Acute stress facilitates trace eyeblink conditioning in C57BL/6 male mice and increases the excitability of their CA1 pyramidal neurons

The effects of stress (restraint plus tail shock) on hippocampus-dependent trace eyeblink conditioning and hippocampal excitability were examined in C57BL/6 male mice. The results indicate that the stressor significantly increased the concentration of circulating corticosterone, the amount and rate of learning relative to nonstressed conditioned mice, and the excitability of CA1 hippocampal pyramidal neurons. Behaviorally, there was no effect of the stressor on control mice that received unpaired presentations of the tone and periorbital shock, i.e., neither stressed nor nonstressed control mice showed an increase in conditioned responding that was above baseline levels. Biophysically, the stressor significantly decreased the amplitude of the post-burst afterhyperpolarization (AHP) and decreased spike frequency accommodation relative to cells from nonstressed control mice. The effect was significant for mice that were stressed either 1 h or 24 h earlier. The results suggest that the stressor increases the excitability of hippocampal pyramidal neurons and that the mechanism underlying this increase may contribute to the more rapid acquisition of hippocampally dependent eyeblink conditioning.     

Hippocampal and cerebellar single-unit activity during delay and trace eyeblink conditioning in the rat

In delay eyeblink conditioning, the CS overlaps with the US and only a brainstem-cerebellar circuit is necessary for learning. In trace eyeblink conditioning, the CS ends before the US is delivered and several forebrain structures, including the hippocampus, are required for learning, in addition to a brainstem-cerebellar circuit. The interstimulus interval (ISI) between CS onset and US onset is perhaps the most important factor in classical conditioning, but studies comparing delay and trace conditioning have typically not matched these procedures in this crucial factor, so it is often difficult to determine whether results are due to differences between delay and trace or to differences in ISI. In the current study, we employed a 580-ms CS-US interval for both delay and trace conditioning and compared hippocampal CA1 activity and cerebellar interpositus nucleus activity in order to determine whether a unique signature of trace conditioning exists in patterns of single-unit activity in either structure. Long-Evans rats were chronically implanted in either CA1 or interpositus with microwire electrodes and underwent either delay eyeblink conditioning, or trace eyeblink conditioning with a 300-ms trace period between CS offset and US onset. On trials with a CR in delay conditioning, CA1 pyramidal cells showed increases in activation (relative to a pre-CS baseline) during the CS-US period in sessions 1-4 that was attenuated by sessions 5-6. In contrast, on trials with a CR in trace conditioning, CA1 pyramidal cells did not show increases in activation during the CS-US period until sessions 5-6. In sessions 5-6, increases in activation were present only to the CS and not during the trace period. For rats with interpositus electrodes, activation of interpositus neurons on CR trials was present in all sessions in both delay and trace conditioning. However, activation was greater in trace compared to delay conditioning in the first half of the CS-US interval (during the trace CS) during early sessions of conditioning and, in later sessions of conditioning, activation was greater in the second half of the CS-US interval (during the trace interval). These results suggest that the pattern of hippocampal activation that differentiates trace from delay eyeblink conditioning is a slow buildup of activation to the CS, possibly representing encoding of CS duration or discrimination of the CS from the background context. Interpositus nucleus neurons show strong modeling of the eyeblink CR regardless of paradigm but show a changing pattern across conditioning that may be due to the necessary contributions of forebrain processing to trace conditioning.     

Effects of Early Hippocampal Lesions on Trace, Delay, and Long-Delay Eyeblink Conditioning in Developing Rats

The effects of bilateral hippocampal aspiration lesions on later acquisition of eyeblink conditioning were examined in developing Long-Evans rat pups. Lesions on postnatal day (PND) 10 were followed by evaluation of trace eyeblink conditioning (Experiment 1) and delay eyeblink conditioning (Experiment 2) on PND 25. Pairings of a tone conditioned stimulus (CS) and periocular shock unconditioned stimulus (US, 100 ms) were presented in one of three conditioning paradigms: trace (380 ms CS, 500 ms trace interval, 880 ms interstimulus interval [ISI]), standard delay (380 ms CS, 280 ms ISI), or long delay (980 ms CS, 880 ms ISI). The results of two experiments indicated that hippocampal lesions impaired trace eyeblink conditioning more than either type of delay conditioning. In light of our previous work on the ontogeny of trace, delay, and long-delay eyeblink conditioning (Ivkovich, Paczkowski, & Stanton, 2000) showing that trace and long-delay eyeblink conditioning had similar ontogenetic profiles, the current data suggest that during ontogeny hippocampal maturation may be more important for the short-term memory component than for the long-ISI component of trace eyeblink conditioning. The late development of conditioning over long ISIs may depend on a separate process such as protracted development of cerebellar cortex.     

Effects of ventral and dorsal CA1 subregional lesions on trace fear conditioning

Recent lines of research have focused on dissociating function between the dorsal and ventral hippocampus along space and anxiety dimensions. In the dorsal hippocampus, the CA1 subregion has been implicated in the acquisition of contextual fear as well as in the trace interval in trace fear conditioning. The present study was designed to test the relative contributions of dorsal (dCA1) and ventral CA1 (vCA1) in trace fear conditioning. Long-Evans rats received ibotenate lesions of the ventral CA1 (n=7), dorsal CA1 (n=9), or vehicle control lesions (n=8) prior to trace fear conditioning acquisition. Results suggest dCA1 and vCA1 groups show no significant deficits during acquisition when compared to control groups. dCA1 and vCA1 both show deficits in the retention of contextual fear when tested 24 h post-acquisition (P<.05 and P<.01, respectively), and vCA1 was impaired relative to dCA1 (P<.05). This is suggestive of a graded involvement in contextual retention between the dorsal and ventral aspects of CA1. dCA1 showed no deficit for retention of conditioned fear to the tone or the trace when tested 48 h post-acquisition, whereas vCA1 did show a significant deficit for the trace interval and a slight, non-significant reduction in freezing to the tone, when compared to the control group (p<.05). Overall the data are suggestive of a graded involvement in retention of fear conditioning between the dorsal and ventral aspects of CA1, but it is likely that vCA1 may be critically involved in retention of trace fear conditioning.     

