Cognitive rigidity and BDNF-mediated frontostriatal glutamate neuroadaptations during spontaneous nicotine withdrawal

Abstract

Cognitive flexibility is the ability to switch strategic responses adaptively in changing environments. Cognitive rigidity imposed by neural circuit adaptations during nicotine abstinence may foster maladaptive nicotine taking in addicts. We systematically examined the effects of spontaneous withdrawal in mice exposed to either nicotine (6.3 or 18 mg/kg/day) or saline for 14 days on cognitive flexibility using an operant strategy set-shifting task. Because frontostriatal circuits are critical for cognitive flexibility and brain-derived neurotrophic factor (BDNF) modulates glutamate plasticity in these circuits, we also explored the effects of nicotine withdrawal on these neurochemical substrates. Mice undergoing nicotine withdrawal required more trials to attain strategy-switching criterion. Error analysis show that animals withdrawn from both nicotine doses committed higher perseverative errors, which correlated with measures of anxiety. However, animals treated with the higher nicotine dose also displayed more strategy maintenance errors that remained independent of negative affect. BDNF mRNA expression increased in the medial prefrontal cortex (mPFC) following nicotine withdrawal. Surprisingly, BDNF protein declined in mPFC but was elevated in dorsal striatum (DS). DS BDNF protein positively correlated with perseverative and maintenance errors, suggesting mPFC-DS overflow of BDNF during withdrawal. BDNF-evoked glutamate release and synapsin phosphorylation was attenuated within DS synapses, but enhanced in the nucleus accumbens, suggesting a dichotomous role of BDNF signaling in striatal regions. Taken together, these data suggest that spontaneous nicotine withdrawal impairs distinct components of cognitive set-shifting and these deficits may be linked to BDNF-mediated alterations in glutamate signaling dynamics in discrete frontostriatal circuits.

Fig. 1

Schematics of the strategy set-shifting task and experimental design. a Mice were initially autoshaped and pretrained to acquire a lever press response within a specified period of time to receive a reward (sweetened water). Animals that reached pretraining criterion were progressed to the spatial discrimination phase in which they were required to adopt an egocentric response strategy to achieve rewards. During each trial, animals were presented with both right and left levers A lever press on the assigned lever (right assignment in this case; lever assignment was counterbalanced within a group) was scored as a “correct response” and was followed by reward delivery. Response on the incorrect lever (left lever in this case) was not rewarded and resulted in a punishment (timeout) phase characterized by a 5 s extinguishing of the house light. After reaching criterion performance, mice progressed to the set-shifting (visual discrimination) phase. In this phase, mice were required to abandon the previously reinforced strategy in favor of a current visual cue-based strategy. A lever press response on the lever associated with the illuminated light was scored as a “correct response’ and was subsequently rewarded. Lever responses to the lever opposite of the light cue were scored as an “error” and resulted in punishment (5 s timeout). b After attaining pretraining criterion, mice were implanted with mini-osmotic pumps delivering saline, 6.3 or 18 mg/kg/day nicotine for 14 days. After 1 week of post-surgery recovery and behavioral retention, mice progressed to the spatial discrimination phase. Animals remained on this phase for 7 days during which they attain criterion. Following the last training session on the 14th day of solution delivery, pumps were removed and spontaneous withdrawal was induced. Mice were then tested on strategy set-shifting (visual discrimination) ~24 h after pump removal and remained on this phase until either criterion performance was reached or 2 weeks whichever was earlier. Tissues were removed for neurochemistry studies after the last behavioral session. c Schematic illustrating discrete components of the frontostriatal circuits examined for the consequences of nicotine withdrawal on BDNF signaling and glutamatergic transmission.

Fig. 2

Spontaneous nicotine withdrawal strategy set-shifting performance. Mice experiencing spontaneous withdrawal following the removal of mini-osmotic pumps that delivered nicotine (both doses: 6.3 and 18 mg/kg/d) for 2 weeks required more trials to reach criterion performance (a) and committed more total errors (b) as compared to saline-exposed mice. Although set-shifting errors incrementally reduced as the animals received more testing sessions; they remain significantly different between the treatment groups (c). Further investigation of error types revealed that withdrawal from either dose of nicotine significantly increased perseverative errors (d); however, maintenance errors significantly increased only in mice that were withdrawn from the higher dose of nicotine (e). Response accuracies for 10 testing sessions depict delayed acquisition of the strategy set shift in nicotine withdrawal animals (f). Correct (g) and incorrect (h) response latencies remained similar across all testing sessions between the groups. The omissions remained similar across all withdrawal conditions (i). Data are mean ± SEM (n = 8 mice per group). Trials to criterion, total errors, perseverative errors, maintenance errors, and omissions were analyzed using one-way ANOVA. Analysis of errors and response latencies for 10 behavioral sessions was conducted using mixed-factor ANOVA. ^#p < 0.001 (main effect of session); ^$p < 0.001 (main effect of treatment); *p < 0.05; ** p < 0.01; ***p < 0.001 (post hoc tests).

