Acute engagement of Gq-mediated signaling in the bed nucleus of the stria terminalis induces anxiety-like behavior.

The bed nucleus of the stria terminalis (BNST) is a brain region important for regulating anxiety-related behavior in both humans and rodents. Here we used a chemogenetic strategy to investigate how engagement of G protein-coupled receptor (GPCR) signaling cascades in genetically defined GABAergic BNST neurons modulates anxiety-related behavior and downstream circuit function. We saw that stimulation of vesicular γ-aminobutyric acid (GABA) transporter (VGAT)-expressing BNST neurons using hM3Dq, but neither hM4Di nor rM3Ds designer receptors exclusively activated by a designer drug (DREADD), promotes anxiety-like behavior. Further, we identified that activation of hM3Dq receptors in BNST VGAT neurons can induce a long-term depression-like state of glutamatergic synaptic transmission, indicating DREADD-induced changes in synaptic plasticity. Further, we used DREADD-assisted metabolic mapping to profile brain-wide network activity following activation of Gq-mediated signaling in BNST VGAT neurons and saw increased activity within ventral midbrain structures, including the ventral tegmental area and hindbrain structures such as the locus coeruleus and parabrachial nucleus. These results highlight that Gq-mediated signaling in BNST VGAT neurons can drive downstream network activity that correlates with anxiety-like behavior and points to the importance of identifying endogenous GPCRs within genetically defined cell populations. We next used a microfluidics approach to profile the receptorome of single BNST VGAT neurons. This approach yielded multiple Gq-coupled receptors that are associated with anxiety-like behavior and several potential novel candidates for regulation of anxiety-like behavior. From this, we identified that stimulation of the Gq-coupled receptor 5-HT2CR in the BNST is sufficient to elevate anxiety-like behavior in an acoustic startle task. Together, these results provide a novel profile of receptors within genetically defined BNST VGAT neurons that may serve as therapeutic targets for regulating anxiety states and provide a blueprint for examining how G-protein-mediated signaling in a genetically defined cell type can be used to assess behavior and brain-wide circuit function.

Extended fear conditioning reveals a role for both N-methyl-D-aspartic acid and non-N-methyl-D-aspartic acid receptors in the amygdala in the acquisition of conditioned fear.

Pavlovian conditioning is a useful tool for elucidating the neural mechanisms involved with learning and memory, especially in regard to the stimuli associated with aversive events. The amygdala has been repeatedly implicated as playing a significant role in the acquisition and expression of fear. If the amygdala is critical for the acquisition of fear, then it should contribute to this processes regardless of the parameters used to induce or evaluate conditioned fear. A series of experiments using reversible inactivation techniques evaluated the role of the amygdala in the acquisition of conditioned fear when training was conducted over several days in rats. Fear-potentiated startle was used to evaluate the acquisition of conditioned fear. Pretraining infusions of N-methyl-d-aspartic acid (NMDA) or non-NMDA receptor antagonists alone into the amygdala interfered with the acquisition of fear early in training, but not later. Pretraining infusions of a cocktail consisting of both an NMDA and non-NMDA antagonist interfered with the acquisition of conditioned fear across all days of training. Taken together these results suggest the amygdala may potentially be critical for the acquisition of conditioned fear regardless of the parameters utilized.

Fear-potentiated startle, but not prepulse inhibition of startle, is impaired in CREBalphadelta-/- mutant mice.

Fear-potentiated startle was assessed in mice with a targeted disruption of the alpha and delta isoforms of the transcription factor cAMP response element binding protein (CREB) 24 hr after 5 tone + shock training trials. Whereas wild-type mice showed fear-potentiated startle that persisted up to 45 days after training, CREBalphadelta-/- mice failed to show fear-potentiated startle. However, CREBalphadelta-/- and wild-type mice had similar startle amplitudes and similar magnitudes of prepulse inhibition of startle, suggesting that CREBalphadelta-/- mice have no obvious sensory or motor deficits. These results add to the literature indicating that CREB-activated transcription plays a critical role in the formation of long-term memory and illustrate the utility of the fear-potentiated startle paradigm for assessing cognition in genetically altered mice.

