Electrolytic post-training lesions of the bed nucleus of the stria terminalis block startle potentiation in a cued fear conditioning procedure.

Existing neuroanatomical models argue that the bed nucleus of the stria terminalis (BST) principally mediates sustained, long-lasting fear or anxiety responses, but not shorter, phasic fear responses, although recent studies paint a more complex picture. In the current study, we evaluated the effect of post-training electrolytic BST lesions in a cued fear conditioning protocol with relatively short (10 s) tones. We hypothesized that the BST would not play a crucial role in the expression of fear upon re-exposure to the conditioned tones. Tone fear memory was primarily assessed through fear-potentiated startle. In addition, freezing measurements were obtained throughout the test sessions. In a series of three experiments, we explored the effects of BST lesions, taking into consideration contextual influences on cued fear expression (using (dis)similar training and test contexts) and temporal involvement of the BST in the consolidation of fear learning (lesion induction 3 or 27 h after fear conditioning). In all three experiments, we found that post-training electrolytic lesions of the BST significantly reduced fear-potentiated startle, implying a deficit in differentiation between tone and context. These results are surprising and challenge the general consensus on the lack of BST involvement in cued fear. We discuss several alternative explanations that may account for these unexpected findings.

The bed nucleus of the stria terminalis is critical for anxiety-related behavior evoked by CO2 and acidosis.

Carbon dioxide (CO2) inhalation lowers brain pH and induces anxiety, fear, and panic responses in humans. In mice, CO2 produces freezing and avoidance behavior that has been suggested to depend on the amygdala. However, a recent study in humans with bilateral amygdala lesions revealed that CO2 can trigger fear and panic even in the absence of amygdalae, suggesting the importance of extra-amygdalar brain structures. Because the bed nucleus of the stria terminalis (BNST) contributes to fear- and anxiety-related behaviors and expresses acid-sensing ion channel-1A (ASIC1A), we hypothesized that the BNST plays an important role in CO2-evoked fear-related behaviors in mice. We found that BNST lesions decreased both CO2-evoked freezing and CO2-conditioned place avoidance. In addition, we found that CO2 inhalation caused BNST acidosis and that acidosis was sufficient to depolarize BNST neurons and induce freezing behavior; both responses depended on ASIC1A. Finally, disrupting Asic1a specifically in the BNST reduced CO2-evoked freezing, whereas virus-vector-mediated expression of ASIC1A in the BNST of Asic1a(-/-) and Asic1a(+/+) mice increased CO2-evoked freezing. Together, these findings identify the BNST as an extra-amygdalar fear circuit structure important in CO2-evoked fear-related behavior.

Excitotoxic lesions of the bed nucleus of the stria terminalis (BNST) attenuate the effects of repeated stress on weight gain: evidence for the recruitment of BNST activity by repeated, but not acute, stress.

Exposure to repeated stress can lead to diverse and widespread behavioral consequences, including reduction in food and water intake and subsequent diminution in weight gain. Many reports have suggested that repeated stress substantially alters the neurochemistry, morphology and physiology of neurons within the bed nucleus of the stria terminalis (BNST). Here we investigate the role of the BNST in mediating the reduced weight gain observed during repeated stress. Rats exposed to a one-week variate stress paradigm exhibited a reduction in weight gain over the course of the 7-day paradigm. Excitotoxic lesions to a subregion of the anterolateral BNST containing the oval nucleus had no effects early in the 7-day paradigm, but significantly attenuated the effects of repeated stress on weight gain by the last day of stress. These data suggest that at least two mechanisms mediate the effects of stress on body weight gain, and that when stressor exposure becomes repeated, the BNST is recruited, worsening the symptoms of stressor exposure.

Recognition of novel objects and their location in rats with selective cholinergic lesion of the medial septum.

