Our findings

provide further evidence for an involvement of a

GABAergic modulation of auditory inputs into the IC

(Li et al., 1998). The possibility that other MT structures

(PAG and SC) show similar effects is open to investigation.

The reduction in the amplitude of the startle reflex

concomitant with the SMC-induced freezing was an

unexpected finding. These effects are the opposite of the

usual increase in startle response induced by aversive

conditioned stimuli. A hypothesis that might account for

Fig. 2. Auditory evoked potentials (AEP) recorded from electrodes implanted into the inferior colliculus and startle responses to pure tones (92.5 dB, 3000 Hz) in rats injected with saline or semicarbazide into

this structure. (A) Photomicrograph of a representative site and location of sites of injections and electrode tips implanted in the inferior colliculus (IC) on cross-sections from the Paxinos and Watson rat brain

atlas (1997). Figures represent the atlas coordinates in mm posterior to bregma. (B) Auditory evoked potentials (AEP) recorded in the central nucleus of the IC of rats. Y axis corresponds to the mean amplitude of

AEPs recorded ipsi and contralateral to the injection sites (mV). X corresponds to the mean latency after the pulse (ms). The graph represents the mean effects of intracerebral injections of saline (sal-thinner line)

and IC microinjections of semicarbazide (Smc—(solid line) on P1 (the first positive wave of the AEP). (C) Averaged collicular auditory evoked potentials following microinjections of saline (sal) and

semicarbazide (Smc—6.0 mg/0.2 ml) or saline into the central nucleus of IC. The recordings were made before, immediately after and 45 min after (recovery) the treatments in the right IC (ipsilateral to the

injection side) and in the left IC (contralateral to the injection side). (D) Effects of microinjections of 6.0 mg/0.2 ml of semicarbazide (Smc) into the central nucleus of the IC on the mean amplitude of startle

response as compared to control animals (sal). The recordings were made before, immediately after and 45 min after (recovery) treatments. *Different from the baseline (p!0.05, Dunnett’s test).

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M.L. Brandao et al. / Neuroscience and Biobehavioral Reviews 29 (2005) 1299–13111304

these striking results is that these effects depend upon the

nature of the stimuli used in these tests. It has been shown that

conditioned stimuli, paired previously with footshocks

during training, lead to increases in fear-potentiated startle

when presented alone during testing. However, this only

occurs as footshock intensity increases from low to moderate

levels. When trained at higher intensities, rats show relatively

poor potentiated startle response (Davis and Astrachan,

1978). In contrast to conditioned stimuli, certain uncondi-

tioned fear stimuli cause a reduction, instead of an increase,

in the startle response. These findings have given rise to the

performance hypothesis, in which startle is suggested to be

non-monotonically related to fear (Davis and Astrachan,

1978; Wecker and Ison, 1986; Walker et al., 1997).

Evidence from several sources has indicated that the

dPAG is involved in the elaboration of potentiated startle.

However, electrical stimulation of the dPAG may increase

motor activity and reduce startle amplitude, simultaneously

(Fendt et al., 1994; Walker et al., 1997). It is also interesting

to note that although electrical stimulation of the IC, by

itself, causes a startle response; prepulse inhibition ensues

when it is applied before a loud sound that itself produces

the startle reflex (Li et al., 1998). Therefore, electrical

stimulation of the IC decreased the startle response in the

same way as SMC microinjections into this region did in our

study. The reduction in startle response during the state of

Fig. 3. Photomicrographs of Fos-immunoreactive cells (dark dots) in coronal

semicarbazide (5 mg/0.2 ml) and bicuculline (40 ng/0.2 ml) into the dPAG. CeA,

dorsomedial part; PMD, dorsal premammillary nucleus of the hypothalamus. Sca

fear induced by SMC observed in this study could result

from an indirect influence of MT mechanisms involved in

active defense on startle, perhaps biasing animals towards

alternative forms of behavior that are incompatible with

startle response. Much evidence exists in support of the

view that MT activation may produce covert preparatory

responses, e.g. changes in muscle tone (Walker et al., 1997;

Watkins et al., 1993), that promote certain classes of

defensive behavior (e.g. freezing) at the expense of others

(e.g. the startle response).

We propose here that GABAergic mechanisms may gate

the processing of aversive sensory information for the

expression of defensive behavior. Injections of SMC into

the dPAG at doses that produce freezing behavior and of

bicuculline that produce escape behavior caused a distinct

pattern of Fos distribution in the brain. SMC-induced

freezing was followed by increased Fos-expression only in

the laterodorsal nucleus of thalamus, while bicuculline

caused a widespread activation in the brain. In the latter

case, the most pronounced labeling was obtained in the CnF,

which is thought to be the main relay station for dPAG- and

SC-mediated defensive responses en route to the medulla

oblongata and spinal cord (Dean et al., 1989; Keay and

Bandler, 2001). The differential distribution of Fos

expression in the brain during freezing and escape is

illustrated in Fig. 3.

sections of the midbrain and diencephalon following microinjections of

central amygdaloid nucleus; vmHdm, ventromedial hypothalamic nucleus,

le bar—200 mm.