Posttraining lesions of the amygdala interfere with fear-potentiated startle to both visual and auditory conditioned stimuli in C57BL/6J mice.

The fear-potentiated startle paradigm has been used with great success to examine conditioned fear in both rats and humans. The purpose of the present experiment was to extend the authors' previous findings and further validate the fear-potentiated startle paradigm in mice. In Experiments 1 and 2, C57BL/6J mice were given Pavlovian fear conditioning with either an auditory or a visual conditioned stimulus. Similar to data collected with rats, fear-potentiated startle was observed for both stimulus modalities. In Experiment 3, posttraining lesions of the amygdala disrupted fear-potentiated startle in both conditioned stimulus modalities. These data are consistent with amygdala lesion studies in rats and suggest that fear-potentiated startle in mice requires an intact amygdala. Together, these results extend the authors' previous results and provide the basis for using this well-understood behavioral paradigm for examining the molecular mechanisms of conditioned fear in transgenic and knockout mice.

The effects of intra-amygdaloid infusions of a D2 dopamine receptor antagonist on Pavlovian fear conditioning.

The present study examined the effects of bilateral intra-amygdaloid infusions of the D2 receptor antagonist, eticlopride, on the acquisition and expression of Pavlovian fear conditioning as measured by freezing to acoustic and background contextual stimuli in the rat. Infusions of eticlopride before acquisition or before both acquisition and retention testing significantly attenuated conditioned freezing to tone presentations during the retention test 24 hr later. No effects, however, were observed on freezing that emerged during acquisition. Furthermore, these effects were not attributable to state-dependent learning effects or alterations in baseline activity or shock reactivity. In conclusion, these results suggest that amygdaloid dopamine transmission at D2 receptors contributes to the formation and/or consolidation of fear memories.

Destruction of the auditory thalamus disrupts the production of fear but not the inhibition of fear conditioned to an auditory stimulus.

The auditory thalamus is part of a neural circuit that mediates the expression of fear to auditory stimuli. Bilateral lesions of the auditory thalamus prevent the expression of fear to an auditory stimulus paired with shock. The present study assessed whether bilateral lesions of the auditory thalamus would also disrupt the inhibition of fear to an auditory stimulus paired with the absence of shock. Rats were given bilateral lesions of the auditory thalamus followed by Pavlovian conditioned inhibition training in which a light was paired with shock and a noise and light compound was presented in the absence of shock. Fear and the inhibition of fear were measured with the fear-potentiated startle effect. Lesions of the auditory thalamus did not disrupt the ability of the noise to inhibit the expression of fear to the light. However, these lesions did disrupt the ability of the noise to produce fear-potentiated startle after it had been subsequently paired with shock. These results suggest that although the auditory thalamus is an essential part of a neural circuit that mediates the expression of fear to auditory stimuli, it is not an essential part of the circuit that mediates the inhibition of fear to auditory stimuli.

The BALB/c mouse as an animal model for progressive sensorineural hearing loss.

To develop the BALB/c mouse strain as an animal model for the study of progressive sensorineural hearing loss, mice ranging in age from young adult through middle age were studied. Auditory brainstem response thresholds, histopathology [cytocochleograms for hair cells, the packing density of spiral ganglion cells (SGCs), the number of neurons and overall size of the anterior ventral cochlear nucleus (AVCN)], and behavioral paradigms (prepulse inhibition, fear-potentiated startle) were compared with previous data from C57BL/6J (C57) and DBA/2J (DBA) mouse strains. Progressive high frequency hearing loss in BALB/c mice was generally more rapid than C57 and slower than DBA (e.g. mean thresholds for 16 kHz: 10-month-old BALB/c mice = 71 dB SPL; 55-day-old DBA mice = 79 dB SPL; 12-month-old C57 mice = 50 dB SPL). Like the other strains, BALB/c exhibited a progressive loss of hair cells and SGCs that was most severe in the cochlear base and least severe in the middle turns; however, BALB/c mice had relatively more SGC loss in the apex. Unlike C57 and DBA, no loss of neurons was observed in the AVCN following cochlear pathology (although AVCN volume was reduced). Like the other strains, successful fear conditioning was obtained with a 12 kHz conditioned stimulus. Prepulse inhibition showed that middle and low frequency tones (4-12 kHz) became more salient as high frequency hearing declined. Similar results had been previously obtained with C57 and DBA mice and were interpreted as reflecting hearing-loss-induced plasticity in the central auditory system.

