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C57BL/6 mice are commonly used as background strains for genetically modified mice, and little attention is usually paid to the notification of the specific substrain. However, it is known that C57BL/6NCrl (B6N) and C57BL/6JOlaHsd (B6JOla) mice differ in the course of extinction of conditioned fear (Stiedl O, Radulovic J, Lohmann R, Birkenfeld K, Palve M, Kammermeier J, et al. Strain and substrain differences in context- and tone-dependent fear conditioning of inbred mice. Behav Brain Res 1999;104:1-12), as well as in the expression of alpha-synuclein (Specht CG, Schoepfer R. Deletion of the alpha-synuclein locus in a subpopulation of C57BL/6J inbred mice. BMC Neurosci 2001;2:11). We tested for a causal relationship between the two findings by employing B6N (expressing alpha-synuclein), B6JOla (not expressing alpha-syn) and the third strain C57BL/6JCrl (B6Jax, expressing alpha-syn). We show that alpha-syn does not account for differences in extinction in a fear conditioning task, as its expression did not covary with the decrease of freezing on repeated non-reinforced tone and context exposure in the three strains: B6Jax exhibited fastest extinction followed by B6JOla. In contrast, B6N showed persistent fear over the course of extinction training. The differences in extinction between B6JOla and B6N were unrelated to sensorimotor processing (pain threshold and basal tone reaction) and innate fear (light-dark test). However, B6Jax displayed less innate fear than B6JOla and B6N. Our results of marked differences in innate and conditioned fear in three B6 substrains illustrate the necessity of a strict adherence to an exact mouse strain nomenclature.

Various lesion studies demonstrated that trace but not delay fear conditioning requires an intact hippocampal formation. Our present study examined the role of NMDA receptor activation and protein synthesis within the dorsal hippocampus for acquisition of fear memories following trace (5-s trace) and delay conditioning. To this end male C57BL/6JOlaHsd mice were chronically implanted with guide cannulae targeting the dorsal hippocampus. Fifteen minutes before conditioning mice received a bilateral intrahippocampal injection of either the NMDA receptor antagonist AP5 (0.5 or 1 microg per 0.5 microl per side) or of anisomycin, an inhibitor of protein synthesis (62.5 microg per 0.5 microl per side). Control mice were treated with vehicle (Ringer's solution). Blocking NMDA receptors before trace but not delay conditioning dose-dependently attenuated the freezing response to the tone as assessed 24 h after conditioning. The same findings were obtained after blocking protein synthesis within the dorsal hippocampus. These data indicate that the hippocampus shows synaptic plasticity during trace conditioning that requires an activation of NMDA receptors and protein synthesis as prerequisites for the acquisition of fear memory.

NCAM, a neural cell adhesion molecule of the immunoglobulin superfamily, is involved in neuronal migration and differentiation, axon outgrowth and fasciculation, and synaptic plasticity. To dissociate the functional roles of NCAM in the adult brain from developmental abnormalities, we generated a mutant in which the NCAM gene is inactivated by cre-recombinase under the control of the calcium-calmodulin-dependent kinase II promoter, resulting in reduction of NCAM expression predominantly in the hippocampus. This mutant (NCAMff+) did not show the overt morphological and behavioral abnormalities previously observed in constitutive NCAM-deficient (NCAM-/-) mice. However, similar to the NCAM-/- mouse, a reduction in long-term potentiation (LTP) in the CA1 region of the hippocampus was revealed. Long-term depression was also abolished in NCAMff+ mice. The deficit in LTP could be rescued by elevation of extracellular Ca2+concentrations from 1.5 or 2.0 to 2.5 mm, suggesting an involvement of NCAM in regulation of Ca2+-dependent signaling during LTP. Contrary to the NCAM-/- mouse, LTP in the CA3 region was normal, consistent with normal mossy fiber lamination in NCAMff+ as opposed to abnormal lamination in NCAM-/- mice. NCAMff+ mutants did not show general deficits in short- and long-term memory in global landmark navigation in the water maze but were delayed in the acquisition of precise spatial orientation, a deficit that could be overcome by training. Thus, mice conditionally deficient in hippocampal NCAM expression in the adult share certain abnormalities characteristic of NCAM-/- mice, highlighting the role of NCAM in the regulation of synaptic plasticity in the CA1 region.

