Cannabinoid CB1 receptors in distinct circuits of the extended amygdala determine fear responsiveness to unpredictable threat.

The brain circuits underlying behavioral fear have been extensively studied over the last decades. Although the vast majority of experimental studies assess fear as a transient state of apprehension in response to a discrete threat, such phasic states of fear can shift to a sustained anxious apprehension, particularly in face of diffuse cues with unpredictable environmental contingencies. Unpredictability, in turn, is considered an important variable contributing to anxiety disorders. The networks of the extended amygdala have been suggested keys to the control of phasic and sustained states of fear, although the underlying synaptic pathways and mechanisms remain poorly understood. Here, we show that the endocannabinoid system acting in synaptic circuits of the extended amygdala can explain the fear response profile during exposure to unpredictable threat. Using fear training with predictable or unpredictable cues in mice, combined with local and cell-type-specific deficiency and rescue of cannabinoid type 1 (CB1) receptors, we found that presynaptic CB1 receptors on distinct amygdala projections to bed nucleus of the stria terminalis (BNST) are both necessary and sufficient for the shift from phasic to sustained fear in response to an unpredictable threat. These results thereby identify the causal role of a defined protein in a distinct brain pathway for the temporal development of a sustained state of anxious apprehension during unpredictability of environmental influences, reminiscent of anxiety symptoms in humans.

Expression of freezing and fear-potentiated startle during sustained fear in mice.

Fear-potentiated acoustic startle paradigms have been used to investigate phasic and sustained components of conditioned fear in rats and humans. This study describes a novel training protocol to assess phasic and sustained fear in freely behaving C57BL/6J mice, using freezing and/or fear-potentiated startle as measures of fear, thereby, if needed, allowing in vivo application of various techniques, such as optogenetics, electrophysiology and pharmacological intervention, in freely behaving animals. An auditory Pavlovian fear conditioning paradigm, with pseudo-randomized conditioned-unconditioned stimulus presentations at various durations, is combined with repetitive brief auditory white noise burst presentations during fear memory retrieval 24 h after fear conditioning. Major findings are that (1) a motion sensitive platform built on mechano-electrical transducers enables measurement of startle responses in freely behaving mice, (2) absence or presence of startle stimuli during retrieval as well as unpredictability of a given threat determine phasic and sustained fear response profiles and (3) both freezing and startle responses indicate phasic and sustained components of behavioral fear, with sustained freezing reflecting unpredictability of conditioned stimulus (CS)/unconditioned stimulus (US) pairings. This paradigm and available genetically modified mouse lines will pave the way for investigation of the molecular and neural mechanisms relating to the transition from phasic to sustained fear.

Standardizing the analysis of conditioned fear in rodents: a multidimensional software approach.

Data comparability between different laboratories strongly depends on the individually applied analysis method. This factor is often a critical source of variation in rodent phenotyping and has never been systematically investigated in Pavlovian fear conditioning paradigms. In rodents, fear is typically quantified in terms of freezing duration via manual observation or automated systems. While manual analysis includes biases such as tiredness or inter-personal scoring variability, computer-assisted systems are unable to distinguish between freezing and immobility. Consequently, the novel software called MOVE follows a semi-automatized approach that prefilters video sequences of interest for the final human judgment. Furthermore, MOVE allows integrating additional data sources (e.g. force-sensitive platform, EEG) to reach the most accurate and precise results. MOVE directly supports multi-angle video recordings with webcams or standard laboratory equipment. The integrated manual key logger and internal video player complement this all-in-one software solution. Calculating the interlaboratory variability of manual freezing evaluation revealed significantly different freezing scores in two out of six laboratories. This difference was minimized when all experiments were analyzed with MOVE. Applied to a genetically modified mouse model, MOVE revealed higher fear responses of CB1 deficient mice compared to their wild-type littermates after foreground context fear conditioning. Multi-angle video analysis compared to the single-camera approach reached up to 15% higher accuracy and two fold higher precision. Multidimensional analysis provided by integration of additional data sources further improved the overall result. We conclude that the widespread usage of MOVE could substantially improve the comparability of results from different laboratories.

Dynamic causal models of steady-state responses.

