Karina Genaro Borelli

Gabaergic regulation of the neural organization of fear in the midbrain tectum

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.

Effects of acute and chronic fluoxetine and diazepam on freezing behavior induced by electrical stimulation of dorsolateral and lateral columns of the periaqueductal gray matter

The defensive responses induced by electrical stimulation of the dorsal periaqueductal gray matter (dPAG) of the rat have been proposed as a model of panic attacks in humans. In the present study we investigated the acute and chronic effects of fluoxetine and diazepam on freezing and escape reactions elicited by electrical stimulation of the dorsolateral (dlPAG) and lateral (lPAG) columns of the periaqueductal gray matter (PAG). The frequencies of crossing, rearing, bouts of micturition and fecal boli were also recorded. Electrodes were unilaterally implanted in the brainstem aimed at the PAG. Drug treatments were given daily for 2 weeks with fluoxetine (5, 10 and 20 mg/kg ip), a selective inhibitor of serotonin reuptake, diazepam (1, 2 and 4 mg/kg ip), or saline. Drug effects were assessed acutely (15 min after the first injection) and chronically (15 min after the 14th injection). Chronic, but not acute, administration of fluoxetine caused a significant increase in the threshold of freezing without affecting the escape response elicited by dlPAG/lPAG stimulation. This characteristic pattern of effects could not be attributed to motor deficit, since this drug did not change the number of crossings and rearings. In contrast, no significant threshold changes were observed following acute and chronic treatment with diazepam. These data give further evidence for (a) an antiaversive effect of chronic treatment with fluoxetine, which caused a selective reduction in freezing behavior and neurovegetative responses associated with fearlike reaction elicited by dlPAG/lPAG electrical stimulation; (b) the involvement of the dlPAG and lPAG in the generation and organization of defensive responses and that freezing may probably be associated with panic attacks; and (c) the lack of effect of diazepam in this model is in line with its inefficacy as a panicolytic drug. The study of the unconditioned freezing behavior evoked by dlPAG/lPAG stimulation may constitute a new and interesting model for the study of panic disorder.

Fos-like immunoreactive neurons following electrical stimulation of the dorsal periaqueductal gray at freezing and escape thresholds

Electrical stimulation of the dorsal regions of the periaqueductal gray (PAG) leads to defensive reactions characterized as freezing and escape responses. Until recently it was thought that this freezing behavior could be due to the recruitment of neural circuits in the ventrolateral periaqueductal gray (vlPAG), while escape would be mediated by other pathways. Nowadays, this view has been changing mainly because of evidence that freezing and escape behaviors thus elicited are not altered after lesions of the vlPAG. It has been suggested that there are at least two pathways for periaqueductal gray-mediated defensive responses, one involving the hypothalamus and the cuneiform nucleus (CnF) which mediates responses to immediate danger and another one involving the amygdala and vlPAG which mediates cue-elicited responses, either learned or innate. To examine this issue further we measured Fos protein expression in brain areas activated by electrical stimulation of the dorsolateral PAG (dlPAG) at the freezing and escape thresholds. The data obtained showed that freezing-provoking stimulation caused increases in Fos expression in the dorsomedial PAG (dmPAG), while escape-provoking stimulation led to increases at both dmPAG and dlPAG. Surprisingly, neither escape- nor freezing-provoking stimulations altered Fos expression in the central nucleus of amygdala (CeA). Escape-provoking stimulation caused increased Fos expression in the ventromedial hypothalamus (VMH), dorsal premammilary nucleus (PMd) and in the cuneiform nucleus. Significant increases in Fos labeling were found in the dmPAG and PMd following freezing-provoking stimulation. Therefore, the present data support the notion of a neural segregation for defensive behaviors in the dorsal columns of PAG, with increased Fos expression in the dmPAG following freezing, while dlPAG is affected by both freezing and escape responses. dlPAG, CnF, VMH and PMd are part of a brain aversion network activated by fear unconditioned stimuli. The present data also suggests that the defensive responses generated at the dlPAG level do not recruit the neural circuits of the vlPAG and CeA usually activated by conditioned fear stimuli.

