Second-by-second analysis of alpha 7 nicotine receptor regulation of glutamate release in the prefrontal cortex of awake rats

Abstract

These experiments utilized an enzyme-based microelectrode selective for the second-by-second detection of extracellular glutamate to reveal the alpha 7-based nicotinic modulation of glutamate release in the prefrontal cortex (PFC) of freely moving rats. Rats received intracortical infusions of the nonselective nicotinic agonist nicotine (12.0 mM, 1.0 microg/0.4 microl) or the selective alpha 7 agonist choline (2.0 mM/0.4 microl). The selectivity of drug-induced glutamate release was assessed in subgroups of animals pretreated with the alpha 7 antagonist, alpha-bungarotoxin (alpha-BGT, 10 microM), or kynurenine (10 microM) the precursor of the astrocyte-derived, negative allosteric alpha 7 modulator kynurenic acid. Local administration of nicotine increased glutamate signals (maximum amplitude = 4.3 +/- 0.6 microM) that were cleared to baseline levels in 493 +/- 80 seconds. Pretreatment with alpha-BGT or kynurenine attenuated nicotine-induced glutamate by 61% and 60%, respectively. Local administration of choline also increased glutamate signals (maximum amplitude = 6.3 +/- 0.9 microM). In contrast to nicotine-evoked glutamate release, choline-evoked signals were cleared more quickly (28 +/- 6 seconds) and pretreatment with alpha-BGT or kynurenine completely blocked the stimulated glutamate release. Using a method that reveals the temporal dynamics of in vivo glutamate release and clearance, these data indicate a nicotinic modulation of cortical glutamate release that is both alpha 7- and non-alpha 7-mediated. Furthermore, these data may also provide a mechanism underlying the recent focus on alpha 7 full and partial agonists as therapeutic agents in the treatment of cortically mediated cognitive deficits in schizophrenia.

Figure 1

The glutamate-sensitive microelectrode array (MEA) and the enzyme scheme used in the detection of glutamate. (A) photograph of the MEA with ceramic wafer and recording channels. (B) high magnification of the tip of the MEA illustrating the 2 pairs of recording sites, one pair sensitive to glutamate and the remaining pair as a sentinel for background (non-glutamate derived) signals. (C, left) coatings on the glutamate-sensitive recording sites allowing for the measurement of glutamate-derived current at the Pt microelectrode. (C, right) coatings on the sentinel recording sites for the measurement of background current (see Method for details).

Figure 2

A representative in vitro calibration of the MEA conducted immediately prior to implantation into prefrontal cortex. The top two tracings originate from the glutamate-sensitive (Glu OX) recording channels and the bottom two tracings are from the sentinel background channels. Arrows correspond to the addition of various substances into the calibration beaker. Current (pAmp) is depicted along the vertical and time (sec) along the horizontal axes. The successive additions of glutamate (raising beaker concentration 20 μM/aliquot) produced a linear increase in glutamate signal. Expectedly, there were no changes in current detected related to glutamate on the two sentinel channels. The calibration also reveals equivalent sensitivities on all four channels to the reporting molecule H₂O₂ and to the potential interferent DA. The effectiveness of the Nafion^®-induced blockade of negatively charged compounds is evident by the minor increase in current depicted following the addition of a large concentration of ascorbic acid (AA) to the beaker.

Figure 3

Photomicrographs illustrating a representative placement of MEA and infusion cannula within prefrontal cortex (PFC). (A) frontal section depicting the position of a microelectrode in the ventral prelimbic region of PFC. The termination of the Pt tip of the MEA is depicted by the arrow above the infra-limbic (IL) cortex. Note the modest tissue disruption produced by the implanted MEA. This is consistent with previous reports from our group (see Rutherford et al., 2007). (B) sagittal section highlighting the relative position of an MEA and the proximity of its infusion cannula. The vertical descent of the MEA can be clearly seen again within pre-limbic (PrL) and infra-limbic (IL) cortex. The modest tissue disruption to the right of the MEA reflects that produced by the infusion cannula and the subsequent infusions of drugs. The close proximity of the end of the infusion cannula and the recording channels of the MEA is evident.

