Hepatocyte transplantation has been used to treat liver disease. The availability of cells for these procedures is quite limited. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) may be a useful source of hepatocytes for basic research and transplantation if efficient and effective differentiation protocols were developed and problems with tumorigenicity could be overcome. Recent evidence suggests that the cell of origin may affect hiPSC differentiation. Thus, hiPSCs generated from hepatocytes may differentiate back to hepatocytes more efficiently than hiPSCs from other cell types. We examined the efficiency of reprogramming adult and fetal human hepatocytes. The present studies report the generation of 40 hiPSC lines from primary human hepatocytes under feeder-free conditions. Of these, 37 hiPSC lines were generated from fetal hepatocytes, 2 hiPSC lines from normal hepatocytes and 1 hiPSC line from hepatocytes of a patient with Crigler-Najjar Syndrome, Type-1. All lines were confirmed reprogrammed and expressed markers of pluripotency by gene expression, flow cytometry, immunocytochemistry, and teratoma formation. Fetal hepatocytes were reprogrammed at a frequency over 50-fold higher than adult hepatocytes. Adult hepatocytes were only reprogrammed with 6 factors, while fetal hepatocytes could be reprogrammed with 3 (OCT4, SOX2, NANOG) or 4 factors (OCT4, SOX2, NANOG, LIN28 or OCT4, SOX2, KLF4, C-MYC). The increased reprogramming efficiency of fetal cells was not due to increased transduction efficiency or vector toxicity. These studies confirm that hiPSCs can be generated from adult and fetal hepatocytes including those with genetic diseases. Fetal hepatocytes reprogram much more efficiently than adult, although both could serve as useful sources of hiPSC-derived hepatocytes for basic research or transplantation.
metabolic liver disease; reprogramming efficiency; induced pluripotent stem cells (iPSCs); fetal hepatocytes
In rat models of drug relapse and craving, cue-induced cocaine seeking progressively increases after drug withdrawal. This ‘incubation of cocaine craving’ is partially mediated by time-dependent adaptations at glutamatergic synapses in nucleus accumbens. However, the circuit-level adaptations mediating this plasticity remain elusive. Here we studied silent synapses—often regarded as immature synapses that express stable NMDA receptors with AMPA receptors either absent or labile—in basolateral amygdala-to-accumbens projection in incubation of cocaine craving. Silent synapses were detected within this projection during early withdrawal from cocaine. As the withdrawal period progressed, these silent synapses became ‘unsilenced’, a process involving synaptic insertion of calcium-permeable AMPA receptors (CP-AMPARs). In vivo optogenetic stimulation-induced downregulation of CP-AMPARs at amygdala-to-NAc synapses, which re-silenced some of the previously silent synapses after prolonged withdrawal, decreased cocaine incubation. Our finding indicates that silent synapse-based reorganization of the amygdala-to-accumbens projection is critical for persistent cocaine craving and relapse after withdrawal.
cocaine; incubation; silent synapse; accumbens; amygdala; NMDA receptor
Medium spiny neurons (MSNs) within the nucleus accumbens shell (NAc) function to gate and prioritize emotional/motivational arousals for behavioral output. The neuronal output NAc MSNs is mainly determined by the integration of membrane excitability and excitatory/inhibitory synaptic inputs. Whereas cocaine-induced alterations at excitatory synapses and membrane excitability have been extensively examined, the overall functional output of NAc MSNs following cocaine exposure still poorly defined because little is known about whether inhibitory synaptic input to these neurons is affected by cocaine. Here, our results demonstrate multidimensional alterations at inhibitory synapses in NAc neurons following cocaine self-administration in rats. Specifically, the amplitude of miniature (m) inhibitory postsynaptic currents (IPSCs) was decreased after 21-d withdrawal from 5-d cocaine self-administration. Upon re-exposure to cocaine after 21-day withdrawal, whereas the amplitude of mIPSCs remained down-regulated, the frequency became significantly higher. Furthermore, the reversal potential of IPSCs, which was not significantly altered during withdrawal, became more hyperpolarized upon cocaine re-exposure. Moreover, the relative weight of excitatory and inhibitory inputs to NAc MSNs was significantly decreased after 1-d cocaine withdrawal, increased after 21-d withdrawal, and returned to the basal level upon cocaine re-exposure after 21-d withdrawal. These results, taken together with previous results showing cocaine-induced adaptations at excitatory synapses and intrinsic membrane excitability of NAc MSNs, may provide a relatively thorough picture of the functional state of NAc MSNs following cocaine exposure.