Fos-like immunoreactivity in the brain associated with freezing or escape induced by inhibition of either glutamic acid decarboxylase or GABAA receptors in the dorsal periaqueductal gray

GABAergic neurons exert tonic control over the neural substrates of aversion in the dorsal periaqueductal gray (dPAG). It has been shown that electrical stimulation of this region at freezing or escape thresholds activates different neural circuits in the brain. Since electrical stimulation activates cell bodies and fibers of passage, it is necessary to use chemical stimulation that activates only post-synaptic receptors. To investigate this issue further, reduction of GABA transmission was performed with local injections of either the GABA-A receptor antagonist bicuculline or the glutamic acid decarboxylase (GAD) inhibitor semicarbazide into the dorsolateral periaqueductal gray (dlPAG). Local infusions of semicarbazide (5.0 microg/0.2 microl) or bicuculline (40 ng/0.2 microl) into this region caused freezing and escape, respectively. The results obtained showed that freezing behavior induced by semicarbazide was associated with an increase in Fos expression in the laterodorsal nucleus of the thalamus (LD) and ventrolateral periaqueductal gray (vlPAG), while bicuculline-induced escape was related to widespread increase in Fos labeling, notably in the columns of the periaqueductal gray, hypothalamus nuclei, the central amygdaloid nucleus (Ce), the LD, the cuneiform nucleus (CnF) and the locus coeruleus (LC). Thus, the present data support the notion that freezing and escape behaviors induced by GABA blockade in the dlPAG are neurally segregated: freezing activates only structures that are mainly involved in sensory processing, and bicuculline-induced escape activates structures involved in both sensory processing and motor output of defensive behavior. Therefore, the freezing elicited by activation of dlPAG appears to be related to the acquisition of aversive information, whereas most brain structures involved in the defense reaction are recruited during escape.

Neural segregation of Fos-protein distribution in the brain following freezing and escape behaviors induced by injections of either glutamate or NMDA into the dorsal periaqueductal gray of rats

Freezing and escape responses induced by gradual increases in the intensity of the electrical current applied to dorsal regions of the periaqueductal gray (dPAG) cause a distinct pattern of Fos distribution in the brain. From these studies, it has been suggested that a pathway involving the dPAG itself, dorsomedial hypothalamus and the cuneiform nucleus (CnF) would mediate responses to immediate danger and another one involving the amygdala and ventrolateral periaqueductal gray (vlPAG) would mediate cue-elicited responses. As electrical stimulation activates body cells and fibers of passage the need of studies with chemical stimulation of only post-synaptic fibers of the dPAG is obvious. To examine further this issue we measured Fos protein expression in brain areas activated by stimulation of the dPAG with glutamate (5 nmol/0.2 microL) and N-methyl-D-aspartate (NMDA) at doses that provoke either freezing (4 nmol/0.2 microL) or escape (7 nmol/0.2 microL) responses, respectively. The results showed that glutamate-induced freezing caused a selective increase in Fos expression in the superior and inferior colliculi as well as in the laterodorsal nucleus of the thalamus. On the other hand, NMDA-induced escape led to widespread increases in Fos labeling in almost all structures studied. Differently from glutamate, NMDA at doses provoking freezing caused significant increase of Fos labeling in the dPAG and CnF. Therefore, the present data support the notion that freezing behavior induced by activation of either non-NMDA or NMDA receptors in the dorsolateral periaqueductal gray (dlPAG) is neurally segregated: glutamate activates only structures that are mainly involved in the sensorial processing and NMDA-induced freezing structures involved in the motor output of defensive behavior. Therefore, the freezing elicited by the activation of non-NMDA receptors seem 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.