Modulation of cholinergic transmission enhances excitability of hippocampal pyramidal neurons and ameliorates learning impairments in aging animals

Four cholinesterase inhibitors have been approved by the US Food and Drug Administration for treating behavioral symptoms of Alzheimer's disease. Here we review our experiences with two cholinesterase inhibitors (metrifonate and galanthamine) and a muscarinic acetylcholine receptor agonist (CI-1017) in behavioral pharmacological and brain slice experiments in aging and young rabbits. Aging rabbits are impaired in their ability to acquire the hippocampus-dependent trace eyeblink conditioning task, as compared to young controls. A large proportion of aging animals cannot reach behavioral criterion in this task. Those that do learn, do so more slowly. In addition, the post-burst afterhyperpolarization and spike frequency accommodation is increased in hippocampal pyramidal neurons from aging animals, i.e., cellular excitability is reduced as compared to those from young animals. Metrifonate, galanthamine, and CI-1017 reduced the learning deficits observed in aging rabbits so that they learned almost as quickly as young controls. These cholinergic compounds also enhanced the postsynaptic excitability of hippocampal pyramidal neurons in vitro. Therefore, we propose that the amelioration of learning impairment with the cholinergic compounds may in part be due to the enhanced excitability of hippocampal pyramidal neurons. The potential relevance of our studies to further understanding the cellular and behavioral changes that occur with normal aging and Alzheimer's Disease is discussed.     

Impairments in trace EB conditioning by knife-cut lesions to the fornix in rabbits: reversal by galantamine

Previous work in our laboratory demonstrated that galantamine, a cholinesterase inhibitor and weak cholinergic agonist, facilitated classical trace eyeblink conditioning in healthy, young rabbits [Simon, B. B., Knuckley, B., & Powell, D. A. (2004). Galantamine facilitates acquisition of a trace-conditioned eyeblink response in healthy, young rabbits. Learning & Memory, 11(1), 116-122.]. The current study investigated the effects of galantamine (0.0 or 3.0mg/kg) in rabbits sustaining knife-cut lesions to the fimbria-fornix, a major projection pathway connecting the hippocampus to cortical and subcortical brain structures involved in the formation of long-term memories. Two experiments were conducted. Experiment one assessed the effects of knife-cut lesions to the fornix or sham surgeries on trace eyeblink (EB) conditioning. Results indicate that fornix lesions significantly retarded EB conditioning when trace parameters were employed. Experiment 2 assessed whether treatment with galantamine would reverse the deficits caused by fornix damage. Results indicate that 3.0mg/kg GAL reversed trace EB conditioning deficits in animals with fornix knife-cut lesions. These findings suggest that galantamine may provide benefit in the reversal of cognitive dysfunction following certain types of brain damage, especially damage involving hippocampal structures.     

Simultaneous training on two hippocampus-dependent tasks facilitates acquisition of trace eyeblink conditioning

A common cellular alteration, reduced post-burst afterhyperpolarization (AHP) in CA1 neurons, is associated with acquisition of the hippocampus-dependent tasks trace eyeblink conditioning and the Morris water maze. As a similar increase in excitability is correlated with these two learning paradigms, we sought to determine the interactive behavioral effects of training animals on both tasks by using either a consecutive or simultaneous training design. In the consecutive design, animals were trained first on either the trace eyeblink conditioning task for six sessions, followed by training on the water maze task for six sessions, or vice versa. The simultaneous design consisted of six or 11 training days; animals received one session/day of both trace eyeblink conditioning and water maze training. Separate groups were used for consecutive and simultaneous training. Animals trained on both tasks simultaneously were significantly facilitated in their ability to acquire the trace eyeblink conditioning task; no effect of simultaneous training was seen on the water maze task. No effect was seen on acquisition for either task when using the consecutive training design. Taken together, these findings provide insight into how the hippocampus processes information when animals learn multiple hippocampus-dependent tasks.     

Opposing effects on muscarinic acetylcholine receptors in the piriform cortex of odor-trained rats

We combined pharmacological studies and electrophysiological recordings to investigate modifications in muscarinic acetylcholine (ACh) receptors (mAChR) in the rat olfactory (piriform) cortex, following odor-discrimination rule learning. Rats were trained to discriminate between positive and negative cues in pairs of odors, until they reached a phase of high capability to learn unfamiliar odors, using the same paradigm (“rule learning”). It has been reported that at 1–3 d after the acquisition of odor-discrimination rule learning, pyramidal neurons in the rat piriform cortex show enhanced excitability, due to a reduction in the spike-activated potassium current I_AHP, which is modulated by ACh. Further, ACh and its analog, carbachol (CCh), lost the ability to reduce the I_AHP in neurons from trained rats. Here we show that the reduced sensitivity to CCh in the piriform cortex results from a decrease in the number of mAChRs, as well as a reduction in the affinity of the receptors to CCh. Also, it has been reported that 3–8 d after the acquisition of odor-discrimination rule learning, synaptic transmission in the piriform cortex is enhanced, and paired-pulse facilitation (PPF) in response to twin stimulations is reduced. Here, intracellular recordings from pyramidal neurons show that CCh increases PPF in the piriform cortex from odor-trained rats more than in control rats, suggesting enhanced effect of ACh in inhibiting presynaptic glutamate release after odor training.     