In this paper, we describe a dynamic causal model (DCM) of steady-state responses in electrophysiological data that are summarised in terms of their cross-spectral density. These spectral data-features are generated by a biologically plausible, neural-mass model of coupled electromagnetic sources; where each source comprises three sub-populations. Under linearity and stationarity assumptions, the model's biophysical parameters (e.g., post-synaptic receptor density and time constants) prescribe the cross-spectral density of responses measured directly (e.g., local field potentials) or indirectly through some lead-field (e.g., electroencephalographic and magnetoencephalographic data). Inversion of the ensuing DCM provides conditional probabilities on the synaptic parameters of intrinsic and extrinsic connections in the underlying neuronal network. This means we can make inferences about synaptic physiology, as well as changes induced by pharmacological or behavioural manipulations, using the cross-spectral density of invasive or non-invasive electrophysiological recordings. In this paper, we focus on the form of the model, its inversion and validation using synthetic and real data. We conclude with an illustrative application to multi-channel local field potential data acquired during a learning experiment in mice.

Axonal connections from posterior paralaminar thalamic neurons to basomedial amygdaloid projection neurons to the lateral entorhinal cortex in rats.

Stimulation of amygdaloid nuclei and emotionally relevant stimuli are known to influence the induction and maintenance of long-term potentiation in the hippocampal formation and the formation of long-term declarative memories. Because the thalamic projection from the posterior paralaminar thalamic nuclei is an important sensory afferent projection to amygdaloid nuclei mediating the fast acquisition of fear-potentiated behavior, we were interested in verifying whether this projection establishes synaptic contacts on amygdala neurons that project to the hippocampal formation. Thalamic afferents were labeled with the anterograde tracer Phaseolus vulgaris leucoagglutinin and amygdalo-hippocampal neurons were identified by injection of the retrograde tracer Fluorogold into the lateral entorhinal cortex. A massive overlap of both projection systems was observed especially in the anterior basomedial nucleus of the amygdala. Light microscopic examination revealed that single anterogradely labeled boutons were in close apposition to retrogradely labeled neurons suggesting synaptic contacts. The occurrence of such synaptic contacts was confirmed with electron microscopy. However, despite the massive overlap of anterogradely labeled axons and retrogradely labeled neurons observed at the light microscopic level, electron microscopy revealed that only 10% of all labeled profiles make direct contacts on each other; anterogradely labeled boutons predominantly contacted unlabeled profiles but synapses with direct contact between labeled profiles were rare. Altogether the findings demonstrate that the thalamic connection with the basomedial nucleus of the amygdala may represent an anatomical substrate for modulating amygdala output to the hippocampal formation.

Contribution of intralaminar thalamic nuclei to spike-and-wave-discharges during spontaneous seizures in a genetic rat model of absence epilepsy.

In an epileptic rat model of generalized absence epilepsies, the genetic absence epilepsy rats from Strasbourg (GAERS), simultaneous recordings of bilateral epidural electroencephalogram (EEG) of the prefrontal cortex and unit activity of neurons in the intralaminar centrolateral (CL) and paracentral thalamic nucleus (PC) were performed under neurolept-anaesthesia (fentanyl-dehydrobenzperidol analgesia). Spike-and-wave (SW) seizures in these rats are characterized by generalized 7-10 Hz spike-and-wave discharges (SWDs) on the EEG. All neurons recorded in intralaminar thalamic nuclei during spontaneous SWDs showed high-frequency (average 368 Hz, range 200-500 Hz), burst-like activity, which occurred in a highly synchronized fashion with every SWD or with alternating SWD-complexes. Burst discharges in intralaminar neurons were delayed by 13.1 ms (CL) and 12.7 ms (PC), with respect to the spike component of a given SWD on the EEG, whereas burst discharges in the ventrobasal thalamus (VB) and in the rostral nucleus reticularis thalami (rRT) preceded the spike component by 17.8 ms and 8.3 ms, respectively. The onset of SWDs on the EEG was preceded by a tonic firing pattern (20-50 Hz) in about one third of CL and PC neurons. Microiontophoretic application of the gamma-aminobutyric acid (GABA)A receptor antagonist bicuculline aggravated, whereas, the glutamate receptor antagonists DNQX and APV dampened, SWD-related discharges in PC and CL; the GABAB receptor antagonist CGP 35347 had no measurable effect. These data indicate that intrathalamic nuclei are recruited rhythmically during SWDs, through mechanisms that seem to rely on a delayed glutamatergic excitation modulated by GABAergic influences, rather than a GABA-mediated rebound burst activity typical of relay cells. The finding of a temporal delay of SWD-related activity in intrathalamic, compared with "specific" thalamic relay nuclei, does not support the notion of a leading or pacemaker role in SWD generation. It is, however, rather suggestive of a function of intrathalamic neurons during synchronization and maintenance of neuronal oscillations, and these intrathalamic neurons may be recruited through glutamatergic corticofugal inputs.