The importance of cholinergic neurons projecting from the medial septum (MS) of the basal forebrain to the hippocampus in memory function has been controversial. The aim of this study was to determine whether loss of cholinergic neurons in the MS disrupts object and/or object location recognition in male Sprague-Dawley rats. Animals received intraseptal injections of either vehicle, or the selective cholinergic immunotoxin 192 IgG-saporin (SAP). 14 days later, rats were tested for novel object recognition (NOR). Twenty-four hours later, these same rats were tested for object location recognition (OLR) (recognition of a familiar object moved to a novel location). Intraseptal injections of SAP produced an 86% decrease in choline acetyltransferase (ChAT) activity in the hippocampus, and a 31% decrease in ChAT activity in the frontal cortex. SAP lesion had no significant effect on NOR, but produced a significant impairment in OLR in these same rats. The results support a role for septo-hippocampal cholinergic projections in memory for the location of objects, but not for novel object recognition.

Timing of impulses from the central amygdala and bed nucleus of the stria terminalis to the brain stem.

The amygdala and bed nucleus of the stria terminalis (BNST) are thought to subserve distinct functions, with the former mediating rapid fear responses to discrete sensory cues and the latter longer "anxiety-like" states in response to diffuse environmental contingencies. However, these structures are reciprocally connected and their projection sites overlap extensively. To shed light on the significance of BNST-amygdala connections, we compared the antidromic response latencies of BNST and central amygdala (CE) neurons to brain stem stimulation. Whereas the frequency distribution of latencies was unimodal in BNST neurons (approximately 10-ms mode), that of CE neurons was bimodal (approximately 10- and approximately 30-ms modes). However, after stria terminalis (ST) lesions, only short-latency antidromic responses were observed, suggesting that CE axons with long conduction times course through the ST. Compared with the direct route, the ST greatly lengthens the path of CE axons to the brain stem, an apparently disadvantageous arrangement. Because BNST and CE share major excitatory basolateral amygdala (BL) inputs, lengthening the path of CE axons might allow synchronization of BNST and CE impulses to brain stem when activated by BL. To test this, we applied electrical BL stimuli and compared orthodromic response latencies in CE and BNST neurons. The latency difference between CE and BNST neurons to BL stimuli approximated that seen between the antidromic responses of BNST cells and CE neurons with long conduction times. These results point to a hitherto unsuspected level of temporal coordination between the inputs and outputs of CE and BNST neurons, supporting the idea of shared functions.

Involvement of the bed nucleus of the stria terminalis in the negative affective component of visceral and somatic pain in rats.

Using a conditioned place paradigm, we examined the involvement of the bed nucleus of the stria terminalis (BST) in the negative affective component of visceral and somatic pain induced by intraperitoneal acetic acid and intraplantar formalin injections, respectively, in rats. Bilateral BST lesions suppressed both the acetic acid- and formalin-induced conditioned place aversion, suggesting the crucial role of the BST in the negative affective component of visceral and somatic pain.

The medial septum acts through the medial preoptic area for thermoregulation and works with it for sleep regulation.

The chronic changes in sleep-wakefulness (S-W), body temperature (Tb), locomotor activity (LMA) and thermal preference were studied in male Wistar rats after the destruction of neurons in both the medial preoptic area (mPOA) and the medial septum (MS) by intracerebral injection of N-methyl-D-aspartic acid. An increase in the Tb, and a preference for higher ambient temperature (Tamb) of 30 degrees C were observed after the combined lesion of the mPOA and the MS. Similar changes were reported to occur after the lesion that was restricted to the mPOA. But these alterations were in contrast to the decrease in Tb and preference for lower Tamb, observed after the MS lesion. The thermostat of the brain would have been reset at a higher level after the combined lesion, as there was an increase in Tb, along with a preference for a higher Tamb, and an increase in LMA. There was a reduction in the frequency and the duration of the slow wave sleep (SWS) episodes, and a reduction in the frequency of the paradoxical sleep (PS) episodes after the combined lesion. The destruction of the MS neurons was probably responsible for the reduction in the frequency of SWS, whereas the loss of mPOA neurons was responsible for the decrease in the duration of SWS and frequency of PS. It can be suggested that the MS exerts its influence on thermoregulation through the mPOA. However, the MS and the mPOA seem to play independent, but complementary roles in sleep promotion.