IPSC; EPSC; GABA; accumbens; cocaine; reversal potential
The neuroadaptation theory of addiction suggests that, similar to the development of most memories, exposure to drugs of abuse induces adaptive molecular and cellular changes in the brain which likely mediate addiction-related memories or the addictive state. Compared to other types of memories, addiction-related memories develop fast and last extremely long, suggesting that the cellular and molecular processes that mediate addiction-related memories are exceptionally adept and efficient. We recently demonstrated that repeated exposure to cocaine generated a large portion of “silent” glutamatergic synapses within the nucleus accumbens (NAc). Silent glutamatergic synapses are synaptic connections in which only N-methyl-D-aspartic acid receptor (NMDAR)-mediated responses are readily detected whereas alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are absent or highly labile. Extensive experimental evidence suggests that silent synapses are conspicuously efficient plasticity sites at which long-lasting plastic changes can be more easily induced and maintained. Thus, generation of silent synapses can be regarded as a process of metaplasticity, which primes the NAc for subsequent durable and robust plasticity for addiction-related memories. Focusing on silent synapse-based metaplasticity, this review discusses how key brain regions, such as the NAc, utilize the metaplasticity mechanism to optimize the plasticity machineries to achieve fast and durable plastic changes following exposure to cocaine. A summary of recent related results suggests that upon cocaine exposure, newly generated silent synapses may prime excitatory synapses within the NAc for long-term potentiation (LTP), thus setting the direction of future plasticity. Furthermore, because cocaine-generated silent synapses are enriched in NMDARs containing the NR2B subunit, the enhanced NR2B-signaling may set up a selective recruitment of certain types of AMPARs. Thus, silent synapse-based metaplasticity may lead to not only quantitative but also qualitative alterations in excitatory synapses within the NAc. This review is one of the first systematic analyses regarding the hypothesis that drugs of abuse induce metaplasticity, which regulates the susceptibility, the direction, and the molecular details of subsequent plastic changes. Taken together, metaplasticity ultimately serves as a key step in mediating cascades of addiction-related plastic alterations.
metaplasticity; silent synapses; cocaine; NMDA; AMPA; membrane excitability
Few behavioral interventions have been conducted to reduce high-risk sexual behavior among HIV-positive Men who have Sex with Men (HIV+MSM). Hence, we lack well-proven interventions for this population.
Positive Connections is a randomized controlled trial (n=675 HIV+MSM) comparing the effects of two sexual health seminars – for HIV+MSM and all MSM – with a contrast prevention video arm. Baseline, 6-, 12- and 18-month follow-up surveys assessed important psychosexual variables and frequency of serodiscordant unprotected anal intercourse (SDUAI).
At post-test, intentions to avoid transmission were significantly higher in the sexual health arms. However, SDUAI frequency decreased equally across all arms, from 15.0 at baseline to 11.5 at 18 months. HIV+MSM engaging in SDUAI at baseline were more likely to leave the study.
Tailoring interventions to HIV+MSM does not appear to increase the effectiveness of HIV prevention. A sexual health approach appears no more effective than video-based HIV prevention.
HIV+MSM; MSM; HIV prevention; behavioral interventions; unsafe sex; prevention for positives
The nucleus accumbens shell (NAc) is a key brain region mediating emotional and motivational learning. In rodent models, dynamic alterations have been observed in synaptic N-Methyl-D-aspartic acid receptors (NMDARs) within the NAc following incentive stimuli, and some of these alterations are critical for acquiring new emotional/motivational states. NMDARs are prominent molecular devices for controlling neural plasticity and memory formation. Although synaptic NMDARs are predominately located postsynaptically, recent evidence suggests that they may also exist at presynaptic terminals and reshape excitatory synaptic transmission by regulating presynaptic glutamate release. However, it remains unknown whether presynaptic NMDARs exist in the NAc to contribute to emotional and motivational learning. In an attempt to identify presynaptically-located NMDARs in the NAc, the present study utilizes slice electrophysiological recording combined with pharmacological and genetic tools to examine the physiological role of the putative presynaptic NMDARs in rats. Our results show that application of glycine, the glycine-site agonist of NMDARs, potentiated presynaptic release of glutamate at excitatory synapses on NAc neurons, whereas application of 5,7-dichlorokynurenic acid (DCKA) or 7-chlorokynurenic acid, the glycine-site antagonists of NMDARs, produced the opposite effect. However, these seemingly presynaptic NMDAR-mediated effects could not be prevented by application of D-APV, the glutamate-site NMDAR antagonist, and were still present in the mice in which NMDAR NR1 or NR3 subunits were genetically deleted. Thus, rather than suggesting the existence of presynaptic NMDARs, our results support the idea that an unidentified type of glycine-activated substrate may account for the presynaptic effects appearing to be mediated by NMDARs.