A cellular analogue of operant conditioning.

Using the hippocampal-slice preparation, we attempted to model operant conditioning in vitro by reinforcing pyramidal cell bursting responses with local micropressure applications of transmitters and drugs. The same injections were administered independently of bursting to provide a "noncontingent" control for direct pharmacological stimulation or facilitation of firing. The results suggested that the bursting responses of individual CA1 pyramidal neurons may be reinforced in a dose-related manner by response-contingent (but not noncontingent) injections of dopamine and the selective dopamine D2 agonist, N-0923. N-0924, a stereoisomer of N-0923 that is largely devoid of D2-agonist activity, failed to reinforce CA1 bursting. Burst-contingent injections of the excitatory neurotransmitter glutamate also failed to reinforce CA1 bursting; indeed, the glutamate applications (whether contingent or random) reduced the likelihood of bursts while increasing the frequency of solitary spikes. Reinforcement delays exceeding 200 ms largely eliminated the reinforcing efficacy of the D2 agonist N-0437 in CA1 operant conditioning. The results are consistent with the suggestion that the behaviorally reinforcing effects of dopaminergic agents can be modeled in vitro in the hippocampal-slice preparation.     

ERK1/2 activation is necessary for BDNF to increase dendritic spine density in hippocampal CA1 pyramidal neurons

Brain-derived neurotrophic factor (BDNF) is a potent modulator of synaptic transmission and plasticity in the CNS, acting both pre- and postsynaptically. We demonstrated recently that BDNF/TrkB signaling increases dendritic spine density in hippocampal CA1 pyramidal neurons. Here, we tested whether activation of the prominent ERK (MAPK) signaling pathway was responsible for BDNF's effects on spine growth. Slice cultures were transfected with enhanced yellow fluorescent protein (eYFP) by particle-mediated gene transfer, and CA1 pyramidal neurons were imaged by laser-scanning confocal microscopy. We confirmed that BDNF (24 h) increases spine density in apical dendrites of CA1 neurons. The MEK (ERK kinase) inhibitors PD98059 and U0126 completely prevented the increase in spine density induced by BDNF, without having an effect on spine density by themselves. In contrast to its actions on cortical pyramidal neurons, BDNF had minor and rather localized effects on dendritic complexity in hippocampal pyramidal neurons, increasing the total length, but not the branching of apical dendrites within CA1 stratum radiatum, without affecting basal dendrites in stratum oriens. Our results support the hypothesis that the ERK-signaling pathway not only mediates long-term synaptic plasticity and hippocampal-dependent learning, but it is also involved in the structural remodeling of excitatory spine synapses triggered by neurotrophins.     

Foreground contextual fear memory consolidation requires two independent phases of hippocampal ERK/CREB activation

Fear conditioning is a popular model for investigating physiological and cellular mechanisms of memory formation. In this paradigm, a footshock is either systematically associated to a tone (paired conditioning) or is pseudorandomly distributed (unpaired conditioning). In the former procedure, the tone/shock association is acquired, whereas in the latter procedure, the context/shock association will prevail. Animals with chronically implanted recording electrodes show enhanced amplitude of the extracellularly recorded field EPSP in CA1 pyramidal cells for up to 24 h after unpaired, but not paired, fear conditioning. This is paralleled by a differential activation of the ERK/CREB pathway in CA1, which is monophasic in paired conditioning (0-15 min post-conditioning), but biphasic (0-1 h and 9-12 h post-conditioning) in unpaired conditioning as revealed by immunocytochemistry and Western blotting. Intrahippocampal injection of the MEK inhibitor U0126 prior to each phase prevents the activation of both ERK1/2 and CREB after unpaired conditioning. Block of any activation phase leads to memory impairment. We finally reveal that the biphasic activation of ERK/CREB activity is independently regulated, yet both phases are critically required for the consolidation of long-term memories following unpaired fear conditioning. These data provide compelling evidence that CA1 serves different forms of memory by expressing differential cellular mechanisms that are dependent on the training regime.     

Learning-dependent potentiation in the vibrissal motor cortex is closely related to the acquisition of conditioned whisker responses in behaving mice

The role of the primary motor cortex in the acquisition of new motor skills was evaluated during classical conditioning of vibrissal protraction responses in behaving mice, using a trace paradigm. Conditioned stimulus (CS) presentation elicited a characteristic field potential in the vibrissal motor cortex, which was dependent on the synchronized firing of layer V pyramidal cells. CS-evoked and other event-related potentials were particular cases of a motor cortex oscillatory state related to the increased firing of pyramidal neurons and to vibrissal activities. Along conditioning sessions, but not during pseudoconditioning, CS-evoked field potentials and unitary pyramidal cell responses grew with a time-course similar to the percentage of vibrissal conditioned responses (CRs), and correlated significantly with CR parameters. High-frequency stimulation of barrel cortex afferents to the vibrissal motor cortex mimicked CS-related potentials growth, suggesting that the latter process was due to a learning-dependent potentiation of cortico-cortical synaptic inputs. This potentiation seemed to enhance the efficiency of cortical commands to whisker-pad intrinsic muscles, enabling the generation of acquired motor responses.     