Serotonergic mechanisms of the median raphe nucleus–dorsal hippocampus in conditioned fear: Output circuit involves the prefrontal cortex and amygdala

Independent studies have shown that the median raphe nucleus (MRN) and dorsal hippocampus (DH) are involved in the expression of contextual conditioned fear (CFC). However, studies that examine the integrated involvement of serotonergic mechanisms of the MRN-DH are lacking. To address this issue, a CFC paradigm was used to test whether the serotonergic projections from the MRN to DH can influence CFC. Serotoninergic drugs were infused either into the MRN or DH prior to testing sessions in which freezing and startle responses were measured in the same context where 6h previously rats received footshocks. A reduction of serotonin (5-HT) transmission in the MRN by local infusions of the 5-HT(1A) agonist 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT) decreased freezing in response to the context but did not reduce fear-potentiated startle. This pattern of results is consistent with the hypothesis that MRN serotonergic mechanisms selectively modulate the freezing response to the aversive context. As for the DH, a decrease in postsynaptic 5-HT receptor activity at projection areas has been proposed to be the main consequence of 5-HT(1A) receptor activation in the MRN. Intra-DH injections of 8-OH-DPAT inhibited both the freezing and fear-potentiated startle response to the context. To reconcile these findings, an inhibitory mechanism may exist between the incoming 5-HT pathway from the MRN to DH and the neurons of the DH output to other structures. The DH-amygdala or medial prefrontal cortex projections could well be this output circuit modulating the expression of CFC as revealed by measurements of Fos immunoreactivity in these areas.

Effects of inactivation of serotonergic neurons of the median raphe nucleus on learning and performance of contextual fear conditioning

Several studies have shown that the median raphe nucleus (MRN) is involved in anxiety. However, no study assessed the role of 5-HT mechanisms of MRN in both freezing and fear-potentiated startle (FPS) within a single form of conditioned learning. In this work we examined the effects of neurotoxic lesions of the MRN with NMDA on freezing and FPS of rats submitted to a contextual fear conditioning paradigm, in which they were tested in the same chamber where they received foot-shocks 24 h before. Compared to controls NMDA-injected rats showed a reduction of freezing and FPS in response to contextual cues. Next, we examined the effects of stimulation of 5-HT1A somatodendritic autoreceptors of the MRN with local injections of 8-OH-DPAT either before training or testing sessions conducted 2 or 24 h post-conditioning. Pre-training injections of 8-OH-DPAT intra-MRN reduced both freezing and FPS whereas post-training injections reduced only freezing to the aversive context without changing the FPS. Thus, freezing is easily disrupted by post-training MRN injections of 8-OH-DPAT while memory for FPS remained unchanged. It is proposed that the consolidation of contextual conditioned fear promoting freezing takes place through a slow mechanism of transference of information through 5-HT mechanisms of the MRN-hippocampus pathway. On the other hand, a rapid fear conditioning process operates for FPS, probably through other pathways.

Distribution of Fos immunoreactivity in the rat brain after freezing or escape elicited by inhibition of glutamic acid decarboxylase or antagonism of GABA-A receptors in the inferior colliculus.

