Jing Liang

Conditional neuroligin-2 knockout in adult medial prefrontal cortex links chronic changes in synaptic inhibition to cognitive impairments

Abnormal activity in the medial prefrontal cortex (mPFC) is consistently observed in neuropsychiatric disorders, but the mechanisms involved remain unclear. Chronic aberrant excitation and/or inhibition of mPFC neurons were proposed to cause cognitive impairments. However, direct evidence for this hypothesis is lacking because it is technically challenging to control synaptic properties in a chronic and locally restricted, yet specific, manner. Here, we generated conditional knockout (cKO) mice of neuroligin-2 (Nlgn2), a postsynaptic cell-adhesion molecule of inhibitory synapses linked to neuropsychiatric disorders. cKO of Nlgn2 in adult mPFC rendered Nlgn2 protein undetectable after already 2–3 weeks, but induced major reductions in synaptic inhibition after only 6–7 weeks, and caused parallel impairments in anxiety, fear memory and social interaction behaviors. Moreover, cKO of Nlgn2 severely impaired behavioral stimulation of immediate-early gene expression in the mPFC, suggesting that chronic reduction in synaptic inhibition uncoupled the mPFC from experience-dependent inputs. Our results indicate that Nlgn2 is required for continuous maintenance of inhibitory synapses in the adult mPFC, and that chronic impairment of local inhibition disengages the mPFC from its cognitive functions by partially uncoupling the mPFC from experience-induced inputs.

Repeated 2 Hz peripheral electrical stimulations suppress morphine-induced CPP and improve spatial memory ability in rats

Our previous studies have shown that 2 Hz peripheral electrical stimulation (PES) can suppress morphine-induced conditioned place preference (CPP) in the rat, although the mechanisms remain unclear. Since CPP involves the mechanism of learning and memory, it is rational to ask whether the suppressive effect of repeated 2 Hz PES on morphine-induced CPP is due to an impairment of the function of spatial learning and memory. Rats were trained with 4 mg/kg morphine, i.p. for 4 days to establish the CPP. Twenty-four hours after the CPP testing, they were given PES at 2 Hz once a day for 1, 3 or 5 days, followed by another CPP testing. The results showed that (1) the morphine-induced CPP was significantly inhibited by 3 or 5 consecutive sessions, but not by single session of 2 Hz PES. (2) A test of spatial leaning and memory ability using the Morris water maze task revealed that 2 Hz PES per se exhibited a promoting, rather than a deteriorating effect on the ability of spatial memory. (3) 2 Hz PES by itself produced a moderate yet significant CPP. The results imply that (a) a low frequency PES can produce a rewarding effect as revealed by the CPP testing, which may account, at least in part, for its suppressive effect on morphine induced CPP, (b) the suppressive effect of PES on morphine induced CPP is not due to a deteriorating effect on the ability of spatial memory.

Roles of BDNF, dopamine D(3) receptors, and their interactions in the expression of morphine-induced context-specific locomotor sensitization.

Drug seeking, craving, and relapse can be triggered by environmental stimuli that acquire motivational salience through repeated associations with the drugs effects. Previous studies indicated that the dopamine D(3) receptor (Drd3) might be involved in the expression of drug-conditioned responses in rats, and brain-derived neurotrophic factor (BDNF) could modulate Drd3 expression in the nucleus accumbens (NAc). However, the involvement of neural regions with Drd3 activation and the underlying interaction between BDNF and Drd3 in the expression of behavioral responses controlled by a drug-associated environment have remained poorly understood. The present study used a conditioning procedure to assess the roles of BDNF, Drd3, and their interactions in the NAc in the expression of morphine-induced context-specific locomotor sensitization. We showed that the expression of locomotor sensitization in the morphine-paired environment was accompanied by significantly increased expression of Drd3 mRNA and BDNF mRNA and protein levels. Both sensitized locomotion in morphine-paired rats and enhanced Drd3 mRNA were suppressed by intra-NAc infusion of anti-tyrosine kinase receptor B (TrkB) IgG. Furthermore, intra-NAc infusion of the Drd3-selective antagonist SB-277011A significantly decreased the expression of context-specific locomotor sensitization and upregulated BDNF mRNA. Altogether, these results suggest that BDNF/TrkB signaling and activation of Drd3 in the NAc are required for the expression of morphine-induced context-specific locomotor sensitization.