Cholinergic modulation of trace conditioning trained in serial compound: A developmental analysis

In four experiments the effects of serial compound conditioning on responding to a trace-conditioned CS were evaluated using a fear conditioning paradigm. The subjects were 18- and 25-day-old Sprague-Dawley rats, previously shown to exhibit little or no trace fear conditioning. Here, animals as young as 18 days of age were shown to be capable of trace conditioning between a visual CS1 and a shock US, provided the trace interval was filled with a non-target CS2 during serial conditioning trials (CS1-->CS2-->US). To explore cholinergic mechanisms involved in trace and serial conditioning, additional experiments assessed conditioned responding following pre-training administration of the muscarinic receptor antagonist scopolamine. Scopolamine produced a dose-dependent reduction in responding to the trace CS1, regardless of whether subjects were trained with standard trace (CS1-->trace interval-->US) or serial (CS1-->CS2-->US) trials. Responding to CS2 was unaffected by scopolamine. These data suggest that central cholinergic systems are functional in the young animals, but are not normally sufficiently activated by standard trace conditioning procedures. The results suggest that serial compound conditioning can promote trace conditioning in young rats, as it does in adults, perhaps by enhancing cholinergic activity during training. Implications for the late ontogenetic emergence of trace conditioning as it relates to maturation of neural pathways and their role in the potentiating effects of a gap filler are discussed.     

Mutation at the TrkB PLC{gamma}-docking site affects hippocampal LTP and associative learning in conscious mice

Previous in vitro studies have characterized the electrophysiological properties and molecular events associated with long-term potentiation (LTP), but as yet there are no in vivo data from molecular-level dissection that directly identify LTP as the biological substrate for learning and memory. Understanding whether the molecular pathways required for learning are also those generating LTP when measured directly on the relevant circuit of a learning animal is clearly important, although so far has proved technically difficult. Here, for the first time, we combine highly defined genetic mouse models with behavior and in vivo recordings. We recorded the activity-dependent changes taking place at the CA3-CA1 synapses during the acquisition and extinction of a simple form of an associative learning task in mice carrying point mutations on specific docking sites of TrkB receptors (trkB(SHC), trkB(PLC)). The learning task consisted of a classical eyeblink conditioning using a trace paradigm. The conditioned stimulus (CS) consisted of a tone and was followed by a periorbital electrical shock as an unconditioned stimulus (US). The US started 500 msec after the end of the CS. We show that a single pulse presented to the Schaffer collateral-commissural pathway during the CS-US interval evoked a monosynaptic field excitatory postsynaptic potential (fEPSP) at the CA1 pyramidal cells, with a slope linearly related to learning evolution in controls and trkB(SHC) mutants, but the relationship was impaired in trkB(PLC) mice. These data support a link between the PLCgamma-docking site downstream of TrkB and the activity-dependent synaptic changes evoked at the CA3-CA1 synapses during associative learning in conscious mice, and indicate that TrkB PLCgamma-site-activated molecular pathway(s) underlie both associative learning and LTP triggered at the CA3-CA1 synapse.     

Selective Abolition of the NMDA Component of Long-Term Potentiation in Mice Lacking mGluR5

The mechanisms underlying the differential expression of long-term potentiation (LTP) by AMPA and NMDA receptors, are unknown, but could involve G-protein-linked metabotropic glutamate receptors. To investigate this hypothesis we created mutant mice that expressed no metabotropic glutamate receptor 5 (mGluR5), but showed normal development. In an earlier study of these mice we analyzed field-excitatory postsynaptic potential (fEPSPs) in CA1 region of the hippocampus and found a small decrease; possibly arising from changes in the NMDAR-mediated component of synaptic transmission. In the present study we used whole-cell patch clamp recordings of evoked excitatory postsynaptic currents (EPSCs) in CA1 pyramidal neurons to identify the AMPAR- and NMDAR-mediated components of LTP. Recordings from control mice following tetanus, or agonist application (IS, 3R-1-amino-cyclopentane 1,3-dicarboxylic acid) (ACPD), revealed equal enhancement of the AMPA and NMDA receptor-mediated components. In contrast, CA1 neurons from mGluR5-deficient mice showed a complete loss of the NMDA-receptor-mediated component of LTP (LTP_NMDA), but normal LTP of the AMPA-receptor-mediated component (LTP_AMPA). This selective loss of LTP_NMDA was seen in three different genotypic backgrounds and was apparent at all holding potentials (−70 mV to +20 mV). Furthermore, the LTP_NMDA deficit in mGluR5 mutant mice could be rescued by stimulating protein kinase C (PKC) with 4β-phorbol-12,13-dibutyrate (PDBu). These results suggest that PKC may couple the postsynaptic mGluR5 to the NMDA-receptor potentiation during LTP, and that this signaling mechanism is distinct from LTP_AMPA. Differential enhancement of AMPAR and NMDA receptors by mGluR5 also supports a postsynaptic locus for LTP.     

Modulation of behavioral inhibition in appetitive extinction following manipulation of adrenal steroids in rats: implications for involvement of the hippocampus.

Corticosterone, the principal glucocorticoid in the rat, binds selectively to the CA1 pyramidal neurons of the hippocampus where the hormone has been demonstrated to exert a moderate chronic suppression of spontaneous activity. In the first experiment of the current study, the functional behavioral significance of this hormone--brain interaction was investigated in the extinction of an appetitive runway response in normal rats and those with lesions of the hippocampus. During extinction, half of the animals in each group were given daily subcutaneous injections of corticosterone. Whie the classical retardation effect of hippocampal lesions on appetitive extinction was replicated, hormone treatment was without effect in normal or hippocampally damaged subjects. The absence of a hormone effect in normals was primarily attributed to a saturated limited-binding system operating in the normal animal. Experiment 2 tested this notion, repeating the first experiment, with adrenal-ectomized (ADX), ADX + corticosterone replacement, and normal groups of animals. Adrenalectomy produced a striking facilitation of extinction which was speculated to be the result of a hyperactive inhibitory neural organ free from an inhibitory endocrine feedback. Corticosterone treatment normalized the progress of extinction in ADX animals, providing support for the afore-mentioned speculation. In the normal animal, it appears that a stress-induced surge in hormone level interacts with a limited-capacity neural binding to produce a transient dynamic range of behavioral disinhibition, perhaps promoting persistence during initial stages of frustrative nonreward in moderate stress tasks.     