Forced ethanol drinking for several days and application of ethanol to cell cultures changes expression levels of various NMDA receptor subunits in rodents. We investigated the influence of long-term voluntary ethanol consumption of moderate ethanol doses on polypeptide levels of the NMDA receptor subunit 2B (NR2B) in various forebrain regions of rats and found a mild increase selectively in the frontal cortex. This result is in accordance with and extends findings from studies using high doses of ethanol for a short period and suggests that the NR2B might be a potential target for an effective treatment of alcoholic patients.

Magnocellular vasopressinergic and oxytocinergic neurons of the hypothalamic supraoptic (SON) and paraventricular nuclei comprise the hypothalamic-neurohypophysial system, which is crucially involved in the regulation of body fluid and electrolyte homeostasis. However, still controversial is to what extent the same system influences the secretion of adrenocorticotropic hormone (ACTH) from the adenohypophysis. Therefore, we selectively stimulated magnocellular neurons of the SON of conscious male Wistar rats via retrodialysis. As expected, dialysis of the SON with hypertonic medium increased both the release of vasopressin within the SON and the secretion of vasopressin and oxytocin into the systemic blood circulation. This activation of the hypothalamic-neurohypophysial system was accompanied by a fivefold increase in plasma ACTH concentration. This effect was observed only if the tip of the microdialysis probe was within the SON. Intravenous infusion of the vasopressin V1 receptor antagonist d(CH2)5Tyr(Me)AVP significantly attenuated the effects of local osmotic stimulation of the SON on ACTH secretion. In contrast, administration of the same antagonist directly into the SON significantly enhanced the osmotically stimulated secretion of ACTH and corticosterone, primarily by delaying the restoration of the hormone secretion to prestimulation levels. We conclude from these findings that vasopressin from the hypothalamic-neurohypophysial system participates in the regulation of the hormonal stress response in a counterbalanced manner at the level of the SON and the adenohypophysis.

Previously, we have demonstrated that forced swimming triggers the release of arginine vasopressin (AVP) within the septum of rats, where AVP modulates stress-coping strategies. The present study was designed to examine the effects of forced swimming on the release of AVP within the amygdala. Therefore, adult male Wistar rats were chronically implanted with a microdialysis probe aimed at the amygdala to monitor the local release of AVP under both resting and stress conditions. A 10-min forced swimming session caused a significant increase in the extracellular AVP concentration (to 366 +/- 90% of baseline; P0.05) within this brain area. In a subsequent experiment we investigated the physiological impact of the stressor-induced release of AVP by administrating the AVP V1 receptor antagonist d(CH2)5Tyr(Me)AVP into the amygdala via inverse microdialysis. Bilateral antagonist treatment modulated the behavioural response acutely by increasing the time the animals spent struggling and by reducing the time the animals floated. Our results demonstrate a significant activation of the vasopressinergic system within the amygdala in response to forced swimming. AVP released within the amygdala seems to be involved in the generation of passive coping strategies in stressful situations. Taken together with previous findings the results of the present study suggest that AVP is released within septum and amygdala to balance the behavioural response during forced swimming.

Many cell adhesion molecules express the HNK-1 carbohydrate involved in formation and functioning of synapses. To assess its role in learning, we injected the monoclonal HNK-1 antibody or nonimmune IgG into the hippocampus of C57BL/6J mice 1 h after training in a step-down avoidance task. In animals treated with the HNK-1 antibody, latencies of step down in a recall session 48 h after injection did not change compared to training values and were significantly shorter versus IgG-treated controls, which acquired the task normally. Similar differences between the two treatments were also observed after a stronger training protocol in a step-down avoidance paradigm. The HNK-1 antibody was effective only when injected 1 h, but not 48 h after training, thus affecting memory consolidation but not memory recall itself. The HNK-1 antibody impaired memory also in tenascin-R knock-out mice, indicating that extracellular matrix molecule tenascin-R, one of the carriers of the HNK-1epitope in the hippocampus, does not mediate the function of the HNK-1 carbohydrate in this task. Our observations show that the HNK-1 carbohydrate is critically involved in memory consolidation in hippocampus-dependent learning in mammals.

