Neural Substrates Of Aversion Research Articles

Fear state induced by diazepam withdrawal may be due to the sensitization of the neural substrates of aversion in the dPAG

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Brainstem areas activated by diazepam withdrawal as measured by Fos-protein immunoreactivity in rats

In the 1970s, chronic treatment with benzodiazepines was supposed not to cause dependence. However, by the end of the decade several reports showed that the interruption of a prolonged treatment with diazepam leads to a withdrawal syndrome characterized, among other symptoms, by an exaggerated level of anxiety. In laboratory animals, signs that oscillate from irritability to extreme fear-like behaviors and convulsions have also been reported. In recent years many studies have attempted to disclose the neural substrates responsible for the benzodiazepines withdrawal. However, they have focused on telencephalic structures such as the prefrontal cortex, nucleus accumbens and amygdala. In this study, we examined the Fos immunoreactivity in brain structures known to be implicated in the neural substrates of aversion in rats under spontaneous diazepam-withdrawal. We found that the same group of structures that originally modulate the defensive responses evoked by fear stimuli, including the dorso-medial hypothalamus, the superior and inferior colliculus and the dorsal periaqueductal gray, were most labeled following diazepam withdrawal. It is suggested that an enhanced neural activation of neural substrates of fear in the midbrain tectum may underlie the aversive state elicited in diazepam-withdrawn rats.

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 μg/0.2 μl) or bicuculline (40 ng/0.2 μl) 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.

P.1.c.032 Regulation of NA and GABA systems on DOI (agonist 5-HT2A/2C) anxiolytic-like effect in the four-plate test and cerebral localization of target receptors

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 μg/0.2 μl) or bicuculline (40 ng/0.2 μl) 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.

Fos-like immunoreactivity in the brain associated with freezing or escape induced by inhibition of either glutamic acid decarboxylase or GABA A 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 μg/0.2 μl) or bicuculline (40 ng/0.2 μl) 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.

Analysis of freezing behavior and ultrasonic vocalization in response to foot-shocks, ultrasound signals and GABAergic inhibition in the inferior colliculus: effects of muscimol and midazolam

Freezing behavior is a common response to distal threatening stimuli. It has been reported that experimenter-presented 20-kHz tones produce freezing in Wistar rats. The present studies were designed to determine the acoustic specificity for induction of the emission of ultrasound vocalizations (USV) and freezing of rats to either ultrasound tones or to a standard stressor, foot-shocks. We also examined whether GABA–benzodiazepine mechanisms, known for modulating anxiety-related processes, are involved in the regulation of defensive responses to these two aversive unconditioned stimuli. It was found that emission of USV was only observed with foot-shocks. Moreover, rats exhibited freezing when they were exposed to foot-shocks and 20–25-kHz ultrasound signals. . Likewise, removal of the GABAergic inhibitory control on the neural substrates of aversion in the inferior colliculus by local microinjections of semicarbazide, a blocker of the glutamic acid decarboxylase, caused freezing behavior without emission of USV. All these responses were significantly reduced by midazolam and muscimol. It is suggested that the neural substrates of aversion in the inferior colliculus are under inhibitory control of GABAergic mechanisms and are different from those triggered by foot-shocks.

Aversive effects of the C-fragment of Substance P in the dorsal periaqueductal gray matter.

There has been an increasing interest in the role of neuropeptides in the integration of brain functions. Besides the well-known positive-reinforcing effects of Substance P (SP) in prosencephalic regions, a role of this neuropeptide in the generation of aversive states in mesencephalic structures has also been envisaged. Evidence from a previous study suggests an involvement of SP in the neural substrates of aversion in the dorsal periaqueductal gray matter (DPAG). In the present study, we investigate whether N- and C-terminal fragments of Substance P are responsible for the effects produced by microinjections of SP into the dorsal periaqueductal gray. The results show that SP and its C-terminal fragment SP(7-11) produced a behavioral activation with increases in locomotor activity, grooming, and rearings, while the N-terminal fragment SP(1-7) produced only an increase in vertical exploratory activity. The effects were more pronounced with intermediate doses of SP and its C-fragment, confirming the characteristic bell-shaped dose-effect function of this neuropeptide. The proaversive effects observed with DPAG microinjections of these neuropeptides in the present study gain further relevance when combined with previous reports showing unconditioned and conditioned aversive effects following DPAG microinjections of SP in the place aversion and the elevated plus maze tests, two widely used animal models of anxiety. These results confirm previous data showing that SP has a modulatory role in the DPAG and that its effects are probably due to its C-terminal fragment.