Fig. 3

Nicotine withdrawal-related alterations in BDNF expression in the frontostriatal regions of task-performing mice. Bar charts illustrate the results of qPCR analysis of the neuronal activity-dependent BDNF exon-IV transcript (a) and BDNF exon IX for total BDNF mRNA (b) in the mPFC. All mRNA data is expressed as fold changes normalized to GAPDH. Mice withdrawn from the higher (18 mg/kg/day) dose of nicotine exhibited significantly higher expression of both BDNF exon IV and total BDNF mRNA as compared to saline. Surprisingly, the expression levels of exon-IV transcript significantly reduced following spontaneous withdrawal from the lower nicotine dose (6.3 mg/kg/day). This effect was not observed in the total BDNF mRNA. Immunoblot analyses showed reduced expression of BDNF protein (mature form of BDNF detected as a 14 kDa immunoreactive band) in the mPFC of mice undergoing withdrawal from the higher nicotine dose as compared to saline-exposed animals (c). Conversely, BDNF protein levels were significantly elevated in the tissue homogenates prepared from the DS region of these mice (d). BDNF protein levels in the mPFC and DS regions remained unaltered at the lower nicotine dose. BDNF protein levels in the NAc remained indifferent between the treatment groups (e). The expression of the high-affinity BDNF receptor, TrkB, remained unaltered with nicotine withdrawal in the DS and NAc (f). All mRNA and protein data are expressed as mean ± SEM (n = 8 mice/group) and analyzed using one-way ANOVA. *p < 0.05; **p < 0.01 (post hoc tests). The results of Pearson’s r correlation analyses between BDNF protein levels and strategy set-shifting errors are depicted in g–l. Prefrontal BDNF protein levels were negatively associated with perseverative errors (g), while a completely opposite pattern was observed with DS BDNF levels (h). No association was observed between NAc BDNF protein levels and behavioral perseveration (i). While prefrontal BDNF levels did not correlate with the strategy maintenance errors (j), DS BDNF levels did show a positive correlation with this behavioral measure (k). NAc BDNF expression was not associated with strategy maintenance errors (l).

Fig. 4

Anxiety-like symptoms associated with cognitive deficits during spontaneous nicotine withdrawal. Spontaneous nicotine withdrawal produced robust anxiety-like effects, indicated by reduce time spent in open arms, on the first and third day of withdrawal, but were absent 7 days after pump removal (a). In general, a negative association was observed between the time spent in open arms and perseverative errors (b), but not strategy maintenance errors (c). Interestingly, PFC BDNF expression positively correlated with increased time spent in the open arm (d). Conversely, DS BDNF protein expression was negatively associated with this behavioral measure (e). Changes in NAc BDNF expression were not associated with anxiety-like symptoms (f). The strength for all associations was measured using Pearson’s r correlations.

Fig. 5

In vivo amperometric recordings striatal glutamatergic transmission. Population traces depicting synaptic glutamate release following brief depolarizing pulses of potassium in the DS (a, upper panel) and NAc (b, upper panel) of mice undergoing saline and nicotine (18 mg/kg/day) withdrawal. Bar charts (lower panel) depict that average glutamate signal amplitudes remained similar between the two groups indicating that nicotine withdrawal neither altered presynaptic release of glutamate in the DS nor NAc. Population traces depicting increases in extracellular glutamate levels following local application of BDNF in the DS (c, upper panel) and NAc (d, upper panel). A significant attenuation of BDNF-evoked glutamate release was observed in the DS of nicotine withdrawal mice compared to the saline-exposed animals treated mice (c, lower panel). On the contrary, glutamate signal amplitudes were significantly higher following local BDNF application in the NAc of nicotine withdrawal mice (d, lower panel). Data are mean ± SEM (n = 5/sal, n = 6/nic) and analyzed using one-way ANOVA. *p < 0.05; **p < 0.01 vs sal.