Normal conditioned inhibition and extinction of freezing and fear-potentiated startle following electrolytic lesions of medical prefrontal cortex in rats.

The authors investigated the role of medial prefrontal cortex (mPFC) in the inhibition of conditioned fear in rats using both Pavlovian extinction and conditioned inhibition paradigms. In Experiment 1, lesions of ventral mPFC did not interfere with conditioned inhibition of the fear-potentiated startle response. In Experiment 2, lesions made after acquisition of fear conditioning did not retard extinction of fear to a visual conditioned stimulus (CS) and did not impair "reinstatement" of fear after unsignaled presentations of the unconditioned stimulus. In Experiment 3, lesions made before fear conditioning did not retard extinction of fear-potentiated startle or freezing to an auditory CS. In both Experiments 2 and 3, extinction of fear to contextual cues was also unaffected by the lesions. These results indicate that ventral mPFC is not essential for the inhibition of fear under a variety of circumstances.

Fear-potentiated startle in two strains of inbred mice.

The fear-potentiated startle paradigm has been used with great success to examine conditioned fear in both rats and humans. The purpose of this study was to examine fear-potentiated startle in inbred mice. One-month-old C57BL/6J (C57) and DBA/2J (DBA) mice were given tone + foot shock training trials. The amplitude of the acoustic startle reflex was measured in the presence and absence of the tone both before and after training. Both strains showed fear-potentiated startle after training as evidenced by larger startle amplitudes in the presence of the tone than in its absence. However, the magnitude of fear-potentiated startle was greater in DBA mice than in C57 mice. These results not only demonstrate fear-potentiated startle in mice for the first time but also suggest that fear-potentiated startle can be influenced by characteristics of the mouse strain.

Lesions of the perirhinal cortex interfere with conditioned excitation but not with conditioned inhibition of fear.

Posttraining lesions of the perirhinal cortex (Prh) have been shown to interfere with the expression of fear. This study assessed whether Prh lesions would also disrupt the inhibition of fear as measured with conditioned inhibition of fear-potentiated startle. Following light + shock, noise-->light-no shock conditioned-inhibition training, rats were given Prh lesions. The lesions interfered with the expression of fear-potentiated startle to the light. To assess whether conditioned inhibition was affected, the rats were given light + retraining without additional noise-->light-training. The noise-conditioned inhibitor retained its ability to inhibit fear-potentiated startle to the retrained light. These results suggest that the areas of the Prh that are essential for the initial expression of conditioned fear are not important for the expression of conditioned inhibition of fear.

Elicitation and reduction of fear: behavioural and neuroendocrine indices and brain induction of the immediate-early gene c-fos.