The naturally occurring social conflict situation to be confronted with an aggressive dominant conspecific was used to study the effects of emotional stress on the release of oxytocin (OXT) and arginine vasopressin (AVP) within the mediolateral septum of the rat brain. Male rats were chronically implanted with a microdialysis probe into this brain area. Local release patterns of both, OXT and AVP were monitored in response to a 30 min social defeat. Social defeat caused a significant increase in the release of OXT (to 254%+/-43%, P

Previous experiments have shown that arginine vasopressin (AVP) released within the septal brain area of adult male rats in response to defined stressor exposure is involved in emotionality-related behavior. We report here that a 10-min forced swimming session stimulated the release of glutamate, aspartate, arginine, γ-aminobutyric acid (GABA) and taurine but not alanine and serine in the medio-lateral part of this brain structure. Local administration of the AVP V1 receptor antagonist d (CH 2 ) 5 Tyr(Me)AVP by inverse microdialysis caused a significant increase in the concentration of taurine in microdialysates under resting conditions that was further potentiated during forced swimming. In contrast, the release of alanine, arginine, GABA and serine was unaffected by antagonist treatment. Taken together with previous data, our results suggest that the effects of intraseptally released AVP on stress-coping strategies might be mediated at least in part via its influence on the local release of taurine.

In addition to its peripheral secretion from the neurohypophysis, the neuropeptide vasopressin (VP) is released within the mammalian brain from probably all parts of the neuronal membrane. In particular the development of brain microdialysis in vivo together with blood microdialysis or blood sampling provides the advantage of being able to reliably compare the dynamic release patterns into different compartments of the organism. The central VP release within hypothalamic (e.g., supraoptic, paraventricular and suprachiasmatic nuclei) and limbic (e.g., septum, amygdala) rat brain areas is stimulated by a variety of substances and stressors, including interleukin-1β, social defeat and forced swimming. Furthermore, it is characterized by positive and negative feedback mechanisms and the capacity of the VP system for co-ordinated or independent release, the latter being observed, for example, during social defeat. This emotional stressor, in contrast to exposure to a novel cage, increased VP release within the supraoptic nucleus, but not into plasma. This failure to release VP peripherally could be observed also during forced swimming, despite a dramatic rise in plasma osmolality and a markedly stimulated central release. In another series of experiments we studied the effects of centrally-released VP on cognitive and emotional aspects of behavior using reverse microdialysis for antagonist administration during the behavioral tests and antisense targeting to downregulate either VP or its local V 1 receptor subtype. In this way, centrally (in particular septally) released VP could be shown to be causally involved in short-term memory and anxiety-related behavior. Furthermore, VP release within the hypothalamic paraventricular nucleus is likely to provide a negative tonus on the activity of the hypothalamic-pituitary-adrenocortical axis. This neuroendocrine effect together with cognitive, emotional and immunological effects of centrally released VP is thought to be essential to ensure adequate behavior of the animal during challenging situations and to contribute to the development of efficient coping strategies.

To investigate the effects of an ethologically-relevant stressor on central and peripheral release of arginine vasopressin and oxytocin, we forced adult male Wistar rats to swim for 10 min and simultaneously measured the release of the two peptides (i) within the hypothalamic supraoptic and paraventricular nuclei (by means of the microdialysis technique) and (ii) into the blood (by chronically-implanted jugular venous catheters). Forced swimming caused a significant rise in the release of arginine vasopressin and oxytocin within both the supraoptic nuclei (four-fold and three-fold, respectively) and the paraventricular nuclei (three-fold and four- to five-fold, respectively). Release patterns measured before, during and after repeated stress exposure on three consecutive days indicated that, at the level of the hypothalamus, the two neuropeptides are critically involved in the rats' stress response in a peptide-, locus- and stress-specific manner. Particularly, despite a general reduction of the recovery of the microdialysis probes over the time, the release of arginine vasopressin within the paraventricular nuclei and of oxytocin within the supraoptic nuclei tended to increase upon repeated stress exposure. Measurement of plasma peptide concentrations revealed that the central release of oxytocin was accompanied by a secretion of this peptide into the systemic circulation. In contrast, arginine vasopressin, assayed in the same plasma samples, failed to respond to the stressor. The latter finding is consistent with a dissociated release of the neuropeptide from different parts of a single neuron (soma/dendrites vs axon terminals). It provides evidence that under physiological conditions plasma hormone levels do not necessarily reflect the secretory activity of central components of the respective neuropeptidergic system.