Figure 4

Representative recordings from animals receiving intra-cortical infusions of nicotine separated by infusion of saline or α₇nACh receptor antagonists. (A) effects of saline: The two infusions of nicotine (marked by TTLs on bottom of graph) produced similar signals on the Glu Ox site (top tracing) as well as increased the non-glutamatergic background signal (middle tracing). The infusion of saline was without effect on any channel. Self-referencing isolated the signal due to glutamate (bottom tracing). This signal rose rapidly to maximum amplitude and then was cleared more gradually. The nicotine-induced signal was similar to that seen following the infusion of an exogenous glutamate standard at the end of the test session. (B) effects of α-BGT: the bottom, self-referenced tracing reveals that the initial infusion of nicotine produced a clear increase (3.0 μM) in glutamate. Infusion of α-BGT, 5 min earlier, markedly attenuated the second nicotine-induced glutamate signal by 64% (1.1 μM). This attenuation did not reflect a loss in the ability of the MEA to detect glutamate as a control infusion of exogenous glutamate still produced a robust (7.0 μM) signal. (C) effects of kynurenine: the bottom self-referenced tracing shows that the initial infusion of nicotine produced a clear increase (5.9 μM) in glutamate. Infusion of kynurenine 40, min earlier, significantly attenuated the second nicotine-induced glutamate signal by 78% (1.3 μM). Kynurenine did not impair the MEA's ability to detect glutamate as the infusion of a glutamate control results in a marked elevation (13.9 μM) in the glutamate signal. (D) Group data: maximum amplitude (μM; mean ± S.E.M.) is depicted following two infusions of nicotine (12 mM in 0.4 μL). The nicotine infusions were conducted both before and after the administration of saline (control), α-BGT, or kynurenine. Separate groups of rats (n = 6/group) were tested in each nicotine-drug combination. * = amplitudes significantly reduced post- relative to pre-α-BGT (P = 0.008) or pre-kynurenine (P = 0.017).

Figure 5

Representative recordings from animals receiving intra-cortical infusions of the α₇ agonist choline separated by a control infusion of saline or α₇nACh receptor antagonists. (A) effects of saline: choline infusions produced marked, symmetrical signals on the glutamate sensitive channel (top tracing). In contrast to nicotine infusions (Figs 4A-C) there was negligible choline-induced activity on the sentinel channel (middle tracing). Application of the self-referencing procedure (bottom tracing) produced highly reproducible choline-induced glutamate signals that rose quickly to maximum amplitude (6.1 μM) and were rapidly cleared to baseline. The infusion of saline did not evoke a glutamate peak nor did it affect the ability of the second choline infusion to stimulate glutamate (6.0 μM). The choline-evoked signals looked very similar to the signal stimulated following a glutamate control infusion (15.0 μM). (B) effects of α-BGT: the initial infusion of choline produced a clear increase (6.0 μM) in glutamate. The infusion of α-BGT, 5 min earlier, completely blocked the ability of a subsequent choline infusion to stimulate glutamate (0 μM). α-BGT had no detrimental effect on the ability of the MEA to detect glutamate as evident by the large signal (13.1 μM) evoked following an infusion of exogenous glutamate. (C) effects of kynurenine: the initial infusion of choline produced a clear increase (7.2 μM) in glutamate. The infusion of kynurenine, 40 min earlier, completely blocked the ability of a subsequent choline infusion to stimulate glutamate (0 μM). Again, kynurenine had no detrimental effect on the ability of the MEA to detect glutamate as evident by the large signal (15.2 μM) evoked following an infusion of exogenous glutamate. (D) Group data: maximum amplitude (μM; mean ± S.E.M.) is depicted following two infusions of choline (2 mM in 0.4 μL). The choline infusions were conducted both before and after the administration of saline (control), α-BGT, or kynurenine. Separate groups of rats (n = 6/group) were tested in each choline-drug combination. * = amplitudes significantly reduced post- relative to pre-α-BGT (P = 0.002) or pre-kynurenine (P = 0.001).