The elicitation and reduction of fear were indexed with fear-potentiated startle and corticosterone release and induction of the immediate-early gene c-fos as a marker of neural activity in male Sprague-Dawley rats. Conditioning consisted of pairing one stimulus with footshock, which was withheld when the conditioned stimulus was preceded by a different modality stimulus, the conditioned inhibitor. On the test day, approximately 60% of the rats were used for c-fos in situ hybridization, and were presented with either the conditioned stimulus alone, the conditioned inhibitor alone, a compound of the two stimuli, or no stimuli, and killed 30 min following the presentation of 10 such stimuli. The remaining rats were tested with the fear-potentiated startle paradigm. Rats displayed reliable fear-potentiated startle and corticosterone release to the conditioned stimulus, and both measures were reduced when the conditioned stimulus was preceded by the conditioned inhibitor. The ventral bed nucleus of the stria terminalis, septohypothalamic nucleus, some tegmental nuclei, and the locus coeruleus had particularly high c-fos induction in rats that received the conditioned inhibitor, providing one of the first functional indication that these nuclei might be important in behavioural or endocrine inhibition. Conditioning specific c-fos induction in the three groups that received a stimulus on the test day was observed in many hypothalamic areas, the medial geniculate body and the central gray, structures previously involved in fear and anxiety. The cingulate, infralimbic and perirhinal cortex, nucleus accumbens, lateral septum, dorsal endopiriform nucleus, and ventral tegmental area had higher c-fos induction in rats presented with the fearful conditioned stimulus, confirming previous studies. The amygdala and hippocampus of conditioned rats did not show higher c-fos induction than in rats repeatedly exposed to the context. Many regions displayed c-fos messenger RNA induction in the control condition, suggesting that processes other than fear and anxiety participate in c-fos induction.

Inhibition of fear-potentiated startle can be detected after the offset of a feature trained in a serial feature-negative discrimination.

Using the fear-potentiated startle paradigm in rats, 4 experiments examined whether the inhibitory effect of a feature is evident after its offset following serial feature-negative discrimination training (A+ and X-->A-). When startle probes were presented shortly after the offset of X on X-->A test trials, the inhibitory properties of X were observed immediately after its offset. Furthermore, trace reinforcement of X (X-->+), but not delay reinforcement (X+), disrupted the ability of X to inhibit fear-potentiated startle on X-->A trials. Trace conditioning to X was also retarded after A+ and X-->A- training. These results suggest that the inhibitory properties of the serially trained feature are present after its offset and raise the possibility that either temporal information regarding nonreinforcement or poststimulus attributes of X acquire inhibitory properties.

Lesions of the central nucleus of the amygdala block conditioned excitation, but not conditioned inhibition of fear as measured with the fear-potentiated startle effect.

Lesions of the amygdala block the expression of fear-potentiated startle following either moderate or extensive light + shock training. The present experiment assessed whether lesions of the amygdala would also block the expression of conditioned inhibition of fear. Rats were given conditioned inhibition training in which a light was paired with shock and a noise and light compound was presented in the absence of shock. Then half of the rats were given bilateral electrolytic lesions of the amygdala and the remaining rats were sham operated. Lesions of the amygdala blocked the expression of fear-potentiated startle to the light. To assess whether conditioned inhibition was disrupted, rats were retrained with light + shock pairings with no further conditioned inhibition training. Amygdala lesioned rats reacquired fear-potentiated startle to the light (Kim & Davis, 1993). Importantly, the noise conditioned inhibitor retained its ability to inhibit fear-potentiated startle to the retrained light. These results indicate that areas of the amygdala critical for initial performance of fear-potentiated startle are not critical for the expression of conditioned inhibition.

Safety signals and human anxiety: a fear-potentiated startle study.

The effect of a safety signal on the magnitude of anticipatory anxiety was investigated using the fear-potentiated startle reflex paradigm in humans. The amplitude of the acoustic startle reflex was measured during the anticipation of unpleasant electric shocks ("threat") and during "safe" conditions. Threat and safe conditions were signaled by three different colored lights. Two lights signaled safe conditions (safe 1, safe 2) and the other light signaled the threat condition (threat). In phase I, the lights alternated, each presentation consisting of one colored light. In phase II, the lights were presented alone or in the two combinations of safe 1 (or safe 2) + threat and safe 1 + safe 2. In both phases, the contingency between the lights and the shock was explained to the subjects. It was emphasized that no shock could be administered when the safe 1 and threat light were simultaneously presented in phase II. Subjects' belief and understanding of the instructions were verified. In Phase I, startle was increased in the threat-alone compared to the safe-alone condition, reflecting increased anticipatory anxiety in the threat-alone condition. In phase II, startle in the safe + threat condition was smaller than in the threat-alone condition, but was larger than in the safe + threat. These results were interpreted as suggesting that the threat signal was still able to elicit anticipatory anxiety despite the fact that it was no longer associated with a threat.

