A clickable analogue of ketamine retains NMDA receptor activity, psychoactivity, and accumulates in neurons

Ketamine is a psychotomimetic and antidepressant drug. Although antagonism of cell-surface NMDA receptors (NMDARs) may trigger ketamine’s psychoactive effects, ketamine or its major metabolite norketamine could act intracellularly to produce some behavioral effects. To explore the viability of this latter hypothesis, we examined intracellular accumulation of novel visualizable analogues of ketamine/norketamine. We introduced an alkyne “click” handle into norketamine (alkyne-norketamine, A-NK) at the key nitrogen atom. Ketamine, norketamine, and A-NK, but not A-NK-amide, showed acute and persisting psychoactive effects in mice. This psychoactivity profile paralleled activity of the compounds as NMDAR channel blockers; A-NK-amide was inactive at NMDARs, and norketamine and A-NK were active but ~4-fold less potent than ketamine. We incubated rat hippocampal cells with 10 μM A-NK or A-NK-amide then performed Cu(2+) catalyzed cycloaddition of azide-Alexa Fluor 488, which covalently attaches the fluorophore to the alkyne moiety in the compounds. Fluorescent imaging revealed intracellular localization of A-NK but weak A-NK-amide labeling. Accumulation was not dependent on membrane potential, NMDAR expression, or NMDAR activity. Overall, the approach revealed a correlation among NMDAR activity, intracellular accumulation/retention, and behavioral effects. Thus, we advance first generation chemical biology tools to aid in the identification of ketamine targets

In utero exposure to transient ischemia-hypoxemia promotes long-term neurodevelopmental abnormalities in male rat offspring

The impact of transient ischemic-hypoxemic insults on the developing fetal brain is poorly understood despite evidence suggesting an association with neurodevelopmental disorders such as schizophrenia and autism. To address this, we designed an aberrant uterine hypercontractility paradigm with oxytocin to better assess the consequences of acute, but transient, placental ischemia-hypoxemia in term pregnant rats. Using MRI, we confirmed that oxytocin-induced aberrant uterine hypercontractility substantially compromised uteroplacental perfusion. This was supported by the observation of oxidative stress and increased lactate concentration in the fetal brain. Genes related to oxidative stress pathways were significantly upregulated in male, but not female, offspring 1 hour after oxytocin-induced placental ischemia-hypoxemia. Persistent upregulation of select mitochondrial electron transport chain complex proteins in the anterior cingulate cortex of adolescent male offspring suggested that this sex-specific effect was enduring. Functionally, offspring exposed to oxytocin-induced uterine hypercontractility showed male-specific abnormalities in social behavior with associated region-specific changes in gene expression and functional cortical connectivity. Our findings, therefore, indicate that even transient but severe placental ischemia-hypoxemia could be detrimental to the developing brain and point to a possible mitochondrial link between intrauterine asphyxia and neurodevelopmental disorders

Motivational disturbances and effects of L-dopa administration in neurofibromatosis-1 model mice.

Children with neurofibromatosis type 1 (NF1) frequently have cognitive and behavioral deficits. Some of these deficits have been successfully modeled in Nf1 genetically-engineered mice that develop optic gliomas (Nf1 OPG mice). In the current study, we show that abnormal motivational influences affect the behavior of Nf1 OPG mice, particularly with regard to their response to novel environmental stimuli. For example, Nf1 OPG mice made fewer spontaneous alternations in a Y-maze and fewer arm entries relative to WT controls. However, analysis of normalized alternation data demonstrated that these differences were not due to a spatial working memory deficit. Other reported behavioral results (e.g., open-field test, below) suggest that differential responses to novelty and/or other motivational influences may be more important determinants of these kinds of behavior than simple differences in locomotor activity/spontaneous movements. Importantly, normal long-term depression was observed in hippocampal slices from Nf1 OPG mice. Results from elevated plus maze testing showed that differences in exploratory activity between Nf1 OPG and WT control mice may be dependent on the environmental context (e.g., threatening or non-threatening) under which exploration is being measured. Nf1 OPG mice also exhibited decreased exploratory hole poking in a novel holeboard and showed abnormal olfactory preferences, although L-dopa (50 mg/kg) administration resolved the abnormal olfactory preference behaviors. Nf1 OPG mice displayed an attenuated response to a novel open field in terms of decreased ambulatory activity and rearing but only during the first 10 min of the session. Importantly, Nf1 OPG mice demonstrated investigative rearing deficits with regard to a novel hanging object suspended on one side of the field which were not rescued by L-dopa administration. Collectively, our results provide new data important for evaluating therapeutic treatments aimed at ameliorating NF1-associated cognitive/behavioral deficits

The investigative rearing deficit in male <i>Nf1</i> OPG mice is not rescued by L-dopa administration.