Effects of bed nucleus of the stria terminalis lesions on conditioned anxiety: aversive conditioning with long-duration conditional stimuli and reinstatement of extinguished fear.

Four experiments investigated the effects of lesions of the bed nucleus of the stria terminalis (BNST) on conditioned fear and anxiety. Though BNST lesions did not disrupt fear conditioning with a short-duration conditional stimulus (CS; Experiments 1 and 3), the lesion attenuated conditioning with a longer duration CS (Experiments 1 and 2). Experiment 3 found that lesions attenuated reinstatement of extinguished fear, which relies on contextual conditioning. Experiment 4 confirmed that the lesion reduced unconditioned anxiety in an elevated zero maze. The authors suggest that long-duration CSs, whether explicit cues or contexts, evoke anxiety conditioned responses, which are dissociable from fear responses to shorter CSs. Results are consistent with behavioral and anatomical distinctions between fear and anxiety and with a behavior-systems view of defensive conditioning.

Aversive stimulus attenuates impairment of acquisition in a delayed match to position T-maze task caused by a selective lesion of septo-hippocampal cholinergic projections.

Infusion of 192 IgG-saporin (SAP) into the medial septum (MS) of rats selectively destroys cholinergic neurons projecting to the hippocampus and impairs acquisition of a delayed matching to position (DMP) T-maze task. The present study evaluated whether introduction of a mild aversive stimulus 30 min prior to training would attenuate the deficit in DMP acquisition caused by the SAP lesions. Male Sprague-Dawley rats received medial septal infusions of either artificial cerebrospinal fluid or SAP (0.22 microg in 1.0 microl). Fourteen days later, all animals were trained to perform the DMP task. Half of the SAP-treated animals and controls received an intraperitoneal injection of saline each day, 30 min prior to training. Results show that intraperitoneal saline attenuated the impairment in DMP acquisition in SAP lesioned rats. These results suggest that a mild aversive stimulus can attenuate cognitive deficits caused by medial septal cholinergic lesions.

Effects of lesions of the bed nucleus of the stria terminalis, lateral hypothalamus, or insular cortex on conditioned taste aversion and conditioned odor aversion.

The effects of permanent forebrain lesions on conditioned taste aversions (CTAs) and conditioned odor aversions (COAs) were examined in 3 experiments. In Experiment 1, lesions of the bed nucleus of the stria terminalis had no influence on CTA or COA acquisition. Although lesions of the lateral hypothalamus induced severe hypodipsia in Experiment 2, they did not prevent the acquisition of CTAs or COAs. Finally, in Experiment 3, lesions of the insular cortex retarded CTA acquisition but had no influence on COA acquisition. The implications of these findings are discussed with regard to the forebrain influence on parabrachial nucleus function during CTA acquisition.

Behavioral effects of basal forebrain cholinergic lesions in young adult and aging rats.

The interactive effects of age and cholinergic damage were assessed behaviorally in young and middle-aged rats. Rats were lesioned at either 3 or 17 months of age by injection of 192 IgG-saporin immunotoxin into the medial septum and the nucleus basalis magnocellularis, and they were then tested on a range of behavioral tasks: a nonmatching-to-position task in a T-maze, an object-recognition task, an object-location task, and an open-field activity test. Depending on the task used, only an age or a lesion effect was observed, but there was no Age X Lesion interaction. Middle-aged and young rats responded to the cholinergic lesions in the same manner. These results show that in the middle-aged rats in which cholinergic transmission was affected, additional injury to the system was not always accompanied by major cognitive dysfunctions.

Involvement of the bed nucleus of the stria terminalis activated by the central nucleus of the amygdala in the negative affective component of morphine withdrawal in rats.