Fear-potentiated startle: a neural and pharmacological analysis.

The fear-potentiated startle paradigm has proven to be a useful system with which to analyze neural systems involved in fear and anxiety. This test measures conditioned fear by an increase in the amplitude of a simple reflex (the acoustic startle reflex) in the presence of a cue previously paired with a shock. Fear-potentiated startle is sensitive to a variety of drugs such as diazepam, morphine, and buspirone that reduce anxiety in people and can be measured reliably in humans when the eyeblink component of startle is elicited at a time when they are anticipating a shock. Electrical stimulation techniques suggest that a visual conditioned stimulus ultimately alters acoustic startle at a specific point along the acoustic startle pathway. The lateral, basolateral and central amygdaloid nuclei and the caudal branch of the ventral amygdalofugal pathway projecting to the brainstem are necessary for potentiated startle to occur. The central nucleus of the amygdala projects directly to one of the brainstem nuclei critical for startle and electrical stimulation of this nucleus increases startle amplitude. Chemical or electrolytic lesions of either the central nucleus or the lateral and basolateral nuclei of the amygdala block the expression of fear-potentiated startle. The perirhinal cortex, which projects directly to the lateral and basolateral amygdaloid nuclei, plays a critical role in the expression of fear-potentiated startle using either visual or auditory conditioned stimuli. These latter amygdaloid nuclei may actually be the site of plasticity for fear conditioning, because local infusion of the NMDA antagonist AP5 into these nuclei blocks the acquisition of fear-potentiated startle. On the other hand, the expression of fear-potentiated startle is blocked by local infusion of the non-NMDA ionotropic antagonist CNQX or the G-protein inactivating toxin, pertussis toxin, but not by AP5. Finally, we have begun to investigate brain systems that might be involved in the inhibition of fear. Local infusion of AP5 into the amygdala was found to block the acquisition of experimental extinction, a prototypical method for reducing fear. We have also established a reliable procedure for producing both external and conditioned inhibition of fear-potentiated startle and hope to eventually understand the neural systems involved in these phenomena.

Visual cortex ablations do not prevent extinction of fear-potentiated startle using a visual conditioned stimulus.

Following observations in the literature that sensory cortex ablations prevent extinction of conditioned fear, the present experiments tested the generality of this finding by examining whether visual cortex ablations would prevent extinction of conditioned fear as assessed by fear-potentiated startle using a visual conditioned stimulus. Consistent with previous reports, visual cortex ablations did not prevent the acquisition or expression of fear-potentiated startle to a visual conditioned stimulus. More importantly, visual cortex ablations did not prevent extinction of fear-potentiated startle to a visual conditioned stimulus, nor did they reverse preoperatively established extinction, indicating that sensory cortex is not required for extinction of conditioned fear in all situations.

Infusion of the non-NMDA receptor antagonist CNQX into the amygdala blocks the expression of fear-potentiated startle.

The involvement of non-N-methyl-D-aspartate receptors in the amygdala in the expression of conditioned fear was examined using the fear-potentiated startle paradigm. Rats implanted with bilateral cannulae in the basolateral amygdaloid nuclei received 10 pairings of either a visual or auditory conditioned stimulus with footshock on each of 2 days. The next day, they were tested by eliciting the acoustic startle reflex in the presence or absence of the conditioned stimulus and divided into groups with equivalent levels of potentiation. One or two days later, rats were tested again following intra-amygdala infusion of vehicle or 0.025, 0.25, or 2.5 micrograms of 6-cyano-7-nitroquinoxaline-2,3-dione. The drug dose-dependently blocked the expression of potentiated startle in both sensory modalities, indicating that activation of non-NMDA receptors in the amygdala is necessary for the expression of conditioned fear.

Lesions of the perirhinal cortex but not of the frontal, medial prefrontal, visual, or insular cortex block fear-potentiated startle using a visual conditioned stimulus.