Memory deficits are associated with impaired ability to modulate neuronal excitability in middle-aged mice

Normal aging disrupts hippocampal neuroplasticity and learning and memory. Aging deficits were exposed in a subset (30%) of middle-aged mice that performed below criterion on a hippocampal-dependent contextual fear conditioning task. Basal neuronal excitability was comparable in middle-aged and young mice, but learning-related modulation of the post-burst afterhyperpolarization (AHP)—a general mechanism engaged during learning—was impaired in CA1 neurons from middle-aged weak learners. Thus, modulation of neuronal excitability is critical for retention of context fear in middle-aged mice. Disruption of AHP plasticity may contribute to contextual fear deficits in middle-aged mice—a model of age-associated cognitive decline (AACD).Plasticity of intrinsic neuronal excitability increases the overall storage capacity of neurons and therefore likely plays a critical role in learning and memory (Zhang and Linden 2003). Increased neuronal excitability via reductions of the post-burst afterhyperpolarization (AHP) is hypothesized as a general mechanism underlying learning and memory tasks (Disterhoft et al. 1986; Disterhoft and Oh 2006). The AHP serves to limit subsequent firing in response to excitation (Madison and Nicoll 1984; Lancaster and Adams 1986; Storm 1990; Sah and Bekkers 1996). Generally speaking, the size of the AHP is inversely related to neuronal excitability, and the measurement of the AHP is routinely used as an index of neuronal excitability.Our laboratory and others have shown that AHP reductions are observed in hippocampal neurons from animals that learn hippocampal-dependent tasks including trace eyeblink conditioning in rabbit and rat (de Jonge et al. 1990; Moyer Jr et al. 1996, 2000; Kuo 2004) and spatial water maze in rat and mouse (Oh et al. 2003; Tombaugh et al. 2005; Ohno et al. 2006b). Learning-related reductions in the AHP have also been observed in cortical neurons following odor discrimination (Saar et al. 1998) and extinction learning (Santini et al. 2008). In vitro, activity-dependent plasticity of the AHP is induced using physiologically relevant stimuli (Kaczorowski et al. 2007). Because the AHP serves to limit subsequent firing, learning-related reductions in the AHP are poised to facilitate mechanisms crucial for information storage, such as long-term potentiation (LTP), synaptic integration (Sah and Bekkers 1996), metaplasticity (Le Ray et al. 2004), and spike-timing dependent plasticity (STDP) (Le Ray et al. 2004).Hippocampal neurons from naïve aged rodents and rabbits show a decrement in basal excitability evidenced by a robust enhancement of the AHP (Landfield and Pitler 1984; Moyer Jr et al. 1992, 2000; Oh et al. 1999; Kumar and Foster 2002, 2004; Power et al. 2002; Hemond and Jaffe 2005; Murphy et al. 2006b; Gant and Thibault 2008). Enhancement of the AHP in hippocampal neurons in aged animals correlates with impaired performance on learning paradigms that depend on a functional hippocampus, such as trace eyeblink and spatial water maze (Moyer Jr et al. 2000; Tombaugh et al. 2005; Murphy et al. 2006a). Pharmaceuticals aimed at reducing the AHP and increasing basal excitability (Moyer Jr et al. 1992; Moyer Jr and Disterhoft 1994) have been successful at restoring performance of aged rats on trace eyeblink conditioning (Deyo et al. 1989; Straube et al. 1990; Kowalska and Disterhoft 1994). Interestingly, AHPs from neurons recorded from aged learners are indistinguishable from young learners; both are reduced compared to that of aged weak-learners (Moyer Jr et al. 2000; Tombaugh et al. 2005). These data suggest that mechanisms that permit learning-related modulation of the AHP are also critical determinants of learning abilities in an aged population. To date, age-related impairments in hippocampal-dependent tasks and biophysical alterations in hippocampal neurons have largely focused on studies that compare animals at extreme ends of the aging spectrum.In an effort to better understand physiological changes that underlie the onset of early cognitive decline, the development of rodent models of “normal” age-associated cognitive decline (AACD), as well as mild cognitive impairment (MCI), is critical (Pepeu 2004). Therefore, we set out to characterize the development of age-related deficits indicative of hippocampal dysfunction in middle-aged C57Bl6/SJL mice and to examine the biophysical changes in hippocampal neurons that accompany such deficits.Recently, age-related deficits in contextual fear memory following trace fear conditioning were reported in a subset of middle-aged rats (Moyer Jr and Brown 2006). Because the dorsal hippocampus is critical for trace and contextual fear conditioning in mice and rats (McEchron et al. 1998; Chowdhury et al. 2005; Misane et al. 2005), trace fear conditioning is an ideal paradigm for exploring cellular mechanisms that underlie early-age-related cognitive decline.Here we investigate the effects of “early” aging on trace fear conditioning by comparing performance outcomes of young (2 mo, n = 7; and 4 mo, n = 8) and middle-aged (8 mo, n = 22) male C57/SJL F1 hybrid mice. Mice were trained and tested singly, and the experimenter was blind to the training and retention status of the mice. All animal procedures were approved by the Northwestern University Animal Care and Use Committee. Preliminary data were reported previously (Kaczorowski 2006).To assess hippocampal function with aging, young and middle-aged mice were trained on a trace fear conditioning task followed by retention tests of the auditory conditioned stimulus (CS) and contextual CS memory. The basic protocol for trace fear conditioning has been described previously (Ohno et al. 2006a). Mice were trained in a Plexiglas conditioning chamber with a stainless-steel floor grid used for shock delivery. After the baseline period (150 sec), mice received four pairings of the CS (tone; 15 sec, 3 kHz, 75 dB) and US (shock; 1 sec, 0.7 mA). The CS and unconditioned stimulus (US) were separated by a 30-sec empty trace interval. The intertrial interval was set at 210 ± 10 sec. The training chamber was wiped with 95% ethyl alcohol, illuminated with a 10-W bulb in an otherwise dark room, and provided with 65-dB white noise to