The central nucleus of the amygdala (Ce) and the bed nucleus of the stria terminalis (BST) are key structures of the extended amygdala, which is suggested to be involved in drug addiction and reward. We have previously reported that the Ce plays a crucial role in the negative affective component of morphine withdrawal. In the present study, we examined the involvement of the neural pathway between the Ce and the BST in the negative affective component of morphine withdrawal in rats. Rats were rendered morphine dependent by s.c. implantation of a 75-mg morphine pellet for 3 days, and morphine withdrawal was precipitated by an i.p. injection of naloxone (0.3 mg/kg). In the place-conditioning paradigm, discrete bilateral excitotoxic lesions of the Ce or the BST significantly reduced naloxone-precipitated morphine withdrawal-induced conditioned place aversion. On the other hand, they had little effect on morphine withdrawal-induced somatic signs. In an immunohistochemical study for c-Fos protein, naloxone-precipitated morphine withdrawal dramatically induced c-Fos-immunoreactive neurons in the capsular part of the Ce, and the lateral and medial divisions of the BST. Bilateral excitotoxic lesion of the Ce reduced the number of morphine withdrawal-induced c-Fos-immunoreactive neurons in the lateral and medial BST, with significant decreases in the posterior, ventral and juxtacapsular parts of lateral division, and anterior part of the medial division, but not in the ventral part of the medial division of the BST. On the other hand, bilateral excitotoxic lesion of the BST had no effect on such c-Fos induction within the capsular part, nor the ventral and medial divisions of the Ce. These results suggest that activation of the BST mediated through the neural pathway from the Ce contributes to the negative affective component of morphine withdrawal.

Effects of hippocampal cholinergic deafferentation on learning strategy selection in a visible platform version of the water maze.

Recent evidence has suggested that the relative levels of acetylcholine (ACh) between brain structures may be an important factor in the choice of behavioral strategy in settings in which either hippocampal or dorsal striatal brain systems can be employed both effectively and independently (McIntyre and Gold. 1999. Soc Neurosci Abs 25:1388). The current investigation used the neurotoxin 192 IgG-saporin to deplete the hippocampus of ACh selectively, while leaving ACh in other brain regions, including dorsal striatum, intact. Rats were then trained on a version of the Morris water maze, in which behavioral strategies attributed to the hippocampus and dorsal striatum are placed in direct competition. It was predicted that rats with hippocampal ACh depletion would display a cue bias. Contrary to this prediction, depleting hippocampal ACh did not bias against and, in fact, promoted use of a hippocampal place strategy in this task, as indicated by choice in competition tests and performance on hidden platform training trials. These data add to a growing literature demonstrating that the septohippocampal cholinergic system is not required for accurate spatial learning and suggest a complex role for basal forebrain projections in processing information about the spatial environment.

Longitudinal axis of the hippocampus: both septal and temporal poles of the hippocampus support water maze spatial learning depending on the training protocol.

It has been suggested previously that 30% sparing of the hippocampus is enough to support spatial learning of a reference memory task in a water maze provided the spared tissue is located septally (Moser et al. 1995, Proc Natl Acad Sci USA 92:9697-9701). Therefore, the temporal hippocampus may not be involved in spatial memory. Place cells are also found in this part of the structure, and it has been suggested that these place cells have larger, less well-tuned place fields than are found in the septal hippocampus. We tested the possibility that the temporal hippocampus might be involved in spatial learning when the animals are required to distinguish between different contexts. Experiment 1 was a replication of the findings reported by Moser et al., using their protocol (8 trials/day, 6 days) and the groups with 20-40% hippocampus spared septally or temporally (volume assessed by quantitative volumetric techniques). In experiment 2, rats with also 20-40% sparing of the hippocampus either septally or temporally were trained in two water maze concurrently (four trials/day/water maze, 8 days). Rats with 20-40% hippocampus spared temporally were able to learn the two water maze tasks normally, and no difference was observed between rats with septal and temporal hippocampus spared across different measures of performance. In experiment 3, rats with 20-40% hippocampus spared septally or temporally were trained in one water maze as in experiment 1, but using a spaced training protocol similar to that of experiment 2 (four trials/day, 8 days). Rats with temporal hippocampus spared developed a preference for the training quadrant and acquired levels of performance indistinguishable from those of rats with septal hippocampus spared. The results suggest that the temporal hippocampus can support the learning of two, but also one, spatial water maze reference memory task, provided the training protocol is adequate.

