Intra hippocampal injection of testosterone impaired acquisition, consolidation and retrieval of inhibitory avoidance learning and memory in adult male rats

The hippocampus is essentially involved in learning and memory, and is known to be a target for androgen actions. Androgen receptors are densely expressed in CA1 of rat hippocampus, and mediate the effects of testosterone (T) on learning and memory. T depletion or administration can modulate neural function and cognitive performance. We conducted series of experiments to further investigate the effect of castration or intra hippocampal injection of T on acquisition, consolidation and retrieval of inhibitory avoidance learning and memory. Male adult rats were bilaterally cannulated into CA1 of hippocampus, and then received T (1, 10, 20, 40 and 80mug/0.5mul/side) or vehicle (DMSO), 30min before training, immediately after training and 30min before retrieval in inhibitory avoidance task. Castration was made by gonadectomy of male rats and behavioral tests performed 4 weeks later. Our results showed that gonadectomy of male rats did not influence performance on inhibitory avoidance task, as compared to sham-operated rats. We have also found that pre-training, post-training and pre-retrieval intra CA1 injections of T significantly decreased step-through latencies in inhibitory avoidance learning at doses 1 and 80, 20, and 20 and 40mug/0.5mul/side, respectively. The data suggest that intra CA1 administration of T could impair learning and memory acquisition, consolidation and retrieval, while systemic androgen's depletion have no effect on memory, in inhibitory avoidance task.     

The effect of intrahippocampal injection of testosterone enanthate (an androgen receptor agonist) and anisomycin (protein synthesis inhibitor) on spatial learning and memory in adult, male rats

In most mammals, the hippocampus has a well-documented role in spatial memory acquisition. High concentration of androgen receptors in fundamental centers of learning and memory in brain such as hippocampus shows that there may be some relationships between androgen receptors and cognitive aspects of brain. Previous studies, which have shown sex-dependent differences in hippocampal morphology and physiology, suggest a modulatory role for sex steroids in hippocampal function. Androgens have been shown to modulate some hippocampal-mediated behaviors including learning and memory. To study the mechanism of action of androgens in processes underlying learning and memory, anisomycin, a protein synthesis inhibitor was used to prevent the genomic effects of testosterone. Therefore, the effects of anisomycin and testosterone together were assessed on rat's performance in MWM. Rats received anisomycin (2.5 microg/0.5 microl), testosterone (80 microg/0.5 microl) or both anisomycin (2.5 microg/0.5 microl) and testosterone (80 microg/0.5 microl) through the connulas in the CA1 region. Anisomycin was injected 20 min and testosterone was injected 35 min before training each day. The results showed that anisomycin (2.5 microg/0.5 microl) and testosterone (80 microg/0.5 microl) increased latencies to find the invisible platform. But the group that received testosterone and anisomycin together was decrease in latency and traveled distance to find the invisible platform.     

The effects of intrahippocampal testosterone and flutamide on spatial localization in the Morris water maze

The high density of the androgen receptors in fundamental centers of learning and memory, such as hippocampus, shows that there must be some relationships between the androgen receptors and cognitive aspects. To determine the role of hippocampal androgen receptors in spatial learning, the current research has been conducted to assess the effect of testosterone enanthate, as the agonist, and flutamide, as the antagonist, of these receptors on spatial discrimination of rats, using the Morris water maze (MWM). Adult male rats were bilaterally cannulated into the CA1 region of their hippocampus. Different groups received different doses of flutamide (2, 5, 10 and 20 microg/0.5 microl) or testosterone enanthate (20, 40 and 80 microg/0.5 microl) through the cannulas 30 min before training for 3 days. The results showed dose-dependent increases in latencies and traveled distances to find the invisible platform both in flutamide- and testosterone-treated groups as compared to the control group, with peak effects at doses of 5 microg/0.5 microl for flutamide and 80 microg/0.5 microl for testosterone. Therefore, it seems that both androgen receptor blockade and exogenous testosterone can effect spatial localization of adult, male rats.     