Morphine-induced conditioned place preference in rats is inhibited by electroacupuncture at 2 Hz: Role of enkephalin in the nucleus accumbens

Our previous studies have demonstrated that morphine-induced conditioned place preference (CPP) can be inhibited by 2 Hz electroacupuncture (EA). This inhibition can be blocked by either the opioid receptor antagonist naloxone (i.p.) or lesion in the nucleus accumbens (NAc), providing evidence that endogenous opioid system in the NAc mediates the effects of EA. Here we report that 1) A single session of 2 Hz EA produced a significant increase of the content of enkephalin in the NAc of morphine-induced CPP rats, and this effect was stronger in three consecutive sessions of EA; 2) Intracerebroventricular injection of the mu-opioid receptor antagonist CTAP or delta-opioid receptor antagonist NTI, but not kappa-opioid receptor antagonist nor-BNI, dose-dependently reversed the inhibitory effects of 2 Hz EA on the expression of morphine-induced CPP; 3) Three consecutive sessions of 2 Hz EA up-regulated the mRNA level of preproenkephalin in the NAc of morphine-induced CPP rats. The results suggest that the inhibitory effects of 2 Hz EA on the expression of the morphine CPP is mediated by mu- and delta-, but not kappa-opioid receptor, possibly via accelerating both the release and synthesis of enkephalin in the NAc. These findings support the possibility of using 2 Hz EA for the treatment of opiate addiction.

Coexistence of Anhedonia and anxiety-independent increased novelty-seeking behavior in the chronic mild stress model of depression

Previous research demonstrated excessive decreases in reward sensitivity and increases in harm avoidance in depressed individuals. These results straightly lead to a hypothesis that depressed patients should avoid novelty or express reduced novelty-seeking behavior. Nevertheless, literature in this regard is inconsistent. Furthermore, whether the potentially altered novelty-associated behavior is dependent on changed anxiety/fear or related to altered goal-directed approaching tendency is unclear. Here, we tested novel object-approaching behavior in a free-exploration paradigm in chronic mild stress (CMS)-induced anhedonic and stress-resistant rats respectively. Other CMS-induced, emotional behaviors were also examined in a battery of behavioral tests including novel cage, exploration, locomotor activity and elevated plus maze (EPM). We found that compared with controls, stress-resistant rats who consistently showed lower anxiety level in EPM (time in open arms) and, open-field (OF) test (time in central area) showed no sign of enhanced novel object approaching behavior. To the contrary, the anhedonic ones who did not express any sign of reduced anxiety showed paradoxically intensified novelty-approaching behavior. We concluded that reduced anxiety would not necessarily lead to enhanced novelty-seeking behavior; anhedonia coexists with anxiety-independent, increased novelty-seeking behavior. The salient paradox of coexistence of anhedonia and increased novelty-seeking behavior was critically discussed.

The possible involvement of endogenous ligands for mu-, delta-and kappa-opioid receptors in modulating morphine-induced CPP expression in rats

Previous studies suggested that electroacupuncture (EA) can suppress opioid dependence by the release of endogenous opioid peptides. To explore the site of action and the receptors involved, we tried to inject highly specific agonists for mu-, delta- and kappa-opioid receptors into the CNS to test whether it can suppress morphine-induced conditioned place preference (CPP) in the rat. Male Sprague-Dawley rats were trained with 4 mg/kg morphine, i.p. for 4 days to establish the CPP model. This CPP can be prevented by (a) i.p. injection of 3 mg/kg dose of morphine, (b) intracerebroventricular (i.c.v.) injection of micrograms doses of the selective mu-opioid receptor agonist DAMGO, delta-agonist DPDPE or kappa-agonist U-50,488H or (c) microinjection of DAMGO, DPDPE or U50488H into the shell of the nucleus accumbens (NAc). The results suggest that the release of endogenous mu-, delta- and kappa-opioid agonists in the NAc shell may play a role for EA suppression of opiate addiction.

N-methyl-D-aspartate receptor-mediated glutamate transmission in nucleus accumbens plays a more important role than that in dorsal striatum in cognitive flexibility.

Cognitive flexibility is a critical ability for adapting to an ever-changing environment in humans and animals. Deficits in cognitive flexibility are observed in most schizophrenia patients. Previous studies reported that the medial prefrontal cortex-to-ventral striatum and orbital frontal cortex-to-dorsal striatum circuits play important roles in extra- and intra-dimensional strategy switching, respectively. However, the precise function of striatal subregions in flexible behaviors is still unclear. N-methyl-D-aspartate receptors (NMDARs) are major glutamate receptors in the striatum that receive glutamatergic projections from the frontal cortex. The membrane insertion of Ca2+-permeable α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptors (AMPARs) depends on NMDAR activation and is required in learning and memory processes. In the present study, we measured set-shifting and reversal learning performance in operant chambers in rats and assessed the effects of blocking NMDARs and Ca2+-permeable AMPARs in striatal subregions on behavioral flexibility. The blockade of NMDARs in the nucleus accumbens (NAc) core by AP5 impaired set-shifting ability by causing a failure to modify prior learning. The suppression of NMDAR-mediated transmission in the NAc shell induced a deficit in set-shifting by disrupting the learning and maintenance of novel strategies. During reversal learning, infusions of AP5 into the NAc shell and core impaired the ability to learn and maintain new strategies. However, behavioral flexibility was not significantly affected by blocking NMDARs in the dorsal striatum. We also found that the blockade of Ca2+-permeable AMPARs by NASPM in any subregion of the striatum did not affect strategy switching. These findings suggest that NMDAR-mediated glutamate transmission in the NAc contributes more to cognitive execution compared with the dorsal striatum.