make it distinct. During training on trace fear conditioning, no effect of age was observed on measures of baseline freezing (F_(2,34) = 2.0, P = 0.15), the expression of freezing during tone (F_(2,34) = 0.6, P = 0.6), or post-shock freezing (F_(2,34) = 0.2, P = 0.8), suggesting that middle-aged and young mice do not differ in measures of anxiolysis or expression of behavioral freezing (measured index of fear) (Fig. 1A).Open in a separate windowFigure 1.Onset of early aging deficits in 8-mo-old middle-aged mice. (A) Baseline (BL) freezing and auditory CS freezing during trace fear conditioning was similar between young (2 mo and 4 mo) and middle-aged (8 mo) mice. (B1) Mean baseline freezing and retention of the auditory CS memory (tones 1–4) were comparable in young (2 mo and 4 mo) and middle-aged (8 mo) mice. (B2) Middle-aged mice showed a significant decrease in freezing compared to young (2 mo and 4 mo) mice when exposed to the original context chamber where they had been trained 1 d earlier; (*) P < 0.05.Retention of the auditory CS:US memory was tested 24 h later in a novel context that differed in its location, size, scent, lighting, background noise, and flooring (bedding) compared to the training chamber. Data from three mice (one young, two middle-aged) were excluded because of video malfunction. Following a 150-sec baseline, mice received four presentations of the tone CS in the absence of footshock. Neither baseline freezing (F_(2,31) = 2.6, P = 0.1) nor conditional freezing in response to the tone CS (F_(2,31) = 1.6, P = 0.2) differed between young and middle-aged mice (Fig. 1B1). Thus, retention of the auditory CS following trace fear conditioning was intact in middle-aged compared to young mice. Although deficits in retention of auditory trace fear have been reported in aged mice and rats (Blank et al. 2003; McEchron et al. 2004; Villarreal et al. 2004), the results herein agree with report of intact trace fear memory in middle-aged rats (Moyer Jr and Brown 2006).One hour after this testing, retention of the contextual fear memory was assessed by placing mice in the original context (in the absence of the tone and footshock) and measuring freezing for 10 min. A subtle but significant difference in freezing was observed as a function of age (F_(2,31) = 4.3, P = 0.02; Fig. 1B ​2).2). A student''s post-hoc t-test revealed that mean freezing (collapsed across 10 min) of middle-aged mice was reduced compared to 2-mo (P < 0.05) and 4-mo (P < 0.05) young mice. Contextual fear memory deficits have been similarly reported in aged mice (Fukushima et al. 2008). Studies that failed to observe contextual fear deficits in aged (>18 mo) mice may result from a floor effect because young mice showed weak conditioning to the context (∼30% freezing) (Feiro and Gould 2005; Gould and Feiro 2005) or employment of delay (Corcoran et al. 2002; Feiro and Gould 2005; Gould and Feiro 2005) compared to trace procedures (Moyer Jr and Brown 2006). Although differences in experimental parameters are plausible, heterogeneity in the performance of aged mice may make detection of age-related impairments difficult owing to increased variability.Open in a separate windowFigure 2.Selective deficits on retention of contextual fear in middle-aged weak-learner mice. (A,B) Summary plot and histogram show young mice (100%, n = 6) at 2 mo of age showed robust recall of contextual fear memory (range 75%–99%) with mean and standard deviation (SD) of 91% ± 10%, whereas retention of middle-aged mice (n = 21) varied to a much greater extent (range 21%–95%; M, SD = 74% ± 19%). Distribution of middle-aged mice relative to their mean percent freezing shows two distinct populations. Middle-aged mice with freezing levels less than 3 SD from the mean freezing in young wild-type (WT) mice (61%, dashed line) were characterized as having weak contextual fear memory (weak learners) and those with freezing levels ≥62% as having strong contextual fear memory (learners). (C) Baseline (BL) freezing, expression of post-shock freezing and freezing during retention tests for auditory CS and trace CS memories, were comparable in both weak-learners and learners. (D) Selective deficits in retention of contextual fear memories were observed in middle-aged weak learners as compared to middle-aged learners; (*) P < 0.05.Previous studies in the rat report heterogeneity in spatial water maze and contextual fear conditioning in middle-aged and/or aged rats compared to young animals (Fischer et al. 1992; Wyss et al. 2000; Moyer Jr and Brown 2006). Therefore, we determined if middle-aged impairments of context fear (Fig. 2A) were driven by a subset of impaired mice. The degree of age-related impairment in each middle-aged mouse was determined by comparison to a reference group of young mice tested concurrently (shown in Fig. 1). The behavioral criterion for retention of contextual fear in middle-aged mice was set at 61%, which was 3 standard deviations (SD) below the mean freezing in young mice (mean and SD, 91% ± 10%; Fig. 1). A bimodal distribution of freezing of middle-aged mice was observed (Fig. 2B), where 70% of middle-aged mice performed above criterion and were labeled learners (n = 14), and 30% of middle-aged mice performed below criterion and were labeled as weak learners (n = 6). Comparison on measures of baseline freezing (F_(1,18) = 1.8, P = 0.2) and expression of post-shock freezing (F_(1,18) = 2.1, P = 0.2) revealed no differences between the groups during auditory trace fear training (Fig. 2C). Similarly, no differences in baseline freezing (F_(1,18) = 0.03, P = 0.9) or acquisition/recall of conditioned auditory trace fear (tone, F_(1,18) = 0.08, P = 0.8; trace, F_(1,18) = 2.4, P = 0.1) were observed 24 h later during retention tests. Thus, deficits ascribed to middle-aged weak learners were limited to contextual processing/retention, where middle-aged weak learners responded to the contextual CS with significantly lower levels of freezing compared to middle-aged learners (F_(1,18) = 47, P = 0.001; Fig. 2D). To summarize, we found that onset of cognitive decline in the C56Bl6/SJL mice was first apparent in a subset of middle-aged mice. Middle-aged weak learners showed a mild but specific deficit in hippocampal-dependent contextual learning/memory (spatial learning) but not hippocampal-dependent