Interactions between anandamide-induced anterograde amnesia and post-training memory modulatory systems

Rats were bilaterally implanted with indwelling cannulae in the CA1 region of the dorsal hippocampus. After recovery from surgery, they were trained in a one-trial, step-down inhibitory avoidance task using a 0.5 mA foot shock. The animals received intrahippocampal infusions of either vehicle or anandamide (100 microM, 0.5 microl/side) 30 min before training. Then, either immediately post-training or 3 h later, they received infusions of saline, noradrenaline (0.5 microg/side), SKF 38393 (1.5 microg/side), oxotremorine (0.6 microg/side) or Sp-cAMPs (0.5 microg/side) also in the hippocampus. All animals were tested for retention 24-h post-training. Anandamide produced anterograde amnesia. Immediate, but not delayed, post-training treatment with Sp-cAMPs and noradrenaline reversed this effect. SKF 38393 and oxotremorine had no influence on the amnesia caused by anandamide either when given immediately or 3 h after training. The results suggest that the amnesic effect of anandamide is related to the known noradrenergic regulation of cAMP-dependent protein kinase (PKA) activity previously described in the hippocampus immediately after avoidance training, which is crucial to long-term memory (LTM) formation.     

BDNF-triggered events in the rat hippocampus are required for both short- and long-term memory formation

Information storage in the brain is a temporally graded process involving different memory types or phases. It has been assumed for over a century that one or more short-term memory (STM) processes are involved in processing new information while long-term memory (LTM) is being formed. Because brain-derived neutrophic factor (BDNF) modulates both short-term synaptic function and activity-dependent synaptic plasticity in the adult hippocampus, we examined the role of BDNF in STM and LTM formation of a hippocampal-dependent one-trial fear-motivated learning task in rats. Using a competitive RT-PCR quantitation method, we found that inhibitory avoidance training is associated with a rapid and transient increase in BDNF mRNA expression in the hippocampus. Bilateral infusions of function-blocking anti-BDNF antibody into the CA, region of the dorsal hippocampus decreased extracellular signal-regulated kinase 2 (ERK2) activation and impaired STM retention scores. Inhibition of ERK1/2 activation by PD098059 produced similar effects. In contrast, intrahippocampal administration of recombinant human BDNF increased ERK1/2 activation and facilitated STM. The infusion of anti-BDNF antibody impaired LTM when given 15 min before or 1 and 4 hr after training, but not at 0 or 6 hr posttraining, indicating that two hippocampal BDNF-sensitive time windows are critical for LTM formation. At the same time points, PD098059 produced no LTM deficits. Thus, our results indicate that endogenous BDNF is required for both STM and LTM formation of an inhibitory avoidance learning. Additionally, they suggest that this requirement involves ERK1/2-dependent and -independent mechanisms.     

Learning-associated activation of nuclear MAPK, CREB and Elk-1, along with Fos production, in the rat hippocampus after a one-trial avoidance learning: abolition by NMDA receptor blockade

It is widely accepted that the formation of long-term memory (LTM) requires neuronal gene expression, protein synthesis and the remodeling of synaptic contacts. From mollusk to mammals, the cAMP/PKA/CREB signaling pathway has been shown to play a pivotal role in the establishment of LTM. More recently, the MAPK cascade has been also involved in memory processing. Here, we provide evidence for the participation of hippocampal PKA/CREB and MAPK/Elk-1 pathways, via activation of NMDA receptors, in memory formation of a one-trial avoidance learning in rats. Learning of this task is associated with an activation of p44 and p42 MAPKs, CREB and Elk-1, along with an increase in the levels of the catalytic subunit of PKA and Fos protein in nuclear-enriched hippocampal fractions. These changes were blocked by the immediate posttraining intra-hippocampal infusion of APV, a selective blocker of glutamate NMDA receptors, which renders the animals amnesic for this task. Moreover, no changes were found in control-shocked animals. Thus, inhibitory avoidance training in the rat is associated with an increase in the protein product of an IEG, c-fos, which occurs concomitantly with the activation of nuclear MAPK, CREB and Elk-1. NMDA receptors appear to be a necessary upstream step for the activation of these intracellular cascades during learning.     