Gene expression of conditioned locomotion and context-specific locomotor sensitization controlled by morphine-associated environment

The nucleus accumbens (NAc) is involved in contextual drug associations, which might be particularly important for environmental cue-induced relapse to drug seeking. In the present study, rats were first administered repeated morphine for 5 days (5 mg/kg, i.p.) in a contextually paired and unpaired design. After reexposure to the morphine-associated environment, which induced conditioned locomotor activity in the morphine-paired group, we performed a rat 27k 70-mer oligo array to profile gene expression in the NAc. One hundred fifty-five upregulated and 88 downregulated genes were found in the paired group compared with the unpaired group. Eight gene transcripts were then selected to confirm their alterations by quantitative real-time polymerase chain reaction (qRT-PCR). The identified genes generally play important roles in neuroactive receptor-ligand interactions, synapse plasticity, ion transport, and protein phosphorylation. Furthermore, the expression of the eight selected genes that were identified and confirmed to show significant fold changes in the first microarray experiment were again measured with qRT-PCR after morphine challenge (2 mg/kg, i.p.). As expected, 2 mg/kg morphine-induced context-specific sensitization. Meanwhile, mRNA expression of the selected genes showed marked upregulation in the morphine-paired group compared with the unpaired and acute groups. These results suggest that alterations in the expression of the identified genes in the NAc may contribute to the neuroplasticity underlying contextual cue-induced relapse to drug use.

Altered phosphorylation of GluA1 in the striatum is associated with locomotor sensitization induced by exposure to increasing doses of morphine

Escalation of drug consumption is involved in the transition from drug use to addiction. Our previous study demonstrated that neuronal activation in ventral tegmental area (VTA) and substantia nigra (SN) was associated with behavioral sensitization induced by increasing doses of morphine. Here we sought to characterize the molecular mechanism underlying this behavioral sensitization. We compared mitogen-activated protein kinase (MAPK) signaling following pretreatment with either increasing doses or fixed doses of morphine before and after behavioral sensitization. We found phospho-MAPK markedly increased in ventral striatum and decreased in dorsal striatum after either pretreatment group, but no further change after sensitization induced by 10mg/kg morphine challenge. Furthermore, we also evaluated the level of phospho-glutamate receptor 1 at serine 845 (pSer845-GluA1) and 831 (pSer831-GluA1) sites in ventral striatum and dorsal striatum. The results showed a significant increase in pSer845-GluA1/GluA1 ratio in ventral striatum but not dorsal striatum after pretreatment with increasing doses of morphine but not after fixed-dose or saline pretreatment. Importantly, pSer845-GluA1/GluA1 ratio was increased exclusively in dorsal striatum and not ventral striatum following acute morphine challenge specifically paired with increasing-dose pretreatment and not fixed-dose or saline. These findings indicate that behavioral sensitization-induced by chronic pretreatment with increasing doses of morphine might be more closely associated with the dynamic GluA1 activity in the striatum rather than the modulation of MAPK signaling. These findings also indicate that GluA1 phosphorylation may occur independent of MAPK activation.

Altered function in medial prefrontal cortex and nucleus accumbens links to stress-induced behavioral inflexibility

HighlightsSingle prolonged stress (SPS) impaired set‐shifting.SPS‐induced set‐shifting impairments coincided with activity reduction in NAc core.Neurons in mPFC exhibited higher activity in SPS‐induced set‐shifting dysfunction.Enhanced phospho‐GluA1‐Ser845 in mPFC was related to the impaired set‐shifting. ABSTRACT The medial prefrontal cortex (mPFC) and its output area, the nucleus accumbens (NAc), are implicated in mediating attentional set‐shifting. Patients with posttraumatic stress disorder (PTSD) exhibit difficulties in the disengagement of attention from traumatic cues, which is associated with impairments in set‐shifting ability. However, unknown is whether alterations in corticostriatal function underlie deficits in this behavioral flexibility in individuals with PTSD. An animal model of single prolonged stress (SPS) has been partially validated as a model for PTSD, in which SPS rats recapitulate the pathophysiological abnormalities and behavioral characteristics of PTSD. In the present study, we firstly found that exposure to SPS impaired the ability in the shift from visual‐cue learning to place response discrimination in rats. Conversely, SPS induced no effect on a place‐to‐cue set‐shifting performance. Based on SPS‐impaired set‐shifting model, we used Western blot and immunofluorescent approaches to clarify SPS‐induced alternations in synaptic plasticity and neuronal activation in the mPFC and NAc. Rats that were subjected to SPS exhibited a large increase in pSer845‐GluA1 and total GluA1 levels in the mPFC, while no significant change in the NAc. We further found that exposure to SPS significantly decreased c‐Fos expression in the NAc core but not the shell after set‐shifting behavior. Whereas, enhanced c‐Fos expression was observed in prelimbic and infralimbic cortices. Collectively, these findings suggest that abnormal hyperactivity in the mPFC and dysfunction in the NAc core underlie long‐term deficits in executive function after traumatic experience, which might play an important role in the development of PTSD symptoms.