auditory trace learning/memory (temporal learning), assessed following trace fear conditioning.Given that contextual fear deficits occurred in a subset of middle-age mice, we were able to directly assess age-related alterations in excitability and AHP plasticity in CA1 neurons as they relate to learning abilities (learners vs. weak learners). Within 1 h of cessation of behavioral tests, middle-aged learners and weak learners were decapitated under deep halothane anesthesia and their brains quickly removed and placed into ice-cold artificial cerebral spinal fluid (aCSF): 125 mM NaCl, 25 mM glucose, 25 mM NaHCO₃, 2.5 mM KCl, 1.25 mM NaH₂PO₄, 2 mM CaCl₂, 1 MgCl₂ (pH 7.5, bubbled with 95%O₂/5%CO₂). Naïve mice were removed from their home cage and underwent identical decapitation procedures. Slices (300 μm) of the dorsal hippocampus and adjacent cortex were made using a Leica vibratome. The slices were first incubated for 30 min at 34°C in bubbled aCSF, and held at room temperature in bubbled aCSF for 1–4 h before use. Recording electrodes prepared from thin-walled capillary glass were filled with potassium methylsulfate-based internal solution and had a resistance of 5–6 MΩ.Whole-cell current-clamp recordings were performed on CA1 hippocampal pyramidal neurons of middle-aged learners (n = 36, 14 mice) and weak-learners (n = 15, 6 mice), as well as middle-aged naïve mice (n = 35 cells, 18 mice). Neuronal excitability was compared by measuring the post-burst AHP generated by 25 action potentials at 50 Hz (Fig. 3A), a stimulus shown to reliably evoke an AHP of sizable—but not maximal—amplitude from hippocampal neurons of mice (Ohno et al. 2006b). A significant difference in the peak amplitude of the AHP from learners, weak learners, and naïve mice was observed (F_(2,83) = 5, P < 0.01). Because the peak AHP and sAHP amplitudes did not differ between neurons from weak-learners and naïve mice (Fig. 3B). No differences in membrane resistance (F_(2,83) = 1.6, P = 0.2) or action potential properties, elicited using a brief (2 msec) near threshold current step (pA), were observed (Open in a separate window^aP < 0.05 compared to Weak L.^bP < 0.05 compared to Naïve.^cP < 0.05 compared to Pooled.Open in a separate windowFigure 3.Learning-related AHP plasticity is impaired in middle-aged weak-learner mice. (A) Representative traces showing the sAHP is reduced in neurons from (black) middle-aged learners compared to (blue) weak-learner mice and (gray) naïve mice. (Inset) The medium AHP (mAHP) of neurons from (black) learner mice was decreased compared to (blue) weak-learner mice and (gray) naïve mice. (B) No differences in the AHP from naïve and weak learners were observed; therefore, their data were pooled, and mean AHP was plotted by time on a log scale. (Inset) The mean amplitude of the peak AHP (1 msec) and sAHP (600 msec) was significantly reduced in neurons from learners compared to AHPs from weak learners and naïve labeled control; (*)P < 0.05.The results presented here are important in two respects. First, we demonstrate that the successful acquisition and recall of trace fear conditioning results in a significant reduction in the AHP in CA1 hippocampal neurons from the mouse. Our data are similar to previous reports showing learning-related reductions of the AHP in hippocampal neurons following training on hippocampal-dependent tasks (Disterhoft and Oh 2007) and thus strengthen the case for neuronal excitability change as a general mechanism underlying hippocampal-dependent learning. Second, we demonstrate that the onset of age-related cognitive decline in the C56Bl6/SJL mouse (termed “weak learners”) first manifests as a specific deficit in spatial associative learning in a subset of middle-age mice. These data, combined with a previous report from middle-aged rats (Moyer Jr and Brown 2006), suggest that initiation of age-related hippocampal dysfunction results in specific spatial—as opposed to temporal—deficits in associative learning and memory during middle age. By combining trace fear conditioning with whole-cell patch-clamp recordings in middle-aged mice, we revealed that “early” age-related impairments in spatial associative learning—like those in the aged hippocampus (Tombaugh et al. 2005)—result in part from an impairment of AHP plasticity of hippocampal neurons. Because AHP reductions are poised to facilitate mechanisms crucial for information storage, it is interesting that trace fear conditioning facilitates the long-term potentiation (LTP) of field excitatory postsynaptic potentials in the CA1 region of the rat hippocampus (Song et al. 2008).Generally speaking, both LTP and activation of AHP currents (I_AHP and sI_AHP) are sensitive to changes in intracellular Ca²⁺ (Storm 1990; Sah 1996; Malenka and Nicoll 1999). Thus, dysregulation of Ca²⁺ homeostasis in the hippocampus of middle-aged rats via enhancement of Ca²⁺-induced Ca²⁺ release (CICR) is an important finding (Gant et al. 2006). Age-related enhancement of Ca²⁺-dependent AHPs has been shown to raise the threshold for induction of LTP (Kumar and Foster 2004). These data support our hypothesis that impairments in contextual fear reported herein, as well as deficits in spatial water maze reported in middle-aged rats (Frick et al. 1995; Markowska 1999; Kadish et al. 2009), result from dysfunction of AHP plasticity.Studies in middle-aged mice have important implications for the treatment of “normal” age-associated cognitive decline (AACD), as well as mild cognitive impairment (MCI) (Pepeu 2004). Further studies aim to examine alterations in cholinergic function in our middle-aged mouse model, as the cholinergic agonist carbachol suppressed the AHP in neurons from naïve middle-aged mice (Supplemental Fig. 1). Activation of cholinergic receptors shape neuronal excitability and synaptic throughput (Tai et al. 2006) through multiple Ca²⁺-dependent processes (Gahwiler and Brown 1987; Tai et al. 2006). Restoration of cholinergic function has been shown to rescue deficits on hippocampal-dependent tasks in aged rodent and mouse models of Alzheimer''s disease (AD) (Disterhoft and Oh 2006), as well as in human AD patients (Cummings et al. 1998; Morris et al. 1998; Pettigrew et al. 1998), and therefore is a potential target aimed at the rescue of early age-related cognitive decline.     