CA3 NMDA receptors are crucial for rapid and automatic representation of context memory

It is argued that the hippocampus contributes to acquisition of context-specific memory although neural mechanisms have not been clarified. To evaluate the role of CA3 in context-specific memory, we developed one-trial context discrimination tasks to test acquisition and retrieval of contextual memory in CA3 pyramidal cell-restricted N-methyl-d-aspartate (NMDA) receptor knockout mice. Mutants were unable to discriminate conditioned and no-shock contexts 3 h after one-trial avoidance training. These phenotypes were not evident 24 h after one-trial training or 3 h after multi-trial training. Following one-trial contextual fear conditioning, mutants showed a selective deficit in context discrimination during a retention test 3 h after acquisition, although overall freezing levels were similar to those of the control mice. As in the avoidance task, this context discrimination impairment was not observed 24 h after initial conditioning. Interestingly, extending the post-shock period to 3 min during the one-trial fear conditioning task eliminated the discrimination deficit observed at the 3 h retention interval. These results suggest that: (i) impaired rapid context discrimination during the recall test is dependent on the duration of post-shock period during conditioning; (ii) CA3 NMDA receptors are critically involved in rapid and automatic formation of a unified context memory representation from the sensory information; (iii) CA3 NMDA receptors support contextual pattern separation; (iv) fear memory to foot-shock is acquired without CA3 NMDA receptors. It appears that rapid and automatic context memory representations from one-time experience are mediated, at least in part, by CA3 NMDA receptors.     

β‐Adrenergic receptors link NO/sGC/PKG signaling to BDNF expression during the consolidation of object recognition long‐term memory

The nitric oxide (NO)/soluble guanylyl cyclase (sGC)/protein kinase G (PKG) pathway is important for memory processing, but the identity of its downstream effectors as well as its actual participation in the consolidation of nonaversive declarative long‐term memory (LTM) remain unknown. Here, we show that training rats in an object recognition (OR) learning task rapidly increased nitrites/nitrates (NOx) content in the CA1 region of the dorsal hippocampus while posttraining intra‐CA1 microinfusion of the neuronal NO synthase (nNOS) inhibitor L‐NN hindered OR LTM retention without affecting memory retrieval or other behavioral variables. The amnesic effect of L‐NN was not state dependent, was mimicked by the sGC inhibitor LY83583 and the PKG inhibitor KT‐5823, and reversed by coinfusion of the NO donor S‐nitroso‐N‐acetylpenicillamine (SNAP) and the PKG activator 8‐bromoguanosine 3′,5′‐cyclic monophosphate (8Br‐cGMP). SNAP did not affect the amnesic effect of LY83583 and KT‐5823. Conversely, 8Br‐cGMP overturned the amnesia induced by LY83583 but not that caused by KT‐5823. Intra‐CA1 infusion of the β‐adrenergic receptor blocker timolol right after training hindered OR LTM and, although coadministration of noradrenaline reversed the amnesia caused by L‐NN, LY83583, and KT5823, the amnesic effect of timolol was unaffected by coinfusion of 8Br‐cGMP or SNAP, indicating that hippocampal β‐adrenergic receptors act downstream NO/sGC/PKG signaling. We also found that posttraining intra‐CA1 infusion of function‐blocking anti‐brain‐derived neurotrophic factor (BDNF) antibodies hampered OR LTM retention, whereas OR training increased CA1 BDNF levels in a nNOS‐ and β‐adrenergic receptor‐dependent manner. Taken together, our results demonstrate that NO/sGC/PKG signaling in the hippocampus is essential for OR memory consolidation and suggest that β‐adrenergic receptors link the activation of this pathway to BDNF expression during the consolidation of declarative memories. © 2009 Wiley‐Liss, Inc.     