Effects of microinjections of the neuropeptide substance P in the dorsal periaqueductal gray on the behaviour of rats in the plus-maze test

Currently, the participation of neuropeptides in the generation of aversive states in the dorsal periaqueductal gray matter (DPAG) is poorly understood. The elevated plus maze (EPM) is widely used for studying the neurobiological mechanisms of anxiety in the laboratory. One difficulty with this test has been to evaluate the involvement of GABA mechanisms in the DPAG substrates of aversion, because microinjections of GABA receptor blockers in this region cause an intense behavioral activation. In this study, we examined in the EPM the effects of semicarbazide, a drug that acts indirectly on GABA neurotransmission through inhibition of the glutamic acid decarboxylase, and substance P (SP) following microinjections into the dorsal periaqueductal gray. Semicarbazide caused a clear decrease in the number of entries and time spent in the open arms. These results confirm previous data showing that GABA has a modulatory role in the DPAG, probably through reduction of tonic inhibitory mechanisms on neural substrates of aversion. A similar pattern of behavioral responses was observed with SP. However, these effects were more pronounced with intermediate doses of SP (25 ng), confirming the characteristic bell-shaped dose-effect function of this neuropeptide. The proaversive effects observed with DPAG microinjections of SP in the present study gain further relevance when combined with previous reports that have shown unconditioned and conditioned aversive effects following DPAG microinjections of SP in other animal models of aversion.

Conditioned place aversion produced by microinjections of substance P into the periaqueductal gray of rats.

The dorsal periaqueductal gray (DPAG) is one of the main structures involved in the integration of defensive behavior in the brain. In order to investigate the participation of neuropeptides in the generation of aversive states, semicarbazide, a glutamic acid decarboxylase inhibitor, and substance P, an active neuropeptide, were injected into the DPAG and their effects evaluated in the open field and the place conditioning tests. While semicarbazide and substance P both increased locomotor activity only substance P increased grooming in the open field. In the place conditioning procedure similar aversion conditioning was produced by both drugs. These results confirm previous data showing that semicarbazide in the DPAG causes place aversion through reduction of tonic inhibitory mechanisms on neural substrates of aversion. Such mechanisms may include substance P neurons as substance P microinjection into the DPAG also functioned as an unconditioned stimulus in the place aversion test.

Mechanisms of defense in the inferior colliculus.

The inferior colliculus (IC) is a well known relay station for auditory pathways in the brainstem. In the present review we are suggesting that aversive states are also generated and elaborated in the inferior colliculus and that this structure may be part of a brain system commanding defensive behavior. The evidences presented in this review have been obtained from experiments carried out with the combined use of intracerebral microinjections and of electrical stimulation of the inferior colliculus. This electrical stimulation caused a behavioral activation together with autonomic reactions usually observed as part of the defense reaction. NMDA--an excitatory amino acid--, or bicuculline--a GABAA antagonist--injected into the IC mimicked the effects of its electrical stimulation. The IC electrical stimulation showed clear aversive properties as rats submitted to a switch-off paradigm quickly learned to interrupt it. Systemic administration as well as IC microinjections of the anxiolytic compound midazolam caused dose-dependent increases in the latency and reductions in the frequency of switch-off responses to the inferior colliculus electrical stimulation. Similar results were obtained following microinjections into this brainstem structure of the GABAA agonist muscimol. These results suggest that neural substrates responsible for defensive behavior in the inferior colliculus may be depressed by benzodiazepines as part of the anxiolytic action of these compounds. This anti-aversive action may be produced by the enhancement of GABAA mechanisms. Serotonergic mechanisms seem also to be involved in the modulation of these aversive states as IC microinjections of zimelidine, a 5-HT uptake blocker, caused a significant inhibition of the switch-off responses in the shuttle-box.(ABSTRACT TRUNCATED AT 250 WORDS)