NMDA receptor; presynaptic; NR3; NR1; d-serine; 5,7-dichlorokynurenic acid
Locomotor sensitization is a common and robust behavioral alteration in rodents whereby following exposure to abused drugs such as cocaine, the animal becomes significantly more hyperactive in response to an acute drug challenge. Here, we further analyzed the role of cocaine-induced silent synapses in the nucleus accumbens (NAc) shell and their contribution to the development of locomotor sensitization. Using a combination of viral vector-mediated genetic manipulations, biochemistry and electrophysiology in a locomotor sensitization paradigm with repeated, daily noncontingent cocaine (15 mg/kg) injections, we show that dominant negative cAMP-element binding protein (CREB) prevents cocaine-induced generation of silent synapses of young (30 d) rats, whereas constitutively active CREB is sufficient to increase the number of NR2B-containing NMDA receptors (NMDAR) at synapses and to generate silent synapses. We further show that occupancy of CREB at the NR2B promoter increases and is causally related to the increase in synaptic NR2B levels. Blockade of NR2B-containing NMDARs by administration of the NR2B-selective antagonist Ro256981 directly into the NAc, under conditions that inhibit cocaine-induced silent synapses, prevents the development of cocaine-elicited locomotor sensitization. Our data are consistent with a cellular cascade whereby cocaine-induced activation of CREB promotes CREB-dependent transcription of NR2B and synaptic incorporation of NR2B-containing NMDARs, which generates new silent synapses within the NAc. We propose that cocaine-induced activation of CREB and generation of new silent synapses may serve as key cellular events mediating cocaine-induced locomotor sensitization. These findings provide a novel cellular mechanism that may contribute to cocaine-induced behavioral alterations.
cocaine; silent synapse; accumbens; NMDA receptor; CREB; NR2B
Studies over the past decade have enunciated silent synapses as prominent cellular substrates for synaptic plasticity in the developing brain. However, little is known about whether silent synapses can be generated post-developmentally. Here, we demonstrate that highly salient in vivo experience, such as exposure to cocaine, generates silent synapses in the nucleus accumbens (NAc) shell, a key brain region mediating addiction-related learning and memory. Furthermore, this cocaine-induced generation of silent synapses is mediated by membrane insertions of new, NR2B–containing N-methyl-D-aspartic acid receptors (NMDARs). These results provide evidence that silent synapses can be generated de novo by in vivo experience and thus may act as highly efficient neural substrates for the subsequent experience-dependent synaptic plasticity underlying extremely long-lasting memory.
silent synapse; NMDA receptor; NR2B; cocaine; accumbens
Head direction (HD) cells discharge as a function of the rat’s directional orientation with respect to its environment. Because animals with posterior parietal cortex (PPC) lesions exhibit spatial and navigational deficits, and the PPC is indirectly connected to areas containing HD cells, we determined the effects of bilateral PPC lesions on HD cells recorded in the anterodorsal thalamus. HD cells from lesioned animals had similar firing properties compared to controls and their preferred firing directions shifted a corresponding amount following rotation of the major visual landmark. Because animals were not exposed to the visual landmark until after surgical recovery, these results provide evidence that the PPC is not necessary for visual landmark control or the establishment of landmark stability. Further, cells from lesioned animals maintained a stable preferred firing direction when they foraged in the dark and were only slightly less stable than controls when they self-locomoted into a novel enclosure. These findings suggest that PPC does not play a major role in the use of landmark and self-movement cues in updating the HD cell signal, or in its generation.
path-integration; vestibular; navigation; anterodorsal thalamic nucleus; spatial orientation
Homeostatic plasticity is a powerful cellular mechanism through which neurons adjust intracellular and intercellular resources to stabilize their functional output through the ever-changing environment. Here, we report a novel form of homeostatic plasticity that nucleus accumbens (NAc) neurons use to regain their functionally active state once it is lost. In vivo, NAc neurons periodically alternate between a functionally active upstate and a functionally quiescent downstate. The upstate of NAc neurons is immediately lost following severe environmental changes, such as deep anesthesia and truncation of excitatory synaptic inputs. Using short-term slice cultures, our current study demonstrates that NAc neurons initially lose but gradually recover their upstate-downstate cycling after shifting to the in vitro condition. Furthermore, we show that this homeostatic recovery of the upstate is mediated by increased synchronization of presynaptic activity. Given that being in the upstate is required for in vivo NAc neurons to fire action potentials, the homeostatic recovery of upstate may underlie an important cellular mechanism for NAc neurons to maintain their functional output against severe environmental fluctuations.