Effects of discrete kainic acid-induced hippocampal lesions on spatial and contextual learning and memory in rats

Substantial information is available concerning the influence of global hippocampal lesions on spatial learning and memory, however the contributions of discrete subregions within the hippocampus to these functions is less well understood. The present investigation utilized kainic acid to bilaterally lesion specific areas of the rat hippocampus. These animals were subsequently tested on a spatial orientation task using a circular water maze, and on an associative/contextual task using passive avoidance conditioning. The results indicate that both the dorsal CA1 and the ventral CA3 subregions play important roles in learning. Specifically, CA1 lesions produced a deficit in the acquisition of the water maze task and a significant memory impairment on the passive avoidance task. CA3 lesions also caused learning deficits in the acquisition of the water maze task, and produced even greater impairments in performance on the passive avoidance task. We conclude that CA1 and CA3 hippocampal subregions each play significant roles in the overall integration of information concerning spatial and associative learning.     

The effect of antagonization of orexin 1 receptors in CA1 and dentate gyrus regions on memory processing in passive avoidance task

The hippocampal formation plays an essential role in associative learning like passive avoidance (PA) learning. It has been shown; orexin-containing terminals and orexin receptors densely are distributed in the hippocampal formation. We have previously demonstrated that antagonization of orexin 1 receptor (OX1R) in CA1 region of hippocampus and dentate gyrus (DG) impaired spatial memory processing. Although, there are few studies concerning function of orexinergic system on memory processing in PA task, but there is no study about physiological function of OX1R on this process. To address this, the OX1R antagonist, SB-334867-A, was injected into DG or CA1 regions of hippocampus and evaluated the influence of OX1R antagonization on acquisition, consolidation and retrieval in PA task. Our results show that, SB-334867-A administration into CA1 region impaired memory retrieval but not PA acquisition and consolidation. However, SB-334867-A administration into DG region impaired acquisition and consolidation but not PA memory retrieval. Therefore, it seems that endogenous orexins play an important role in learning and memory in the rat through OX1Rs.     

Cortistatin modulates memory processes in rats

Cortistatin (CST) is a recently described neuropeptide with high structural homology with somatostatin. Its mRNA is restricted to gamma amino butyric acid (GABA)-containing cells in the cerebral cortex and hippocampus. CST modulates the electrophysiology of the hippocampus and cerebral cortex of rats; hence, it may be modulating mnemonic processes. In this study, we have evaluated the effect of CST and somatostatin (SS) on short- and long-term memory (STM and LTM, respectively), as well as on the extinction of the behavior by using the footshock passive avoidance behavioral test. In addition, we tested the ability of both neuropeptides to affect the generation of cAMP in hippocampal neurons in culture. Results showed that the administration of either CST or SS into the hippocampal CA1 deteriorates memory consolidation in a dose-response fashion and facilitates the extinction of the learned behavior. CST was more potent than SS. Likewise, CST increases cAMP while SS decreases it. These results strongly support a modulatory role for CST in memory processes.     

BDNF mRNA expression in rat hippocampus following contextual learning is blocked by intrahippocampal IL-1beta administration

The present study examined the modulating effects of an intrahippocampal injection of interleukin-1beta (IL-1beta) on brain-derived neurotrophic factor (BDNF) mRNA expression 0.5, 2, 4, and 6 h following contextual fear conditioning, a task known to increase BDNF mRNA, in rats. Contextual fear conditioning produced a time-dependent increase in BDNF mRNA that varied by region of hippocampus. IL-1beta blocked or reduced these increases in BDNF mRNA in the CA1, CA2, and dentate gyrus regions of the hippocampus, but had no effect in cortical regions. These data support the idea that IL-1beta-produced memory deficits may be mediated via BDNF mRNA reductions in hippocampus.     