M.L. Brandao et al. / Neuroscience and Biobehavioral Reviews 29 (2005) 1299–1311 1305

The present findings add to those of other studies that

also claim a distinct biological meaning for the various

components of the defensive behavioral repertoire

generated in the MT level. It has been reported that

depending on the nature of the aversive stimulus (whether

conditioned or unconditioned), the intensity (moderate or

intense) or the distance (distal or proximal), distinct kinds of

freezing can be obtained by activation of the PAG (for a

review see Vianna and Brandao, 2003). Freezing induced by

stimulation of its ventrolateral column tends to disappear

when the stimulation is terminated, whereas freezing

induced by stimulation of its dorsolateral column remains

high in the absence of stimulation. In view of these results, it

has been suggested that unconditioned fear induced by the

direct electrical stimulation of the dPAG is downstream of

the prosencephalic control, but the post-stimulation freezing

may reflect the process of acquisition of information of

aversive nature that ascends to the amygdala (Martinez

et al., submitted for publication).

The neural substrates of defensive behavior in the dPAG

deserve a deeper discussion with respect to their biological

meaning. Some 25 years ago, Adams (1979) suggested that

the PAG might contain two different, parallel neural

substrates for defense and submission. Defense is the

behavior that includes lunge-and-bite attack, squealing,

upright posture, fleeing, freezing, warning noises and

vocalizations. On the other hand, submission is the behavior

usually observed in laboratory animals under attack by

conspecifics and includes the full submissive posture

(lying on the back) and many of the patterns of defense.

The defensive animal is dangerous, while the submissive

one is vulnerable.

As noted earlier, during freezing induced by activation of

the MT the animals even show a reduction in biting and

attacks, which suggests that this particular form of freezing

behavior may be a submissive posture. Due to similarities in

the motor patterns of submission and defense, their

motivational mechanisms may consist of sets of homo-

geneous neurons with similar neural connections. Although

they have the same locus, submission may have evolved as a

subset of defense during the course of phylogeny. Defense

probably evolved first to deal with predators, and the

submission system may have evolved later to modify

defense behavior when the animal is confronted with a

conspecific, whose offensive behavior would be inhibited by

submissive postures. Thus, while submission clearly

involves integration of aversive information in higher

brain structures, defense does not require this refinement.

Indeed, it has been shown that escape behavior is still

induced by MT stimulation in thalamic rats (Tomaz et al.,

1988). Adams (1979) suggested that the ventromedial

nucleus of the hypothalamus, which interconnects heavily

with the amygdala, seems to possess a mechanism that

switches the behavior of an animal from defense to

submission in particularly dangerous situations. Likely,

concomitant with freezing behavior, ascending information

is relayed in the dPAG column on its way up to the thalamus

and from there to forebrain structures, probably the

amygdala, which may trigger the switch mechanisms at

the hypothalamic level. In support of this, the dPAG sends

information to the laterodorsal nucleus of the thalamus,

from which forebrain structures are called into play

(Ferreira-Netto et al., 2005).

4. Regulation from substantia nigra pars reticulata

The substantia nigra pars reticulata (SNpr) plays an

important role in the inhibitory control of motor behavior

(Chevalier et al., 1981; Chevalier and Denniau, 1990). SNpr

neurons use GABA as a transmitter and project to the SC,

dPAG and IC (Chevalier et al., 1981; Di Chiara et al., 1979;

Imperato and DiChiara, 1981; Yasui et al., 1991). Inhibitory

control of the dPAG and IC may be provided by these

GABAergic projections from the SNpr (Coimbra and

Brandao, 1993; Nobre et al., 2004). Lesions of the SNpr

or local injections of muscimol into the SNpr increase

defensive responses (e.g. escape threshold) induced by

electrical or chemical stimulation of these structures

(Coimbra and Brandao, 1993; Nobre et al., 2004). It has

also been shown that defensive behavior induced by

unilateral injections of NMDA into the IC is ipsilaterally

modulated by prior microinjections of muscimol into the

SNpr (Fig. 4). The results obtained show that unilateral

microinjections of muscimol into the SNpr caused a

significant increase in turning, freezing and jumping

following microinjection of the excitatory amino acid

NMDA into the IC (Nobre et al., 2004). These effects are

probably due to the removal of the inhibitory control exerted

by GABA on defensive circuitry in the MT (Coimbra et al.,

1993; Nobre et al., 2004). Together, these results support the

proposal that nigrocollicular GABAergic fibers constitute a

tonically active inhibitory control for the fear-related

processes organized at the MT level (Brandao et al., 1999,

2003; Huston et al., 1980, 1990).

