The age of an experimental animal can be a critical variable, yet age matters are often overlooked within neuroscience. Many studies make use of young animals, without considering possible differences between immature and mature subjects. This is especially problematic when attempting to model traits or diseases that do not emerge until adulthood. In this commentary we discuss the reasons for this apparent bias in age of experimental animals, and illustrate the problem with a systematic review of published articles on long-term potentiation. Additionally, we review the developmental stages of a rat and discuss the difficulty in using the weight of an animal as a predictor of its age. Finally, we provide original data from our laboratory and review published data to emphasise that development is an ongoing process that does not end with puberty. Developmental changes can be quantitative in nature, involving gradual changes, rapid switches, or inverted U-shaped curves. Changes can also be qualitative. Thus, phenomena that appear to be unitary may be governed by different mechanisms at different ages. We conclude that selection of the age of the animals may be critically important in the design and interpretation of neurobiological studies.

Repeated non-contingent cocaine injections, which lead to behavioral sensitization, increase α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor (AMPAR) transmission in the rodent nucleus accumbens (NAc) in a withdrawal-dependent manner. On withdrawal days (WD) 10-21, this is attributable to upregulation of GluA1A2-containing AMPARs. However, synaptic incorporation of GluA2-lacking/Ca2+ permeable AMPARs (CP-AMPARs) was observed after longer withdrawal (WD35) from repeated non-contingent cocaine injections in young mice (Mameli et al., 2009). CP-AMPARs had previously been observed in NAc synapses only after prolonged (WD30-47) withdrawal from extended access cocaine self-administration. Our goal was to determine if rats receiving repeated non-contingent cocaine injections during adulthood similarly exhibit CP-AMPARs in the NAc after prolonged withdrawal. For comparison, we began by evaluating CP-AMPARs on WD35-49 after extended access cocaine self-administration. Confirming our previous results, whole cell recordings revealed inwardly rectifying AMPAR excitatory postsynaptic currents (EPSCs), a hallmark of CP-AMPARs. This was observed in both core and shell. Next, we conducted the same analysis in adult rats treated with 8 daily non-contingent cocaine injections and recorded on WD35-49. AMPAR EPSCs in core and shell did not show inward rectification, and were insensitive to 1-naphthylacetylspermine (Naspm; a selective antagonist of CP-AMPARs). Locomotor sensitization could still be demonstrated after this long withdrawal period, although the upregulation of GluA1A2-containing AMPARs observed at earlier withdrawal times was no longer detected. In conclusion, in adult rats, accumulation of synaptic CP-AMPARs in the NAc occurs after prolonged withdrawal from extended access cocaine self-administration but not after prolonged withdrawal from non-contingent cocaine injections.

Following prolonged withdrawal from extended access cocaine self-administration in adult rats, high conductance Ca2+-permeable AMPA receptors (CP-AMPARs) accumulate in nucleus accumbens (NAc) synapses and mediate the expression of “incubated” cue-induced cocaine craving. Using patch clamp recordings from NAc slices prepared after extended access cocaine self-administration and >45 days of withdrawal, we found that group I metabotropic glutamate receptor (mGluR) stimulation using 3,5-dihydroxyphenylglycine (DHPG; 50μM) rapidly eliminates the postsynaptic CP-AMPAR contribution to NAc synaptic transmission. This is accompanied by facilitation of Ca2+-impermeable AMPAR (CI-AMPAR)-mediated transmission, suggesting that DHPG may promote an exchange between CP-AMPARs and CI-AMPARs. In saline controls, DHPG also reduced excitatory transmission but this occurred through a CB1 receptor-dependent presynaptic mechanism rather than an effect on postsynaptic AMPARs. Blockade of CB1 receptors had no significant effect on the alterations in AMPAR transmission produced by DHPG in the cocaine group. Interestingly, the effect of DHPG in the cocaine group was mediated by mGluR1 whereas its effect in the saline group was mediated by mGluR5. These results indicate that regulation of synaptic transmission in the NAc is profoundly altered after extended access cocaine self-administration and prolonged withdrawal. Furthermore, they suggest that activation of mGluR1 may represent a potential strategy for reducing cue-induced cocaine craving in abstinent cocaine addicts.