Opposite effects of low and high doses of morphine on neural substrates of aversion in the inferior colliculus

The inferior colliculus (IC) is a well known relay station for auditory pathways in the brainstem. Much evidence has implicated this structure as part of a brain system mediating aversive states, and changes in its functioning as responsible for the occurrence of audiogenic seizures. Rats were implanted with chemitrodes, devices which allow electrical stimulation and microinjections of drugs in the same brain sites. Stepwise increases in the intensity of electrical stimulation of the IC of rats placed inside a circular arena allowed the determination of thresholds for freezing and escape behavior. Morphine (10-40nmol) caused dose-dependent increases in both aversive thresholds. A systemic injection of naloxone reversed the effects of morphine on the centrally induced aversive responses. These data suggest that neural substrates controlling defensive behavior in this structure are under opioid inhibitory control. Higher doses of morphine (80nmol) induced a non-naloxone reversible fearful hyper activity. It is suggested that opioid mechanisms exert an inhibitory control on the neural substrates of aversion in the IC and that high doses of morphine microinjected into IC cause pro-aversive actions probably through non-opioid mechanisms.

Mediation by serotonin of the antiaversive effect of zimelidine and propranolol injected into the dorsal midbrain central grey

Previously reported results indicate that serotonin (5-HT) inhibits the neural sub strate of aversion in the dorsal midbrain central grey (DCG) of the rat. In addition, the present results show that microinjection of the 5-HT uptake inhibitor zimelidine (100 nmol) into the DCG of rats with chronically implanted chemitrodes raised the threshold of aversive electrical stimulation. This antiaversive effect of zimelidine was antagonized by pretreatment with the 5-HT(2) receptor blocker ritanserin (10 nmol), also microinjected into the DCG. In contrast, the antiaversive effect of the benzodiazepine agonist midazolam (40 nmol) was unaffected by ritanserin. Propranolol (2.2, 4.4 and 8.8 nmol) raised the aversive threshold in a dose-depen dent way following its injection into the DCG. The antiaversive effect of 4.4 nmol of propranolol was antagonized by previous administration of ritanserin (10 nmol). Moreover, combined administration of zimelidine (100 nmol) followed by propranolol (4.4 nmol) caused an anti aversive effect which was equivalent to the sum of the effect of each drug alone. These results indicate that the antiaversive effect of intracerebrally injected zimelidine and propranolol is mediated by endogenous 5-HT, through activation of 5-HT(2) receptors.

Unit activity alterations induced in the mesencephalic periaqueductal gray by local electrical stimulation

In the framework of a series of investigations concerning the neural substrate of aversion, electrophysiological methods were used in order (1) to specify, within the rat's periaqueductal gray (PAG), functional properties, viz. conduction velocity and refractory period, of PAG neurons already assessed in previous studies by means of behavioral methods, and (2) to gather data on their local synaptic relationships. Unit activities were recorded from the periaqueductal gray with the aim of analyzing those alterations that would be induced by locally applying an electrical stimulation with parameters shown to elicit escape behavior. An implanted row of electrodes allowed the application of a stimulation to several sites aligned along a mediolateral or a rostrocaudal axis through the periaqueductal gray. The results indicate that an electrical stimulation applied to the periaqueductal gray may induce its effects through the activation of a number of dendrites and many slow conducting fibers running in a great variety of directions and branching within the periaqueductal gray. Their refractory period was surprisingly low (0.6 ms) for slow conducting fibers (below 1 m/s). The local circuitry appears to include many inhibitory connections. Their organization is assumed to be partly recurrent. Stimulation-induced inhibition becomes predominant when the stimulation is moved away from the recorded neuron along the mediolateral axis, but not along the rostrocaudal axis.