Reinforcement of early long-term potentiation (early-LTP) in dentate gyrus by stimulation of the basolateral amygdala: heterosynaptic induction mechanisms of late-LTP.

The basolateral amygdala (BLA) can influence distinct learning and memory formation. Hippocampal long-term potentiation (LTP), the most prominent cellular model of memory formation, can be modulated by stimulation of the BLA in its induction and early maintenance. However, it is not known how the late maintenance of LTP beyond its initial phases might be affected. Behavioral stimuli have been shown to result in a reinforcement of a transient early-LTP into a lasting potentiation. Here we show that BLA stimulation mimics the behavioral effects on early-LTP in freely moving rats when the BLA is activated within a time window of 30 min before or after tetanization of the perforant path. The reinforcement of LTP was blocked by inhibitors of muscarinergic and beta-adrenergic but not dopaminergic receptors and was dependent on translation. Through these heterosynaptic associative interactions, hippocampal sensory information can be stabilized by amygdaloidal influences.

Cellular processes in the amygdala: gates to emotional memory?

The amygdala is considered a core structure of the so-called limbic system and has been implicated in a variety of functions, including emotional interpretation of sensory information, emotional arousal, emotional memory, fear and anxiety, and related clinical disorders. Despite the clinical and functional importance of the amygdala, it is only recently that some general principles of intra-amygdaloid mechanisms of signal processing that are relevant for fear behavior and memory have emerged from behavioral, anatomical, electrophysiological, and neurochemical studies performed in the amygdala of various mammalian species in vivo, in situ and in vitro.

Relations between cortical and thalamic cellular activities during absence seizures in rats.

In a rat model of generalized absence epilepsies (Genetic Absence Epilepsy Rats from Strasbourg, GAERS), multiunit activity was recorded simultaneously at different sites of the thalamocortical system under neurolept anaesthesia (fentanyl-droperidol). Under these conditions, bilaterally synchronized spike-and-wave-discharges (SWDs) occurred spontaneously on the electroencephalogram (EEG) that were in principle identical to those reported earlier from unanaesthetized preparations. The generation of SWDs on the EEG was associated with spike-concurrent, rhythmic burst-like activity in (mono-)synaptically connected regions of specific (somatosensory) thalamic regions and layers IVN of the somatosensory cortex, and the reticular thalamic nucleus. Precursor activity was typically recorded in cortical units, concomitant with 'embryonic' SW seizures on the EEG, before the paroxysm was evident on the gross EEG and in the thalamus. On average, SWD-correlated activity in layers IVN of the somatosensory cortex started significantly earlier than correlated burst-like firing in reticular and in ventrobasal thalamic neurons. Cellular peak firing in thalamus and cortex during bilaterally synchronized SWDs was related to the spike component on the gross EEG with the temporal rank order ventroposteromedial > ventrolateral > or = ventroposterolateral thalamic > > rostral reticular thalamic nuclei > or = cortex (layers IVN) = caudal reticular thalamic nucleus. A spike-related depression and wave-related increase in firing was recorded in anteroventral ventrolateral thalamic areas, presumably reflecting their peculiar anatomical arrangement within the thalamus. These results from an in vivo preparation with intact synaptic connections that spontaneously produces SWDs indicate that SWDs spread within the thalamocortical network, involving short and long delays. The order of concurrent rhythmic firing observed in thalamocortical circuits during SW seizures are supportive of the hypothesis that the processes of rhythmogenesis recruit local thalamic networks, while cortical mechanisms appear to synchronize rhythmic activities on a larger spatiotemporal scale, thereby providing an important contribution to the generalization of epileptiform activity and expression of SWDs on the EEG.

A post-tetanic time window for the reinforcement of long-term potentiation by appetitive and aversive stimuli.

Current theories on the encoding and storage of information in the brain commonly suppose that a short-term memory is converted into a lasting one; thus, it becomes consolidated over time. Within a finite period after training, such a short-term memory can be reinforced by behavioral and humoral stimuli. We have found that, long-term potentiation (LTP), a likely candidate for a memory-encoding mechanism at the cellular level, displays similar features. LTP in the dentate gyrus of freely moving rats was reinforced after its induction by appetitive and aversive stimuli. The efficacy of these stimuli terminates about 1 h after tetanization, which may reflect the time constants of the mechanisms underlying the consolidation that takes place. The reinforcement by appetitive and aversive stimulation was blocked by the beta-adrenergic antagonist propranolol, implicating norepinephrine in the underlying cellular processes.

Ultrastructural distribution of NADPH-diaphorase in the normal hippocampus and after long-term potentiation.