5. Regulation by basolateral amygdala

Besides sending projections to lower brainstem regions,

the MT also sends information to structures located

rostrally. Reciprocal connections between the dPAG and

the central nucleus of the amygdala have already been

demonstrated (Rizvi et al., 1991).

It has been proposed that distinct circuits exist for

anticipatory responses to distal or conditioned stimuli, and

for fear responses to proximal or unconditioned danger

stimuli (Blanchard and Blanchard, 1969, 1972, 1988; Davis

et al., 1994). It seems that the dPAG is implicated in active

responses to threatening stimuli while the ventrolateral

periaqueductal gray (vPAG) is involved in more passive

responses (De Oca et al., 1998; Fanselow, 1984; Walker and

Fig. 4. Regulation of the defensive behavior elicited by NMDA injections into the central nucleus of the inferior colliculus by GABAergic fibers from the substantia nigra pars reticulata-SNpr. (A and B)

Representative photomicrograph of injection sites into the inferior colliculus (IC) and SNpr, respectively. CG, central gray; PnO, pontine reticular nucleus, pars oralis; MnR, median raphe nucleus; Pn,

pedunculopontine nucleus; SC, superior colliculus; ML, medial lemniscus; MGB, medial geniculate nucleus; Rn, red nucleus. (C) Frequency of rearings, crossings, groomings, jumps and turnings as well as

duration of freezing behavior displayed by rats placed in a circular arena for 30 min after being injected with saline (Sal) or muscimol (Mus-1 nmol/0.2 ml) into the SNpr and 5 min later with saline, muscimol

(1 nmol/0.2 ml) or NMDA (7 nmol/0.2 ml) into the inferior colliculus (meanGSEM). *p!0.05 in relation to the control group (sal–sal). #p!0.05 in relation to salCNMDA group.

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Fig. 5. Schematic illustrating possible modulatory mechanisms of sensory

input to the dorsal periaqueductal gray. The basolateral nucleus of

amygdala (BLA) receives sensory input probably from the dorsal raphe

nucleus (DRN) and exerts an inhibitory control on the output neurons in the

central nucleus of amygdala (CeA). Projection neurons from the CeA may

have direct or indirect (through substantia nigra pars reticulata-SNpr)

influence on the neural substrates of aversion in the dorsal periaqueductal

gray (dPAG). The projection neurons from amygdala activate the

ventrolateral periaqueductal gray (vPAG).

M.L. Brandao et al. / Neuroscience and Biobehavioral Reviews 29 (2005) 1299–1311 1307

Carrive, 2003). Vianna et al. (2001a,b) found that lesions of

the vPAG reduce the expression of conditioned freezing

behavior but leave unaffected the expression of active

defensive patterns of behavior induced by electrical or

chemical activation of the dorsal parts of this structure.

Much evidence exists for the expression of the

conditioned freezing through vPAG being driven by the

amygdala. Electrolytic or chemical lesions of the amygdala

reduce freezing and other behavioral components of

conditioned fear such as potentiated startle and hypoalgesia

(Helmstetter, 1992; De Oca et al., 1998; Carrive et al.,

2000). Taken together, these data support the notion that the

transition from passive to active behaviors as the threat

levels increase is mediated neurally by a shift in activity

from the ventral to the dorsal MT regions. The amygdala

and other higher brain structures are involved in the first

condition while a group of MT structures made up of the

dPAG, IC and SC act in concert under the second condition

generating primitive forms of fear related to panic attacks

(see for instance Brandao et al., 1999; Vianna and Brandao,

2003).

Another distinct behavioral stage in the defense reaction

is generated when the animal is in contact with the predator

and it may be too late to perform successful antidetection

behavior (Klemm, 1976). If this were the case, then in close

proximity to danger signals, the rat should implement

another defensive strategy—such as moving to a safe place

or preparing a counterattack. However, such strategies

would be difficult to recognize when testing the rat in a

conditioning chamber that has no escape routes or places of

safety. In many animal species, the response to this

threatening condition is the so-called tonic immobility. In

natural environments, this response is a component of

antipredator behavior and the last resource to reduce the

probability of attacks from the predator. Evidence gathered

on the mediation of tonic immobility points to intrinsic

mechanisms in the dPAG and vPAG acting in concert,

which are probably regulated by GABAergic, cholinergic

and serotonergic mechanisms (Monassi and Menescal-deO-

liveira, 2004).

The amygdala is critically involved in the modulation

of innate and conditioned reactions to threatening stimuli

(Blanchard and Blanchard, 1972; Davis et al., 1994;

LeDoux et al., 1988). The basolateral nucleus of the

amygdala (BLA) receives relevant information from the

environment via the dorsal raphe nucleus, hippocampal,

thalamic and cortical afferents (McDonald, 1998;

Stutzmann and LeDoux, 1999), and is predominantly

involved in the filtering of such aversive information.

