Imaging the spread of reversible brain inactivations using fluorescent muscimol

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Abstract

Muscimol is a GABAA-agonist that causes rapid and reversible suppression of neurophysiological activity. Interpretations of the effects of muscimol infusions into the brain have been limited because of uncertainty about spread of the drug around the injection site. To solve this problem, the present study explored the use of a fluorophore-conjugated muscimol molecule (FCM). Whole-cell recordings from horizontal brain slices demonstrated that bath-applied FCM acts like muscimol in reversibly suppressing excitatory synaptic transmission. Two types of in vivo experiments demonstrated that the behavioral effects of FCM infusion are similar to the behavioral effects of muscimol infusion. FCM infusion into the rat amygdala before fear conditioning impaired both cued and contextual freezing, which were tested 24 or 48 h later. Normal fear conditioning occurred when these same rats were subsequently given phosphate-buffered saline infusions. FCM infusion into the dorsomedial prefrontal cortex impaired accuracy during a delayed-response task. Histological analysis showed that the region of fluorescence was restricted to 0.5–1 mm from the injection site. Myelinated fiber tracts acted as diffusional barriers, thereby shaping the overall spread of fluorescence. The results suggest that FCM is indeed useful for exploring the function of small brain regions.

Introduction

Reversible inactivations of small volumes of brain tissue have been used to explore fundamental questions in behavioral and systems neuroscience (Majchrzak and Di Scala, 2000). Reversible inactivations are an important alternative to permanent brain lesions (Jarrard, 2002) in assessing structure–function relationships. The obvious advantage is that reversible brain inactivations allow within-subject designs. One common disadvantage is that this method typically leaves considerable uncertainty about the size and shape of the volume of the drug-infused tissue.

Muscimol, a GABAA-agonist (Beaumont et al., 1978), is commonly and productively used for reversible inactivations (Krupa and Thompson, 1997, Lomber, 1999, Martin and Ghez, 1999). However, the spatial extent of a muscimol infusion is indeterminate without the use a radioactive tracer ([3H]muscimol; Edeline et al., 2002, Martin, 1991). Indirect estimates about the spread of muscimol have been obtained by measuring its anticipated functional consequences on glucose uptake (Martin, 1991), evoked field potentials (Wilensky et al., 2006), and multiunit activity levels (Arikan et al., 2002, Edeline et al., 2002). Practical considerations have precluded the routine use of either radioactive or functional techniques (but see Krupa and Thompson, 1997).

Here we evaluated the use of a fluorophore-conjugated muscimol molecule (FCM) for producing local and reversible brain inactivations. FCM was first used in conjunction with muscimol to assess the role of dorsomedial prefrontal cortex (dmPFC) in a delayed-response task (Narayanan and Laubach, 2006, Narayanan et al., 2006). Our evaluation of FCM made use of whole-cell recordings, well-characterized brain–behavior relationships, and optical quantification methods. The results show that FCM can serve as a stand-alone and practical substitute for muscimol.

Section snippets

Subjects

This study used a total of 15 rats, described for each experiment below. Procedures were approved by the Yale University Animal Care and Use Committees (whole-cell, acute infusion, and fear conditioning) and the John B. Pierce Laboratory (delayed-response task).

Drugs

Muscimol was acquired from Sigma–Aldrich. FCM (commercially designated as muscimol, BODIPY® TMR-X conjugate) was acquired from Molecular Probes (product #M23400, www.molecularprobes.com). FCM is a conjugate of muscimol and the Bodipy®

Effects of FCM on excitatory postsynaptic potentials and input resistance

Horizontal rat brain slices were used to verify that FCM, like unlabeled muscimol, does in fact suppress neuronal and synaptic function. Whole-cell recordings were made from neurons in layer V of PR. Synaptic responses were evoked in these neurons by stimulating layer I of PR. The complete experimental protocol was conducted on 11 healthy cells recorded from 3 rat pups. In each case, bath application FCM (10 μM in aCSF) caused an immediate and rapid suppression of excitatory synaptic

FCM reversibly depresses synaptic transmission

Whole-cell recordings in PR showed that bath application of FCM decreased the mean amplitude of EPSPs and also decreased RN (Fig. 2). These results show that the desired neuropharmacological action persists after conjugating muscimol with a large fluorophore. Following washout of FCM from the bath, the recovery of neural function required hours (Fig. 2). The slow reversal may result from the lipophilic properties of the Bodipy-TMR-X fluorophore®, as discussed below.

FCM spread is not always uniform

The spread of FCM can be

Acknowledgements

This work was supported by a National Institute of Health grant (MH58405) and Yale University to Thomas H. Brown; supported by a National Science Foundation grant (0642951) and the Kavli foundation awarded to Mark Laubach.

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