The distribution of the enzyme nitric oxide synthase (NOS) was investigated at the ultrastructural level in synaptic structures of the hippocampal formation in relation to long-term potentiation (LTP), based on the histochemical NADPH-diaphorase (NADPH-d) staining with the tetrazolium salt BSPT. BSPT-formazan, the osmiophilic reaction product, was found to be selectively distributed and predominantly attached to membranes of the endoplasmic reticulum. In synaptic regions mainly the presynaptic sides showed labeling. Although several groups have demonstrated a principal involvement of NO in the LTP-mechanism, we found only a low, statistically insignificant increase in NADPH-d stained presynaptic areas of the dentate gyrus, where LTP was evoked. Postsynaptic elements also did not show any noticeable differences. Based on the present results, the predominantly presynaptic localization of NOS should be preferably considered in models describing a functional role of NO in LTP formation, despite the fact that we failed to reveal any indications for an LTP-related change in synaptically located NADPH-d.

Task-relevant late positive component in rats: is it related to hippocampal theta rhythm?

Long-latency components of event-related potentials (like the P300 or P3) correlate with the ability of subjects to detect and process unexpected, novel or task-relevant events. Task-relevant late positive components were recorded in the neocortex and hippocampus of rats performing an auditory discrimination task, similar to the "odd-ball" paradigm used in human experiments. Surface and depth electrodes were implanted in anaesthetized rats at frontal, temporal and anterior occipital neocortical regions and the hippocampus. After recovery from surgery rats were trained to discriminate two auditory signals, a frequent irrelevant tone and a rare tone related to water reward. In response to the task-relevant tone but not the irrelevant tone, P300-like late positive components (mean latency of 274 ms) were recorded throughout the surface of the neocortex. The largest amplitudes were found at the anterior occipital cortex situated above the hippocampal CA1 region. The amplitude of the task-relevant positive component increased further with cortical depth without reversing its polarity. An amplitude maximum was found in the CA1 region with a polarity reversal at the pyramidal cell layer and the largest negative amplitude in stratum radiatum. Power spectra of differences between responses evoked by task-relevant tones and those evoked by irrelevant tones revealed peaks in the theta range (4-12 Hz). It is suggested that the P300-like component in rats corresponds to a theta wave out of a burst of hippocampal theta cycles.

Asymptotic hippocampal long-term potentiation in rats does not preclude additional potentiation at later phases.

Hippocampal long-term potentiation may serve as an elementary process underlying certain forms of learning and memory in vertebrates. As is the case with behavioural memory, hippocampal long-term potentiation in the CA1 region and in the dentate gyrus exhibits distinct phases. These comprise a short-term early potentiation which lasts one to three hours and is independent of protein synthesis and is characterized in general by the activation of N-methyl-D-aspartate receptors and protein kinases; and a later, longer lasting phase, which can be separated by inhibitors of protein synthesis. Here, we report that the prior induction of long-term potentiation, both in the dentate gyrus in vivo and in the CA1-region in vitro, precludes further long-term but not short-term potentiation by means of a newly delivered conditioning stimulus to a subset of the same activated synapse population during the early stage (approximately 1-3 hours post tetanus). In contrast, a subsequent, long-lasting potentiation can be induced after the establishment of the late phase of potentiation (> 4 h). Thus, the system preserves the capacity for short-term potentiation immediately after potentiation, but the capacity for the induction of longer lasting plastic changes is recovered only after about four hours. Our results demonstrate that, once long-term potentiation has been established, hippocampal neurons do not lose their capacity for functional plasticity during certain phases of the maintenance of long-term potentiation.

Drinking after water deprivation prolongs "unsaturated" LTP in the dentate gyrus of rats.

The role of different behavioral states was tested with respect to induction and duration of long-term potentiation (LTP), using strong or weak tetanic stimuli of the perforant path input to the dentate gyrus of freely moving male Wistar rats. Recording and stimulating electrodes were chronically implanted into the granule cell layer of dentate gyrus and the perforant path, respectively. The effect of tetanic stimulation during three different behavioral states was compared: (a) highly motivated drinking (HMD): Animals (water deprived before experiment 23 h per day on four consecutive days) were tetanized during drinking; (b) high motivation (HM): Animals were treated as the HMD group but received water 1 h after tetanization; and (c) nondeprived controls (NDC). A strong tetanization (10 bursts, 200 Hz) produced a "saturated" LTP for more than 24 h, which was not affected by the behavioral state. LTP induced by weak tetanization (3 bursts, 200 Hz), which usually declines to baseline after at most 7 h, was clearly prolonged by HMD (persisting for at least 24 h), but facilitated to a lesser extent by HM. We suggest that a psychological component that arises during drinking after water deprivation triggers mechanisms resulting in an overt strengthening of an "unsaturated" LTP after weak tetanization, but without having any effect on a "saturated" LTP induced by strong tetanization.