<p>(A–C) No significant overall effects of Group were found in the third cohort of mice for ambulatory activity (A), rearing frequency (B), or time spent rearing (C) during a 30-min habituation trial in the open-field apparatus, which was conducted the day before the test session and did not include any drug/vehicle injections. (D) In contrast, planned comparisons showed that the CON+SAL mice spent significantly more time rearing to investigate the hanging object (ball) compared to the time spent rearing in the same area at the opposite end of the field (BALL vs OPP; *p = 0.017), while the <i>Nf1</i> OPG+SAL group did not show significantly different rearing times with regard to the ball versus the opposite area. The <i>Nf1</i> OPG+LDOPA mice reared for substantially longer times in investigating the ball versus the opposite area but these differences were not statistically significant (p = 0.064). (E) Similar results were found for the rearing frequency data where planned comparisons revealed that the CON+SAL mice spent significantly more time rearing to investigate the hanging ball versus the time spent rearing in the opposite area of the field (BALL vs OPP; *p = 0.010), while the <i>Nf1</i> OPG+SAL group did not. Again, the <i>Nf1</i> OPG+LDOPA mice showed a trend toward greater investigative rearing toward the ball versus the opposite area but these differences were not statistically significant (p = 0.063). (F) Although the <i>Nf1</i> OPG+SAL mice tended to spend less time rearing in general throughout the field compared to the CON+SAL and <i>Nf1</i> OPG+LDOPA groups, no statistically significant effects were observed for this variable. The male mice in cohort 3 were 3.5–4.5 months old and the sample size for each of the three groups was the same (n = 12).</p

Habituation increases exploratory hole poking in male <i>Nf1</i> OPG mice and L-dopa administration rescues normal olfactory preference behaviors.

<p>(A–C) In cohort 3, no differences were observed between saline-treated WT control mice (CON+SAL), saline-treated <i>Nf1</i> OPG mice (<i>Nf1</i> OPG+SAL) or <i>Nf1</i> OPG mice treated with L-dopa (<i>Nf1</i> OPG+LDOPA) in terms of total hole pokes (A), total side pokes (B), or general ambulatory activity (C). (D) However, planned comparisons showed that the CON+SAL and <i>Nf1</i> OPG+LDOPA groups displayed a significant preference (increased poke frequency) for the odorant-containing versus the empty corner holes (EMP vs ODR; **p = 0.003 and *p = 0.020, respectively), while the <i>Nf1</i> OPG+SAL mice did not show a significant preference. (E) Similarly, the CON+SAL and <i>Nf1</i> OPG+LDOPA mice exhibited a significant preference for the familiar (fresh homecage bedding) versus the novel (coconut) odorant (NOV vs FAM; *p = 0.0001 and **p<0.00005, respectively), in contrast to the <i>Nf1</i> OPG+SAL group which again did not show a significant preference. In addition, the <i>Nf1</i> OPG+SAL mice poked significantly more often into the hole containing the novel odorant compared to the levels observed in the <i>Nf1</i> OPG+LDOPA group (^†p = 0.016). (F) The average hole poke durations were significantly greater for the odorant-containing versus the empty holes in both the CON+SAL and <i>Nf1</i> OPG+LDOPA groups (EMP vs ODR; **p = 0.001 and *p = 0.041, respectively), while the <i>Nf1</i> OPG+SAL mice did not show significant differences in poke durations for the different types of holes. The mice in cohort 3 were all males that were 3.5–4.5 months of age and each of the three groups had the same sample size (n = 12).</p

Mouse cohorts used for behavioral tests.

<p>Ages of mice at testing, order of behavioral tests and sex distribution for each mouse cohort used in the present study.</p

<i>Nf1</i> OPG mice exhibit an abnormal response to novel environmental stimuli in an open field.