Infusions of allopregnanolone into the hippocampus and amygdala,but not into the nucleus accumbens and medial prefrontal cortex,produce antidepressant effects on the learned helplessness rats

Patients with depression showed a decrease in plasma and cerebrospinal fluid allopregnanolone (ALLO). But antidepressants increased the contents of ALLO in the rat brain. We examined the antidepressant‐like effects of infusion of ALLO into the cerebral ventricle, hippocampus, amygdala, nucleus accumbens, or prefrontal cortex of learned helplessness (LH) rats (an animal model of depression). Of these regions, infusions of ALLO into the cerebral ventricle, the CA3 region of hippocampus, or the central region of amygdala exerted antidepressant‐like effects. Infusion of ALLO into the hippocampal CA3 region or the central amygdala did not produce memory deficits or locomotor activation in the passive avoidance and open field tests. It is well documented that ALLO exerts its effects through GABA receptors. Therefore, we examined the antagonistic effects of flumazenil (a GABA receptor antagonist) on the antidepressant‐like effects of ALLO. Coinfusion of flumazenil with ALLO into the hippocampal CA3 region, but not into the central amygdala, blocked the antidepressant‐like effects of ALLO. However, coinfusion of (+)MK801 (an NMDA receptor antagonist), but not cycloheximide (a protein synthesis inhibitor), blocked the antidepressant‐like effects of ALLO in the central amygdala. These results suggest that ALLO exerts antidepressant‐like effects in the CA3 region of hippocampus through the GABA system and in the central region of amygdala, dependently on the activation of the glutamatergic mechanisms. © 2010 Wiley‐Liss, Inc.     

Functional deficits after sustained stimulation of the perforant path

Several reports have implicated the overactivity of hippocampal glutaminergic systems in neurodegenerative conditions including Senile dementia of the Alzheimer's type (SDAT). The neurobiological effects of hippocampal glutaminergic hyperactivity were studied by perforant pathway stimulation. Forty-five minutes of sustained perforant pathway stimulation produced a 50% or greater increase in motor activity 1, 2, and 3 weeks after stimulation. Robust retention deficits in a 48-h step-through passive avoidance task were evident 2 weeks post-stimulation. Furthermore, animals receiving stimulation were impaired in the acquisition of a spatial task in the Morris water maze. Stimulated animals exhibited little reduction in their escape latencies over the testing period. The learning and memory deficits were associated with a loss of CA1 and CA3 pyramidal cells and pretreatment with the N-methyl-D-aspartate antagonist MK-801 reduced this cell loss, particularly in the CA1 region of the hippocampus. These results suggest that sustained stimulation of the perforant pathway may be useful in studying neurological deficits associated with glutaminergic hyperfunction.     

Different hippocampal molecular requirements for short- and long-term retrieval of one-trial avoidance learning

Rats were trained in one-trial step-down inhibitory avoidance and tested either 3 h or 31 days later. Ten minutes prior to the retention test, through indwelling cannulae placed in the CA1 region of the dorsal hippocampus, they received 0.5 microl infusions of: saline, a vehicle (2% dimethylsulfoxide in saline), the glutamate NMDA receptor blocker, aminophosphonopentanoic acid (AP5) (5.0 microg), the AMPA/kainate receptor blocker, cyanonitroquinoxaline dione (CNQX) (0.25 or 1.25 microg), the metabotropic receptor antagonist, methylcarboxyphenylglycine (MCPG) (0.5 or 2.5 microg), the inhibitor of calcium/calmodulin-dependent protein kinase II (KN62) (3.5 microg), the inhibitor of cAMP-dependent protein kinase (PKA), Rp-cAMPs (0.1 or 0.5 microg), the stimulant of the same enzyme, Sp-cAMPs (0.1 or 0.5 microg), or the inhibitor of the mitogen-activated protein kinase (MAPK) kinase, PD098059 (10 or 50 microM). CNQX, KN62 and PD098059 were dissolved in the vehicle; the other drugs were dissolved in saline. All these drugs, at the same doses, had been previously found to affect short- and long-term memory formation of this task. Retrieval measured 3 h after training (short-term memory) was blocked by CNQX and MCPG, and was unaffected by all the other drugs. In contrast, retrieval measured at 31 days was blocked by MCPG, Rp-cAMPs and PD098059, enhanced by Sp-cAMPs, and unaffected by CNQX, AP5 or KN62. The results indicate that, in CA1, glutamate metabotropic receptors are necessary for the retrieval of both short- and long-term memory; AMPA/kainate receptors are necessary for short-term but not long-term memory retrieval, and NMDA receptors are uninvolved in retrieval. Both the PKA and MAPK signalling pathways are required for the retrieval of long-term but not short-term memory.     