The BLA projects to the central nucleus of the amygdala,

which in turn projects to areas of the brainstem involved

in the coordination of endocrine, somatic and behavioral

responses to aversive stimulation characteristic of fear

and anxiety (Charney and Deutch, 1996; LeDoux et al.,

1988, 1990; Davis et al., 1994; Maisonnette et al., 1996;

Stutzmann and LeDoux, 1999).

To study the functional role of the BLA, we selectively

inhibited the activity of this structure by local administration

of muscimol. Muscimol injections into the BLA increased the

aversiveness of IC stimulation (Macedo et al., 2005). These

findings confirm previous reports with electrolytic or

neurotoxic lesions showing that the BLA regulates the

defensive behavior generated at the IC level (Macedo et al.,

2002; Maisonnette et al., 1996, 2000). On the other hand,

electrolytic lesionsof the BLA or local injectionsofmuscimol

into the BLA did not change the freezing and escape

thresholds determined by stepwise increases in the current

of the electrical stimulation of the dPAG (Oliveira et al.,

2004). Thus, the disruption of the modulatory mechanisms of

the BLA might greatly amplify or facilitate the occurrence of

defensive behaviors induced by stimulation of the IC, but not

of the dPAG. These latter findings thus point to a different

functional role for BLA mechanisms in the regulation of

unconditioned fear generated either in the dPAG or IC.

The anatomical circuitry of the amygdala is such that it

enables this region to integrate emotional information in a

unique manner. It has direct input from all sensory modalities

via the thalamus and the neocortex (Tigges et al., 1982, 1983;

Amaral and Price, 1984). The BLA is an integration center

for sensory information and memories, while the central

nucleus is an important output center for behavioral and

neurovegetative responses during anxiety (Campeau and

Davis, 1995; LeDoux et al., 1988, 1990). Thus, changes in the

functioning of the BLA resulting in abnormal sensory

interpretation and emotional responses could conceivably

lead to pathological states.

M.L. Brandao et al. / Neuroscience and Biobehavioral Reviews 29 (2005) 1299–13111308

The possibility of conjoint inhibitory influences of the

BLA and SNpr on the neural substrates of aversion in the

MT still remains. The SNpr-MT pathway might well be

modulated by descending projections from the amygdaloid

nuclei (Hopkins and Holstege, 1978; Shinonaga et al.,

1992). A diagram illustrating possible modulatory

connections involving amygdaloid nuclei, SNpr, and PAG

is presented in Fig. 5.

6. Final comments

In MT structures such as the dPAG, SC and IC,

GABAergic neurons exert tonic control over the neural

substrates involved in the generation and expression of

defensive reactions. It is still unclear how these

mechanisms are called into play during the processing

of information of an aversive nature when sensory

influences trigger the motor expression of defense. We

can infer that such induced states and behaviors must

either be a precondition for the expression of fear, or a

consequence thereof. We can thus conclude that the MT

‘defense-aversion systems’ as they are called, also

comprise a substrate for, or source of information for

state properties that decide the animal’s behavior on a

scale of anxiety as defined by performance in anxiety

tests. Thus, while freezing behavior is the usual response

when the animal perceives the danger, escape is the

natural reaction when the predator is close. This line of

research has evolved to disclose the structures and the

neural mechanisms that could participate in the acqui-

sition of aversive information and expression of the

different types of defensive behavior.

Studies using electrical stimulation of the MT have

shown that defensive strategies as distinct as avoidance,

freezing and escape are likely to be organized by

different networks. Studies aimed at examination of

both the processing of sensory information and their

behavioral counterparts during the reduction of GABA

transmission caused by local injections of GABA

receptor blockers (e.g. BIC) or GAD inhibitors (e.g.

SMC) into the MT have shown that they induce freezing

and escape behavior, respectively. Moreover, SMC

enhances auditory evoked potential and impair the startle

reaction to loud sound. These results suggest that

reduction of the GABAergic control of the MT results

in an enhanced processing of information of aversive

nature along with defensive behaviors that are incompa-

tible with the startle response. Also, we present evidence

here that Fos expression in the laterodorsal nucleus of

the thalamus is increased during freezing induced by

stimulation of the dPAG suggesting that aversive

information ascends to higher structures during this

emotional condition.

Acknowledgements

This work was supported by FAPESP (Proc. no.

02/03705-0) and CNPq (301069/81–6). KG Borelli, JM

Santos and RC Martinez are recipients of doctorate

scholarships from CAPES. AR Oliveira and L Albrechet-

Souza are recipients of master scholarships from FAPESP

and CAPES, respectively. I also thank Dr Silvio Morato for

the helpful contribution to the writing style of this

manuscript.

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