It has been shown that electrical stimulation of the central nucleus of the inferior colliculus (IC) at freezing or escape thresholds activates different neural circuits in the brain. Since electrical stimulation activates cell bodies and fibers of passage it is necessary to use chemical stimulation that activates only post-synaptic receptors. To examine this issue in more detail, we took advantage of the fact that GABAergic neurons exert tonic control over the neural substrates of aversion in the IC. Reduction of GABA transmission in this structure was performed with the use of semicarbazide - an inhibitor of the GABA synthesizing enzyme glutamic acid decarboxylase (GAD) - and the GABA-A receptor antagonist bicuculline. Depending on the dose employed local infusions of semicarbazide (6.0 microg/0.2 microl) or bicuculline (40 ng/0.2 microl) into this region caused freezing and escape, respectively. The results obtained showed that freezing behavior induced by semicarbazide was associated with an increase in Fos expression in the dorsomedial column of the PAG (dmPAG) only, while bicuculline-induced escape was related to widespread increase in Fos labeling, notably in the periaqueductal gray, hypothalamus nuclei, amygdaloid nuclei, the laterodorsal nucleus of thalamus (LD), the cuneiform nucleus (CnF) and the locus coeruleus (LC). Thus, the present data support the notion that freezing and escape behaviors induced by GABA blockade in the IC are neurally segregated: acquisition of aversive information of acoustic nature from the IC probably uses the dmPAG column as a relay station to higher brain centers whereas bicuculline-induced escape activates structures involved in both sensory processing and motor output of defensive behavior. These results support the existence of distinct neural circuits mediating the sensory and motor responses of the defense reaction. The extent of the brain activation during freezing appears to be limited to the anatomical connections of the dmPAG, whereas an overall activation of the limbic system predominates during escape behavior induced by IC stimulation.

Apomorphine enhances conditioned responses induced by aversive stimulation of the inferior colliculus

Consistent evidence has shown that learning may be produced in paradigms using electrical stimulation of the inferior colliculus (IC) as unconditioned stimulus (UCS). Recent reports have also demonstrated that aversive stimulation of the IC, at the escape threshold, enhances dopamine (DA) release in the prefrontal cortex. The purpose of the present study was to determine whether dopaminergic mechanisms are involved in the Pavlovian conditioning and latent inhibition using IC stimulation as UCS and light as conditioned stimulus (CS). Rats were placed inside a shuttle box and subjected to a two-way avoidance paradigm. IC aversive electrical stimulation was used as UCS and shuttle box illumination as CS. The rats quickly learned to avoid or interrupt the IC stimulation. Apomorphine injections produced a dose-dependent increase in the number of avoidance responses. On the other hand, chlorpromazine administration promoted a dose-dependent reduction of the avoidance responses. Previous injections of chlorpromazine inhibited the effects of apomorphine. Also, previous exposure to unreinforced light weakened the strength of the conditioning. Apomorphine blocked this latent inhibition effect, which was antagonized by previous injections of chlorpromazine. These findings bring evidence for the involvement of DA in the setting up of adaptive responses to aversive states generated at the IC level, which may underlie stressful situations present in anxiety.

Anxiety-like symptoms induced by morphine withdrawal may be due to the sensitization of the dorsal periaqueductal grey

Withdrawal from morphine leads to the appearance of extreme anxiety accompanied of several physical disturbances, most of them linked to the activation of brainstem regions such as the locus coeruleus, ventral tegmental area, hypothalamic nuclei and periaqueductal grey (PAG). As anxiety remains one of the main components of morphine withdrawal the present study aimed to evaluating the influence of the dorsal aspects of the PAG on the production of this state, since this structure is well-known to be involved in defensive behaviour elicited by anxiety-evoking stimuli. Different groups of animals were submitted to 10 days of i.p. morphine injections, challenged 2 h after with an i.p. injection of naloxone (0.1 mg/kg), and submitted to the plus-maze, open-field and light-dark transition tests. The effects of morphine withdrawal on anxiety-induced Fos immunolabelling were evaluated in four animals that passed by the light-dark transition test randomly chosen for Fos-protein analysis. Besides the PAG, Fos neural expression was conducted in other brain regions involved in the expression of anxiety-related behaviours. Our results showed that morphine withdrawn rats presented enhanced anxiety accompanied of few somatic symptoms. Increased Fos immunolabelling was noted in brain regions well-known to modulate these states as the prelimbic cortex, nucleus accumbens, amygdala and paraventricular hypothalamus. Increased Fos labelling was also observed in the ventral and dorsal aspects of the PAG, a region involved in anxiety-related processes suggesting that this region could be a common neural substrate enlisted during anxiety evoked by dangerous stimuli as well as those elicited by opiate withdrawal.