5,7-DHT-induced hippocampal 5-HT depletion attenuates behavioural deficits produced by 192 IgG-saporin lesions of septal cholinergic neurons in the rat.

Adult Long-Evans male rats sustained injections of 5,7-dihydroxytryptamine into the fimbria-fornix (2.5 microg/side) and the cingular bundle (1.5 microg/side) and/or to intraseptal injections of 192 IgG-saporin (0.4 microg/side) in order to deprive the hippocampus of its serotonergic and cholinergic innervations, respectively. Sham-operated rats were used as controls. The rats were tested for locomotor activity (postoperative days 18, 42 and 65), spontaneous T-maze alternation (days 20-29), beam-walking sensorimotor (days 34-38), water maze (days 53-64) and radial maze (days 80-133) performances. The cholinergic lesions, which decreased the hippocampal concentration of ACh by about 65%, induced nocturnal hyperlocomotion, reduced T-maze alternation, impaired reference-memory in the water maze and working-memory in the radial maze, but had no effect on beam-walking scores and working-memory in the water maze. The serotonergic lesions, which decreased the serotonergic innervation of the hippocampus by about 55%, failed to induce any behavioural deficit. In the group of rats given combined lesions, all deficits produced by the cholinergic lesions were observed, but the nocturnal hyperlocomotion and the working-memory deficits in the radial maze were attenuated significantly. These results suggest that attenuation of the serotonergic tone in the hippocampus may compensate for some dysfunctions subsequent to the loss of cholinergic hippocampal inputs. This observation is in close concordance with data showing that a reduction of the serotonergic tone, by pharmacological activation of somatodendritic 5-HT(1A) receptors on raphe neurons, attenuates the cognitive disturbances produced by the intrahippocampal infusion of the antimuscarinic drug, scopolamine. This work has been presented previously [Serotonin Club/Brain Research Bulletin conference, Serotonin: From Molecule to the Clinic (satellite to the Society for Neuroscience Meeting, New Orleans, USA, November 2-3, 2000)].

Crossed unilateral lesions of temporal lobe structures and cholinergic cell bodies impair visual conditional and object discrimination learning in monkeys.

Monkeys with excitotoxic lesions of the CA1/subiculum region in the right hemisphere and with immunotoxic lesions of the cholinergic cells of the diagonal band in the left hemisphere were impaired on a visual conditional task. In this task, correct choice of one of two objects depends on which of two background fields both objects are presented against, irrespective of the spatial positions of the objects. They were not impaired on simple object or shape discrimination tasks. The pattern of impairments is the same as that seen after bilateral excitotoxic lesions of CA1/subiculum, implying that the diagonal band lesion disables the ipsilateral CA1/subiculum. It also argues that CA1/subiculum, sustained by its cholinergic input, is necessary for some forms of nonspatial conditional learning. Addition of an inferotemporal (IT) cortical ablation to the left hemisphere did not affect simple visual discrimination learning, although all the monkeys then failed to learn a new visual conditional task. This demonstrates that intact IT cortex in only one hemisphere is sufficient to sustain simple visual discrimination learning but implies that the cholinergic input and the inferotemporal cortical input to the hippocampus both contribute to visual conditional learning. The subsequent addition of an immunotoxic lesion of the basal nucleus of Meynert in the right hemisphere resulted in an additional impairment on a difficult shape discrimination. This argues that it is the cholinergic projection to the inferotemporal cortex, rather than to the rest of the cortex, which contributes to visual discrimination learning and memory.