Phasic changes in dopamine activity play a critical role in learning and goal-directed behavior. Unpredicted reward and reward predictive cues evoke phasic increases in the firing rate of the majority of midbrain dopamine neurons – results that predict uniformly broadcast increases in dopamine concentration throughout the striatum. However, measurement of dopamine concentration changes during reward has cast doubt on this prediction. We systematically measured phasic changes in dopamine in four striatal subregions (nucleus accumbens shell (Shell) and core (Core), dorsomedial (DMS) and dorsolateral striatum (DLS)) in response to stimuli known to activate a majority of dopamine neurons. We used fast-scan cyclic voltammetry in awake and behaving rats, which measures changes in dopamine on a similar timescale to the electrophysiological recordings that established a relationship between phasic dopamine activity and reward. Unlike the responses of midbrain dopamine neurons, unpredicted food reward and reward-predictive cues evoked a phasic increase in dopamine that was subregion specific. In rats with limited experience, unpredicted food reward evoked an increase exclusively in the Core. In rats trained on a discriminative stimulus paradigm, both unpredicted reward and reward-predictive cues evoked robust phasic dopamine in the Core and DMS. Thus, phasic dopamine release in select target structures is dynamic and dependent on context and experience. Since the four subregions assayed receive different inputs and have differential projection targets, the regional selectivity of phasic changes in dopamine has important implications for information flow through the striatum and plasticity that underlies learning and goal-directed behavior.

SK channels are Ca2؉ -activated K ؉ channels that underlie after hyperpolarizing (AHP) currents and contribute to the shaping of the firing patterns and regulation of Ca 2؉ influx in a variety of neurons. The elucidation of SK channel function has recently benefited from the discovery of SK channel enhancers, the prototype of which is 1-EBIO. 1-EBIO exerts profound effects on neuronal excitability but displays a low potency and limited selectivity. This study reports the effects of DCEBIO, an intermediate conductance Ca 2؉ -activated K ؉ channel modulator, and the effects of the recently identified potent SK channel enhancer NS309 on recombinant SK2 channels, neuronal apamin-sensitive AHP currents, and the excitability of CA1 neurons. NS309 and DCEBIO increased the amplitude and duration of the apamin-sensitive afterhyperpolarizing current without affecting the slow afterhyperpolarizing current in contrast to 1-EBIO. The potentiation by DCEBIO and NS309 was reversed by SK channel blockers. In current clamp experiments, NS309 enhanced the medium afterhyperpolarization (but not the slow afterhyperpolarization sAHP) and profoundly affected excitability by facilitating spike frequency adaptation in a frequency-independent manner. The potent and specific effect of NS309 on the excitability of CA1 pyramidal neurons makes this compound an ideal tool to assess the role of SK channels as possible targets for the treatment of disorders linked to neuronal hyperexcitability.In hippocampal pyramidal neurons voltage-independent, Ca 2ϩ -activated K ϩ channels are responsible for the generation of two distinct afterhyperpolarizing currents, I AHP 5 and sI AHP (1-4). I AHP is characterized by a time constant of decay of ϳ100 ms and by its sensitivity to the bee venom toxin, apamin, and to the scorpion toxins, scyllatoxin and tamapin (5-7). sI AHP is characterized by a slower time course (in the range of seconds), by its lack of sensitivity to apamin or any other classical K ϩ channel blocker, and by its modulation by several neurotransmitters (1-3, 8). Based on their kinetic and pharmacological features and on the results obtained from genetically manipulated mice, SK channels mediate I AHP , whereas the molecular correlate for sI AHP is still unknown (2-4, 9, 10). In addition to the use of selective blockers, an important contribution to the elucidation of the physiological role of SK and IK channels has arisen from the use of a small organic compound that enhances channel activity, the benzimidazolinone 1-EBIO (11-15). 1-EBIO enhances the activity of SK channels in the presence of the physiological activator, intracellular Ca 2ϩ , by increasing the apparent sensitivity of SK channels to Ca 2ϩ (14). As a consequence, 1-EBIO increases the amplitude of SK-mediated AHP currents and their duration in a variety of neurons, leading to profound changes in neuronal activity and firing patterns (14, 16 -18). Although 1-EBIO has been a useful tool to elucidate the function of SK channels in their native context, it has some important li...

Brief, high-concentration (phasic) spikes in nucleus accumbens dopamine critically participate in aspects of food reward. Although physiological state (e.g., hunger, satiety) and associated hormones are known to affect dopamine tone in general, whether they modulate food-evoked, phasic dopamine specifically is unknown. Here, we used fast-scan cyclic voltammetry in awake, behaving rats to record dopamine spikes evoked by delivery of sugar pellets while pharmacologically manipulating central receptors for the gut "hunger" hormone ghrelin. Lateral ventricular (LV) ghrelin increased, while LV ghrelin receptor antagonism suppressed the magnitude of dopamine spikes evoked by food. Ghrelin was effective when infused directly into the lateral hypothalamus (LH), but not the ventral tegmental area (VTA). LH infusions were made in close proximity to orexin neurons, which are regulated by ghrelin and project to the VTA. Thus, we also investigated and found potentiation of food-evoked dopamine spikes by intra-VTA orexin-A. Importantly, intra-VTA blockade of orexin receptors attenuated food intake induced by LV ghrelin, thus establishing a behaviorally relevant connection between central ghrelin and VTA orexin. Further analysis revealed that food restriction increased the magnitude of dopamine spikes evoked by food independent of any pharmacological manipulations. The results support the regulation of food-evoked dopamine spikes by physiological state with endogenous fluctuations in ghrelin as a key contributor. Our data highlight a novel mechanism by which signals relating physiological state could influence food reinforcement and food-directed behavior.