Cortical barrel lesions impair whisker-CS trace eyeblink conditioning

Whisker deflection is an effective conditioned stimulus (CS) for trace eyeblink conditioning that has been shown to induce a learning-specific expansion of whisker-related cortical barrels, suggesting that memory storage for an aspect of the trace association resides in barrel cortex. To examine the role of the barrel cortex in acquisition and retrieval of trace eyeblink associations, the barrel cortex was lesioned either prior to (acquisition group) or following (retention group) trace conditioning. The acquisition lesion group was unable to acquire the trace conditioned response, suggesting that the whisker barrel cortex is vital for learning trace eyeblink conditioning with whisker deflection as the CS. The retention lesion group exhibited a significant reduction in expression of the previously acquired conditioned response, suggesting that an aspect of the trace association may reside in barrel cortex. These results demonstrate that the barrel cortex is important for both acquisition and retention of whisker trace eyeblink conditioning. Furthermore, these results, along with prior anatomical whisker barrel analyses suggest that the barrel cortex is a site for long-term storage of whisker trace eyeblink associations.     

Hippocampal CA1 spiking during encoding and retrieval: relation to theta phase

The hippocampal theta rhythm is a prominent oscillation in the field potential observed throughout the hippocampus as a rat investigates stimuli in the environment. A recent computational model [Hasselmo, M. E., Bodelon, C., & Wyble, B. P. (2002a). A proposed function for hippocampal theta rhythm: separate phases of encoding and retrieval enhance reversal of prior learning. Neural Computation, 14, 793-817. Neuromodulation, theta rhythm and rat spatial navigation. Neural Networks, 15, 689-707] suggested that the theta rhythm allows the hippocampal formation to alternate rapidly between conditions that promote memory encoding (strong synaptic input from entorhinal cortex to areas CA3 and CA1) and conditions that promote memory retrieval (strong synaptic input from CA3 to CA1). That model predicted that the preferred theta phase of CA1 spiking should differ for information being encoded versus information being retrieved. In the present study, the spiking activity of CA1 pyramidal cells was recorded while rats performed either an odor-cued delayed nonmatch-to-sample recognition memory test or an object recognition memory task based on the animal's spontaneous preference for novelty. In the test period of both tasks, the preferred theta phase exhibited by CA1 pyramidal cells differed between moments when the rat inspected repeated (match) and non-repeated (nonmatch) items. Also in the present study, additional modeling work extended the previous model to address the mean phase of CA1 spiking associated with stimuli inducing varying levels of retrieval relative to encoding, ranging from novel nonmatch stimuli with no retrieval to highly familiar repeated stimuli with extensive retrieval. The modeling results obtained here demonstrated that the experimentally observed phase differences are consistent with different levels of CA3 synaptic input to CA1 during recognition of repeated items.     

The role of awareness in delay and trace fear conditioning in humans

Expression of conditional fear without awareness has been previously demonstrated during delay conditioning, a procedure in which the conditioned stimulus (CS) and unconditioned stimulus (UCS) overlap. However, less is known about the role of awareness in trace fear conditioning, where an interval of time separates the CS and UCS. The present study assessed skin conductance response (SCR) and UCS expectancy during delay and trace conditioning. UCS predictability was varied on a trial-bytrial basis by presenting perithreshold auditory CSs. Differential UCS expectancies were demonstrated only on perceived delay and trace trials. Learning-related SCRs were observed during both perceived and unperceived delay CSs. In contrast, differential SCRs were demonstrated only for perceived trace CSs. These data suggest that awareness is necessary for conditional responding during trace, but not delay, fear conditioning.