Aging and neuronal plasticity: lessons from a model.

In spite of many well-documented examples of age-related reductions in neuronal plasticity, the causes of such changes remain largely unknown. One example of age-reduced plasticity involves an aberrant sprouting response of mature rat sympathetic neurons into the CNS (hippocampal formation). This phenomenon has proven to be useful for exploring the relative contribution of target aging (extrinsic influences) versus neuronal aging (intrinsic influences) to reduced sprouting. Aged sympathetic neurons mount a robust growth response when confronted with young target tissue or when exposed to exogenous trophic factor in vivo. In contrast, the aged target tissue (the hippocampal formation in this example) exhibits reduced receptivity for sympathetic sprouting. This change in the target does not appear to be due to alterations in baseline levels of trophic or substrate support for axonal growth. Rather, aging appears to dampen the consequences of target denervation so that the aged target elicits less sprouting. Age-related reductions in neuronal sprouting are speculated to reflect increasing commitment to information storage at the expense of neuronal plasticity.

Peripheral blood natural killer cell cytotoxicity after damage to the limbic system in the rat.

The present work was aimed at examining the possible involvement of different parts of the septal area (dorsal, medial, lateral, and septohypothalamic nucleus), the basolateral amygdala, and the bed nucleus of the stria terminalis (BNST) in the regulation of the cytotoxic activity of NK cells (NKCC). The experimental approach included performing electrolytic (or sham) lesions in the tested brain areas and to measuring the peripheral blood NKCC (chromium-51 release assay), the number of leukocytes and lymphocytes, and the plasma corticosterone levels both before and at different time points after the lesion. Lesions were also induced in the three extralimbic structures: the paraventricular hypothalamic nucleus (PVN), the dorsal caudate-putamen, and the cerebellum. To test for a possible effect on NKCC of stress associated with blood collection, anesthesia, cranial surgery, and passing electric current through the brain the proper control experiments were also performed. Lesions of the medial septum and BNST caused gradual depression of NKCC, which peaked on the 10th day after the lesion, followed by a recovery to the baseline on days 21 (medial septum) and 42 (BNST) postinjury. In the respective sham-lesioned groups, mere insertion of electrodes into the medial septum and BNST evoked transient enhancement of NKCC (on the 3rd postlesion day), probably resulting from mechanical stimulation of the nervous tissue. Destruction of the other limbic and extralimbic structures appeared ineffective. After PVN lesions NKCC remained unchanged, despite an approximately 60% decrease in the basal corticosterone level. No adverse effects of the experimental and surgical procedures on NKCC, leukocyte and lymphocyte number, and corticosterone level were found, indicating that electrolytic lesions and other stereotaxic techniques can be safely used to study the brain-immune system interactions. The results obtained raise the question about the interrelationship between the medial septum and the hippocampal formation, BNST, the medial amygdala, and the hypothalamus (both medial and lateral) as a possible circuit involved in the regulation of cellular immune functions.

Cholinergic and noncholinergic septal neurons modulate strategy selection in spatial learning.

Rats solving a simple spatial discrimination task in a plus maze initially employ a place-learning strategy, then switch to a motor response strategy. The hippocampus is required for the use of a place-learning strategy in this task. Rats with 192 IgG-saporin lesions of the medial septum/vertical limb of the diagonal band (MS/VDB), that selectively removed cholinergic neurons projecting to the hippocampus, were significantly facilitated in acquisition of the spatial discrimination, and switched from place to response strategies just as control rats did. Rats with ibotenic acid lesions of the MS/VDB, that produced cell loss in the MS/VDB but little damage to cholinergic neurons, were significantly impaired in acquiring the spatial discrimination and did not reliably employ either a place or response strategy at any point in training. This suggests that the MS/VDB modulates hippocampal involvement in place learning, but that cholinergic MS/VDB neurons are neither necessary nor sufficient for using a place strategy to solve a spatial discrimination.