The hypothalamic suprachiasmatic nucleus (SCN) is the predominant pacemaker of the mammalian brain that generates and controls circadian rhythms of various endocrine and behavioral processes. Different lines of evidence suggest that stress interferes with the maintenance of such rhythms. As a first approach to investigate whether the neuropeptide arginine vasopressin (AVP), which shows circadian rhythms of synthesis and release within the SCN, might contribute to this stress-induced alterations in circadian rhythms, we monitored acute effects of swim stress on the intra-SCN release of AVP in male rats by means of the microdialysis technique. A 10-min forced swimming session triggered a marked but relatively short-lasting increase in the intranuclear release of AVP (to approx. 440%). This effect was restricted to the area containing predominantly somata and dendrites of vasopressinergic neurons, since no changes in AVP release could be measured in one of their major projection areas, the nucleus of the dorsomedial hypothalamus. Our data provide evidence that the amount of AVP released within the SCN can vary widely not only in accordance with AVP's intrinsically regulated circadian rhythm but also in response to a physiologically relevant stressor. In this way, the neuropeptide may contribute to the regulation of endocrine and behavioral rhythms particularly in challenging situations associated with resettings of the endogenous clock.

It is important to realize that neuropeptides such as vasopressin (AVP) and oxytocin (OXT) only become biologically active after their release into the extracellular space. To measure this release, the majority of previous studies have relied on post-mortem methods, which only offer limited snapshots at selected time frames in a dynamic process. Thus, although measurements of AVP and OXT content in brain tissue, of neuropeptide concentrations in the cerebrospinal fluid and attempts to reflect central fluctuations in AVP and OXT just by determining plasma peptide levels have all substantially contributed to our present knowledge, they have to be interpreted with extreme caution.

I.A. Ionescu1 °, J. Dine2, J. Stepan2, Y.C. Yen3, L. Herrmann1, F. Holsboer4, C.T. Wotjak3, R. Landgraf5, M. Eder2, U. Schmidt1. 1Max-Planck-Institut fur Psychiatrie, RG Molecular Psychotraumatology, Munchen, Germany; 2Max-Planck-Institut fur Psychiatrie, RG Neuronal Networks Dynamics, Munchen, Germany; 3Max-Planck-Institut fur Psychiatrie, RG Neuronal Plasticity, Munchen, Germany; 4Max-Planck-Institut fur Psychiatrie, MPI of Psychiatry, Munchen, Germany; 5Max-Planck-Institut fur Psychiatrie, RG Behavioral Neuroendocrinology, Munchen, Germany

Arginine vasopressin (AVP) or its V1 receptor antagonist d(CH2)5Tyr(Me)AVP was administered directly into the septal brain area of adult male rats by means of inverse microdialysis. Immediately after a 30-min dialysis period, during which either approximately 0.25 ng AVP or 5 ng of the V1 antagonist were delivered into the brain tissue, anxiety-related behaviour of the animals was measured on an elevated plus-maze apparatus. While synthetic AVP failed to alter plus-maze behaviour compared to vehicle-treated controls, animals treated with the V1 receptor antagonist made more entries into (P < 0.01) and spent more time on the open arms (P < 0.05), indicating reduced anxiety. Since administration of neither AVP nor the V1 antagonist significantly influenced general locomotor activity of the rats on the plus-maze and in an open field, these data point towards a critical involvement of intraseptally released AVP in the emotional evaluation of novel situations.

Impaired cognitive function and enhanced activity of the hypothalamic-pituitary-adrenocortical system are among the cardinal symptoms of major depression in humans that resolve after successful antidepressant treatment. We used a transgenic mouse model expressing antisense RNA complementary to that of glucocorticoid receptor (GR) mRNA to test the hypothesis that reduced GR function can cause these clinical disturbances. The transgenic mice show profound behavioural changes in a number of animal tests that are indicative of cognitive impairment. These mice also have elevated plasma corticotropin concentrations in response to stress. After long-term treatment with moclobemide, a reversible inhibitor of monoamine oxidase type A that acts clinically as an antidepressant, both the behavioural deficits and the hormonal alterations disappeared. These observations suggest that a transgenic mouse with GR dysfunction may be a useful model for investigation of drug effects on the cognitive and neuroendocrine aspects of depression.

Diurnal changes in the intranuclear release of vasopressin (VP) and oxytocin (OT) in the suprachiasmatic (SCN), paraventricular (PVN) and supraoptic nuclei (SON) of the rat were studied by means of brain microdialysis. A significant diurnal variation in VP release in the SCN was detected, with the highest levels occurring during midday and a trough around midnight. OT release from the SCN was below detection limit. The release of neither of these neurohypophysial peptides showed diurnal variations within the PVN or SON.