Adaptive motivated behavior requires rapid discrimination between beneficial and harmful stimuli. Such discrimination leads to the generation of either an approach or rejection response, as appropriate, and enables organisms to maximize reward and minimize punishment. Classically, the nucleus accumbens (NAc) and the dopamine projection to it are considered an integral part of the brain’s reward circuit, i.e., they direct approach and consumption behaviors and underlie positive reinforcement. This reward-centered framing ignores important evidence about the role of this system in encoding aversive events. One reason for bias toward reward is the difficulty in designing experiments in which animals repeatedly experience punishments; another is the challenge in dissociating the response to an aversive stimulus itself from the reward/relief experienced when an aversive stimulus is terminated. Here, we review studies that employ techniques with sufficient time resolution to measure responses in ventral tegmental area and NAc to aversive stimuli as they are delivered. We also present novel findings showing that the same stimulus – intra-oral infusion of sucrose – has differing effects on NAc shell dopamine release depending on the prior experience. Here, for some rats, sucrose was rendered aversive by explicitly pairing it with malaise in a conditioned taste aversion paradigm. Thereafter, sucrose infusions led to a suppression of dopamine with a similar magnitude and time course to intra-oral infusions of a bitter quinine solution. The results are discussed in the context of regional differences in dopamine signaling and the implications of a pause in phasic dopamine release within the NAc shell. Together with our data, the emerging literature suggests an important role for differential phasic dopamine signaling in aversion vs. reward.

Phasic dopamine signaling participates in associative learning by reinforcing associations between outcomes (unconditioned stimulus; US) and their predictors (conditioned stimulus; CS). However, prior work has always engendered these associations with innately rewarding stimuli. Thus, whether dopamine neurons can acquire prediction signals in the absence of appetitive experience and update them when the value of the outcome changes remains unknown. Here, we used sodium depletion to reversibly manipulate the appetitive value of a hypertonic sodium solution while measuring phasic dopamine signaling in rat nucleus accumbens. Dopamine responses to the NaCl US following sodium depletion updated independent of prior experience. In contrast, prediction signals were only acquired through extensive experience with a US that had positive affective value. Once learned, dopamine prediction signals were flexibly expressed in a state-dependent manner. Our results reveal striking differences with respect to how physiological state shapes dopamine signals evoked by outcomes and their predictors.nucleus accumbens | dopamine | voltammetry | learning | motivation R econciling differences between anticipated and experienced outcomes is fundamental for how an organism learns about the world. A key component of temporal difference (TD) learning models is the reward prediction error (RPE) term (1, 2), which is thought to be represented by phasic activity of midbrain dopamine neurons (3-5). Indeed, conditioned stimulus (CS)-related dopamine activity correlates with multiple behavioral indices of learning (6-8), and phasic dopamine signaling is sufficient to drive CSunconditioned stimulus (US) learning (9).In much of the supportive empirical work, food-or fluidrestricted animals first experience and then learn to anticipate an innately appetitive US (e.g., sucrose, juice, water). Thus, the US always has an inherent caloric, nutritive, or positive affective value to the organism. Consequently, it is uncertain whether dopamine neurons can acquire CS-US associations without first experiencing the US as a reward. Resolving this question is critical, because the striatal underpinnings of goal-directed behavior may encompass both RPE and experience-independent, model-based strategies (10, 11). One way to delineate dopamine's role in these different learning strategies would be to promote associations between a CS and a neutral or normally avoided US whose affective value could be manipulated and then determine the experience dependency of dopamine CS responses.Sodium appetite is an ideal platform on which to address this question. Sodium depletion induces a powerful sodium hunger and radically but reversibly alters the rewarding value of hypertonic NaCl solutions (12, 13). The appetite is highly selective for sodium and manifests independent of prior experience with either sodium solutions or sodium deficiency (14, 15). Therefore, sodium appetite facilitates the delivery of a US (hypertonic NaCl) that is rewarding only in a specific physiolog...