Mechanisms of hippocampal long-term depression are required for memory enhancement by novelty exploration

It is well known that novel environments can enhance learning and memory. However, the underlying mechanisms remain poorly understood. Here, we report that, in freely moving rats, novelty exploration facilitates the production of hippocampal CA1 long-term depression (LTD), a well characterized form of synaptic plasticity believed to be a cellular substrate of spatial learning, and thereby converts short-term memory (STM) into long-term memory (LTM) in an inhibitory avoidance learning procedure. Blocking the induction or the expression of CA1 LTD with two mechanistically and structurally distinct inhibitors prevents not only novelty acquisition but also the novelty exploration-promoted conversion of STM into LTM. Moreover, production of LTD with a strong electrical stimulation induction protocol or facilitation of hippocampal LTD by pharmacological inhibition of glutamate transporter activity mimics the behavioral effects of novelty exploration, sufficiently promoting the conversion of STM into LTM. Together, our findings suggest that induction of LTD may play an essential role not only in novelty acquisition but also in novelty-mediated memory enhancement.     

Activation of hippocampal postsynaptic muscarinic receptors is involved in long-term spatial memory formation

Spatial memory has been strongly associated with hippocampal function. There are several reports of the participation of this structure in acquisition and consolidation of spatial tasks. In this study, we evaluated the effects of selective and non-selective muscarinic antagonists in the dorsal hippocampus of rats during acquisition and encoding of a spatial task. Rats were trained in a Morris water maze for 4 days with identical daily sessions, and tested for long-term memory (LTM) 1 week after training. The animals were injected bilaterally in the dorsal hippocampus 20 min before the start of every day of training. The results showed that the non-selective muscarinic antagonist, scopolamine, disrupted acquisition of water maze memory formation. Moreover, microinjections of a selective postsynaptic muscarinic antagonist, pirenzepine, disrupted LTM, whereas it did not affect acquisition. Conversely, a selective presynaptic muscarinic antagonist, AFDX-116, did not disrupt either water maze acquisition or LTM formation. Combination of AFDX-116 and pirenzepine had similar effects as scopolamine, partially blocking acquisition and impairing long-term spatial memory. These results support the view that muscarinic receptors are involved in spatial learning and that postsynaptic muscarinic receptors in the dorsal hippocampus are particularly involved in long-term spatial memory formation.     

The ubiquitin-proteasome cascade is required for mammalian long-term memory formation

It has been recently demonstrated that ubiquitin-proteasome-mediated proteolysis is required for long-term synaptic facilitation in Aplysia. Here we show that the hippocampal blockade of this proteolytic pathway is also required for the formation of long-term memory in the rat. Bilateral infusion of lactacystin, a specific proteasome inhibitor, to the CA1 region caused full retrograde amnesia for a one-trial inhibitory avoidance learning when given 1, 4 or 7h, but not 10 h, after training. Proteasome inhibitor I produced similar effects. In addition, inhibitory avoidance training resulted in an increased ubiquitination and 26S proteasome proteolytic activity and a decrease in the levels of IkappaB, a substrate of the ubiquitin-proteasome cascade, in hippocampus 4 h after training. Together, these findings indicate that the ubiquitin-proteasome cascade is crucial for the establishment of LTM in the behaving animal.