This study examined the effects of direct osmotic stimulation of the hypothalamic paraventricular nucleus (PVN) on grooming behavior of adult male rats. Animals chronically fitted with a microdialysis probe in the right PVN responded to dialysis with hypertonic (containing 1 M NaCl) artificial cerebrospinal fluid (aCSF) with grooming behavior (to approx. 500%, P

Fear memory formation critically relies on the function of the lateral amygdala (LA) and changes in synaptic transmission of its sensory inputs. There is evidence that signaling through NO/cGMP in the amygdala contributes to these processes [1-3]. The cGMP-dependent protein kinases (cGK) type I and II serve as universal effectors of the NO/cGMP signaling cascade. Recently, we established a functional role of cGKI for synaptic plasticity and fear memory consolidation in the amygdala. First, we demonstrated that the cGKIβ isoform is highly expressed in the LA. Furthermore, we showed that cGKI-deficient mice revealed deficits in amygdala-dependent fear memory consolidation. In contrast, short-term memory and hippocampus-dependent memory was intact in these mice. In line with the behavioral phenotype of impaired auditory long-term memory, we found reduced long-term potentiation (LTP) in the LA of cGKI-deficient mice, which is considered as a cellular mechanism for learning induced synaptic plasticity. Unlike cGKI-deficient mice, cGKII-knockout mice lack these phenotypes. These results clearly suggest a crucial role of cGKI for consolidation of amygdala-dependent fear memory, a process by which newly learned information is stabilized into long-term memory (LTM). It is a well known fact that memory consolidation strictly depends on transcriptional and translational processes which is also proved essential for amygdala-dependent fear memory consolidation [4,5]. A functional link between cGKI activity and gene transcription could already be demonstrated in vitro [6]. The upcoming question is if cGKI-induced transcription also takes place in amygdala neurons recruited during memory formation. Preliminary data indicate a role for cGKI in cAMP-response element binding protein (CREB) signaling in the LA, a prominent transcription factor involved in learning and memory.

Marijuana and its main psychotropic ingredient Δ9-tetrahydrocannabinol (THC) exert a plethora of psychoactive effects through the activation of the neuronal cannabinoid receptor type 1 (CB1), which is expressed by different neuronal subpopulations in the central nervous system. The exact neuroanatomical substrates underlying each effect of THC are, however, not known. We tested locomotor, hypothermic, analgesic, and cataleptic effects of THC in conditional knockout mouse lines, which lack the expression of CB1 in different neuronal subpopulations, including principal brain neurons, GABAergic neurons (those that release γ aminobutyric acid), cortical glutamatergic neurons, and neurons expressing the dopamine receptor D1, respectively. Surprisingly, mice lacking CB1 in GABAergic neurons responded to THC similarly as wild-type littermates did, whereas deletion of the receptor in all principal neurons abolished or strongly reduced the behavioural and autonomic responses to the drug. Moreover, locomotor and hypothermic effects of THC depend on cortical glutamatergic neurons, whereas the deletion of CB1 from the majority of striatal neurons and a subpopulation of cortical glutamatergic neurons blocked the cataleptic effect of the drug. These data show that several important pharmacological actions of THC do not depend on functional expression of CB1 on GABAergic interneurons, but on other neuronal populations, and pave the way to a refined interpretation of the pharmacological effects of cannabinoids on neuronal functions., Author Summary Marijuana and its main psychoactive component, THC, exert a plethora of behavioural and autonomic effects on humans and animals. Some of these effects are the cause of the widespread illicit use of marijuana, while others might be involved in the potential therapeutic use of this drug for the treatment of several neuronal disorders. The great majority of these effects of THC are mediated by cannabinoid receptor type 1 (CB1), which is abundantly expressed in the central nervous system. The exact anatomical and neuronal substrates of each action are, however, not clearly known at the moment. We addressed this issue by using an advanced genetic approach. Control and conditional mutant mice, lacking CB1 expression in defined neuronal subpopulations but not in others, were treated with THC, and typical effects of the drug on motor behaviour, pain, and thermal sensation were scored. Our results show that different neuronal subpopulations mediate different effects of THC and could lead to a refined interpretation of the pharmacological actions of cannabinoids. Moreover, these data might provide the rationale for the development of drugs capable of selectively activating CB1 in specific neuronal subpopulations, thereby better exploiting cannabinoids' potential therapeutic properties., Advanced genetics techniques reveal that the effects of cannabinoids on motor behavior and thermal and pain sensation are mediated by distinct populations of glutamatergic and dopaminergic neurons, not GABAergic neurons as previously thought.