<p>(A) In the cohort 2 mice, locomotor and exploratory activity were quantified over a 30-min period in an open field. An rmANOVA and pair-wise comparisons revealed that <i>Nf1</i> OPG mice showed significantly (beyond Bonferroni correction: p<0.017) reduced total ambulations (whole body movements) compared to WT littermate controls but only during the first 10-min block of the open-field test (*p = 0.015), although large differences were also observed during Block 2 (^†p = 0.038) (Genotype effect: ^††p = 0.027; Genotype by Time interaction: ^††p = 0.033). (B) Similarly, <i>Nf1</i> OPG mice exhibited significantly decreased numbers of vertical rearings during only the first 10-min time block as well (*p = 0.010). (Genotype by Time interaction: ^†p = 0.043). (C) The <i>Nf1</i> OPG mice also spent significantly less total time rearing in the open field compared to control mice (*p = 0.005) during the first time block with large differences also being observed for the second time block (^†p = 0.046), (Genotype effect: F(1,16) = 5.00, **p = 0.040). (D) <i>Nf1</i> OPG mice displayed significantly reduced rearing to investigate an object (ball) suspended on one side of the open field apparatus relative to control mice (*p = 0.014) although the groups did not differ in the time spent rearing in the same area on the opposite side of the field. In addition, the WT control mice showed significantly increased rearing times to investigate the ball relative to the amount of rearing time displayed on the opposite side of the field (BALL vs OPP; **p = 0.0001), while no significant differences were found in terms of the rearing times between the two areas in <i>Nf1</i> OPG mice. (E) <i>Nf1</i> OPG mice spent significantly less time rearing in the open field in general (*p = 0.014) compared to the control group. (F) When rearing to investigate the hanging object and rearing displayed in the same area on the opposite side of the field were calculated as percentages of the total rearing time, the WT control mice, but not the <i>Nf1</i> OPG mice, showed significant differences in rearing to investigate the ball versus rearing on the opposite side of the field (BALL vs OPP; **p = 0.001). During the open-field testing, the cohort 2 groups were 5.5 months old and consisted of the same sample sizes and sex distribution (n = 10: M = 4; F = 6).</p

<i>Nf1</i> OPG mice display context-dependent alterations in activity in the elevated plus maze (EPM).

<p>No differences were observed in the distances traveled by the <i>Nf1</i> OPG mice compared to the WT littermate control group from cohort 2 (4.5 months old) in either the open arms (A) or in the center area of the EPM (B). (C) However, the <i>Nf1</i> OPG mice traveled a significantly shorter total distance throughout the entire EPM compared to the control group (Genotype effect: ^††p = 0.009) with significant differences between groups occurring on Test Days 2 (*p = 0.015) and 3 (**p = 0.009). (D) The differences in total distance traveled were found to be mostly due to differences between the two groups in distance traveled in the relatively non-threatening closed arms. Specifically, the <i>Nf1</i> OPG mice, on average, traveled a significantly shorter distance in the closed arms compared to the WT controls (Genotype effect: ^††p = 0.0015), with significant differences being found on Test Days 2 (**p = 0.003) 3 (*p = 0.006), although large differences were also found on Test Day 1 as well (^†p = 0.024). For both groups in cohort 2 the sample sizes were the same (n = 10), as was the sex distribution (M = 4; F = 6).</p

<i>Nf1</i> OPG mice show decreased spontaneous alternations in a Y-maze but no deficits in spatial working memory or long-term depression (LTD) in hippocampal slices.

<p>(A–B) In cohort 1, <i>Nf1</i> OPG mice (n = 20; M = 8; F = 12) made significantly fewer alternations (A; *p = 0.008) and arm entries (B; *p = 0.011) compared to WT control mice (n = 17; M = 10; F = 7) suggesting a diminished response to novelty in the <i>Nf1</i> mutant mice. (C) To control for differences in activity, alternation scores were transformed with reference to the number of arm entries in calculating the percentage of spontaneous alternations, and no significant differences in performance were observed between groups suggesting intact spatial working memory in the <i>Nf1</i> OPG mice. The mice in cohort 1 were 5 months of age. (D) The graph shows the time course of change in EPSP slope in response to 900 pulse LFS delivered at 1 Hz (connected arrows). LFS produced robust LTD in hippocampal slices from both WT control and <i>Nf1</i> OPG mice. Traces to the right of the graph show representative EPSPs from control and <i>Nf1</i> OPG slices during baseline (dashed traces) and 60 min following LFS (solid traces). Scale = 1 mv, 5 ms.</p