The present study is part of an ongoing series of experiments aimed at delineation of the neural pathways that mediate fear-potentiated startle, a model of conditioned fear in which the acoustic startle reflex is enhanced when elicited in the presence of a light previously paired with shock. A number of cortical areas that might be involved in relaying information about the visual conditioned stimulus (the light) in fear-potentiated startle were investigated. One hundred thirty-five rats were given 10 light-shock pairings on each of 2 consecutive days, and 1-2 d later electrolytic or aspiration lesions in various cortical areas were performed. One week later, the magnitude of fear-potentiated startle was measured. Complete removal of the visual cortex, medial prefrontal cortex, insular cortex, or posterior perirhinal cortex had no significant effect on the magnitude of fear-potentiated startle. Lesions of the frontal cortex attenuated fear-potentiated startle by approximately 50%. However, lesions of the anterior perirhinal cortex completely eliminated fear-potentiated startle. The effective lesions included parts of the cortex both dorsal and ventral to the rhinal sulcus and extended from approximately 1.8 to 3.8 mm posterior to bregma. Lesions slightly more posterior (2.3-4.8 mm posterior to bregma) or lesions that included only the perirhinal cortex dorsal to the rhinal sulcus had no effect. The region of the perirhinal cortex in which lesions blocked fear-potentiated startle projects to the amygdala, and thus may be part of the pathway that relays the visual conditioned stimulus information to the amygdala, a structure that is also critical for fear-potentiated startle. In addition, the present findings are in agreement with numerous studies in primates suggesting that the perirhinal cortex may play a more general role in memory.

Involvement of pertussis toxin sensitive G-proteins in conditioned fear-potentiated startle: possible involvement of the amygdala.

The present study evaluated the effects of intraventricular or intracerebral administration of pertussis toxin on fear-potentiated startle (a measure of conditioned fear) and shock sensitization (a measure of unconditioned fear). In Experiment 1 all animals were unilaterally implanted with cannulae into the lateral ventricle 1 week prior to 2 days of fear conditioning (ten light-shock pairings on each of 2 days). Five days later, animals were infused with either 1 microgram pertussis toxin or saline and tested for fear-potentiated startle 24 h after infusion and tested for shock sensitization 26 or 50 h after infusion. Pertussis toxin blocked the ability of a light conditioned stimulus to facilitate startle but did not alter the ability of acute footshock to increase startle amplitude in the same animals. In Experiment 2 bilateral infusion of 1 microgram pertussis toxin into the basolateral nuclei of the amygdala, but not the interpositus nuclei of the cerebellum, also blocked fear-potentiated startle when animals were tested 6 h after infusion. These findings suggest a role for pertussis toxin sensitive G-proteins, perhaps within the amygdala, in the expression of conditioned but not unconditioned fear.

Lesions of the central nucleus of the amygdala, but not the paraventricular nucleus of the hypothalamus, block the excitatory effects of corticotropin-releasing factor on the acoustic startle reflex.

Intracerebroventricular (icv) infusion of corticotropin-releasing factor (CRF) was previously found to produce a long-lasting, dose-dependent (0.1-1.0 microgram) increase in the amplitude of the acoustic startle reflex. The present study sought to determine where in the CNS CRF acts to increase startle. Intracisternal infusion of CRF (0.1-1.0 microgram) increased startle with a time course and magnitude similar to that produced by icv CRF, unlike intrathecal infusion, which produced a small, more rapid enhancement of startle. While lesions of the paraventricular nucleus of the hypothalamus had no effect on icv CRF-enhanced startle, bilateral lesions of the central nucleus of the amygdala significantly attenuated the excitatory effect of icv CRF but had no effect on intrathecal CRF-enhanced startle. Even though lesions of the amygdala blocked icv CRF-enhanced startle, local infusion of CRF into the amygdala did not significantly elevate startle. The present data indicate that the amygdala is part of the neural circuitry required for icv CRF to elevate startle, but does not appear to be the primary receptor area where CRF acts. The involvement of the amygdala in icv CRF-enhanced startle is consistent with the hypothesis that both the amygdala and CRF are critically involved in fear and stress.