LTP in hippocampal CA1 of urethane-narcotized rats requires stronger tetanization parameters.

Rats with chronically implanted electrodes in the hippocampal CA1 region were tested in their capacity to express and maintain long-term potentiation (LTP) of the population spike (PS) or of the field excitatory postsynaptic potential (fEPSP). Two different states were compared: a) freely moving animals; b) urethane-anesthetized animals (1 g/kg, IP). We found that a short, high-frequency tetanus (six bursts of 15 pulses; 200 Hz; double-pulse width; interburst interval 10 s) increased PS amplitudes and fEPSP slopes up to 300% in response to test stimuli in double-pulse width; interburst interval 10 s) increased PS amplitudes and fEPSP slopes up to 300% in response to test stimuli in the awake rat. The PS amplitude slowly decreased in time, returning to baseline levels 4 h post-tetanically, whereas the fEPSP slope remained at higher values for 24 h. Urethane injection reduced the fEPSP slope and abolished the PS to normal test pulses. We thus increased the strength of the test stimuli until we again recorded magnitudes of PSs and fEPSPs comparable to those in the awake animal. In conjunction with these stronger stimuli, tetanus-induced LTP was elicited that for the PS was increased in magnitude and prolonged in duration compared to the untreated control group. Although, stronger tetanic stimuli were applied to the narcotized fEPSP group too, no difference was found compared to controls. These results suggest that urethane narcosis influences the sensitivity of CA1 neurons to express LTP. Stronger stimulation was required to induce and maintain a long-lasting potentiation of the fEPSP slope and PS amplitude.

Neuronal transmission of hippocampal CA1 neurones is modulated by corticotropin-like intermediate lobe peptide [CLIP; ACTH(18-39)].

The study was conducted to test whether CLIP [ACTH(18-39)] influences the neuronal transmission and the induction of long-term potentiation (LTP) in the hippocampus. The population spike was recorded in the hippocampal CA1 region of freely moving rats before and after intracerebroventricular (ICV) administration of CLIP in comparison to ACTH and saline (controls). After infusion of CLIP, the population spike amplitude (PSA) rose to about 200% of baseline values. After reaching this level, it was impossible to induce a further increase of PSA by tetanization. However, if the stimulus intensity was reduced to a new baseline level, electrically induced LTP could be observed. There were no significant changes after infusion of ACTH. Our results indicate that the ICV administration of CLIP leads to an enhancement of excitability in the hippocampal CA1 region, which might be independent of LTP.

Central substance P increased blood pressure, heart rate and splanchnic nerve activity in anaesthetized rats without impairment of the baroreflex regulation.

In anaesthetized rats the baroreflex was checked before and 15 min after i.c.v. administration of 10 micrograms SP. The baroreflex was checked indirectly by relating both the reflex prolongation in heart period (inter-beat-interval: IBI) and the reflex inhibition of SNA to a pharmacologically induced BP rise. After i.c.v. administration of SP (n = 10) the resting values of the BP increased significantly from 73 +/- 16 mm Hg to 86 +/- 9 mm Hg (diastolic pressure) and from 98 +/- 20 mm Hg to 113 +/- 14 mm Hg (systolic pressure) whilst in the control group (n = 14) the BP remained constant (63 +/- 9 vs 63 +/- 7 mm Hg diastolic pressure and 106 +/- 12 vs 106 +/- 9 mm Hg systolic pressure). In the experimental group the resting value in IBI was shortened significantly from 218 +/- 40 ms to 167 +/- 28 ms (controls: 218 +/- 22 ms vs 218 +/- 18 ms) and the SNA (estimated in arbitrary units) rose significantly by about 50% in relation to the reference period before i.c.v. SP (3.31 +/- 0.11 vs 6.27 +/- 0.17 arbitrary units per IBI). In contrast, the baroreflex behaved similarly before and after any treatment, i.e. both the reflex prolongation in IBI (1.34 +/- 0.75 vs 1.39 +/- 0.95 ms/mm Hg) and the reflex inhibition of SNA (0.0312 +/- 0.01 vs 0.0555 +/- 0.015 arbitrary units/mm Hg) caused by that pharmacologically induced BP rise were comparable before and after i.c.v. SP.(ABSTRACT TRUNCATED AT 250 WORDS)