Muscimol Diffusion after Intracerebral Microinjections: A Reevaluation Based on Electrophysiological and Autoradiographic Quantifications

Abstract

Intracerebral muscimol injection is widely used to inactivate discrete brain structures during behavioral tasks. However, little effort has been made to quantify the extent of muscimol diffusion. The authors report here electrophysiological and autoradiographic results obtained after muscimol injection (1 microg/microl) either into the nucleus basalis magnocellularis (0.1-0.4 microl) or into the thalamic reticular nucleus (RE, 0.05-0.1 microl). In 52 rats, multiunit recordings were collected either in the RE or in the auditory thalamus during the 2 h following muscimol injection. Decreases in neuronal activity were observed up to 3 mm from the injection site; their time of occurrence was a function of the distance between the injection and recording sites. Because these decreases cannot be explained by physiological effects, they likely reflected muscimol diffusion up to the recording sites. Autoradiographic studies involved 25 rats and different experimental conditions. Optical density (OD) measures indicated that after a survival time of 15 min, a 0.05 microl injection produced a labeled area of 5.25 mm(2) at the injection site and a rostrocaudal labeling of 1.7 mm. Increasing the survival time to 60 min, or increasing the injected volume to 0.1 microl, systematically led to a larger labeled area at the injection site (8-12 mm(2)) and to a larger rostrocaudal diffusion (2.0-2.5 mm). Direct quantifications of radioactivity by a high-resolution radioimager validated the OD measures and even indicated a larger muscimol diffusion (up to 3.25 mm). Thus, these data point out that muscimol diffusion after intracerebral microinjection is larger than usually supposed. The relationships between these results and those obtained in behavioral studies are discussed.

Temporary inhibition of dorsal or ventral hippocampus by muscimol: Distinct effects on measures of innate anxiety on the elevated plus maze, but similar disruption of contextual fear conditioning

“…Using a small infusion volume (0.5 µl) and fine infusion cannulae (34 g) as in the present study, the infused drug is estimated to spread 1 mm or less in any direction (Myers, 1966). While one study suggested that the spread of muscimol may be quite wide-spread following infusion into the nucleus basalis magnocellularis or the reticular nucleus of the thalamus (Edeline et al, 2002), recent studies using infusion of fluorescent muscimol into prefrontal cortex (Allen et al, 2008) or into dorsal and ventral hippocampus (Jacobs et al, 2013) with doses and infusion volumes similar to the present study suggested that spread of muscimol is largely restricted to 0.5-1 mm. In addition, the densely packed fiber bundles surrounding the hippocampus also seem to prevent diffusion out of the hippocampus (Morris et al, 1998).…”

The Effects of Temporary Inactivation of the Core and the Shell Subregions of the Nucleus Accumbens on Prepulse Inhibition of the Acoustic Startle Reflex and Activity in Rats

“…In addition, it gives rise to a labeled area with a mean width of 1.9 or 1.3 mm at the injection site when infused into either the core or the shell, respectively (notably, the mean width of the core and the shell as determined from Figures 11 and 12 of Paxinos and Watson's Rat Brain Atlas (1997) is approximately 1.8 and 1.0 mm, respectively). These findings are in accordance with Martin (1991) and Edeline et al (2002), who obtained comparable values of muscimol diffusion after infusion into the cortex, the nucleus basalis magnocellularis and the thalamic reticular nucleus. Hence, our findings indicate that although the use of an injected volume of 0.2 ml already produced a considerable diffusion radius, it nonetheless did not exceed the anatomical borders of either the core or the shell.…”

“…used in drug-infusion studies (0.5 ml or larger) to selectively stimulate or inactivate either one of the accumbal subregions (eg Maldonado-Irizarry and Kelley, 1994;Reijmers et al, 1995) or to investigate functional segregations within one accumbal subregion (eg Reynolds and Berridge, 2003;Zhang and Kelley, 2000). Although sparse data are available on quantification of muscimol diffusion after intracerebral microinfusions in the rodent brain, they do suggest that the diffusion of muscimol is larger than initially assumed (Edeline et al, 2002;Martin, 1991).…”

“…Using the software, the area of labeled signal was delineated on the digitally acquired images, and the following parameters from each labeled area were measured: (a) optical density, (b) width (medial-lateral extent), (c) height (dorsal-ventral extent) and (d) surface area (cf Edeline et al, 2002;Martin, 1991). All sections (on average 23 per brain) covering the total injection site along its rostral-caudal axis were measured, yielding a final section interval of 100 mm.…”

Time course of dorsal and ventral hippocampal involvement in the expression of trace fear conditioning

“…Inactivation or damage to VH prior to testing in trace conditioning has been observed to dramatically impair expression of acquired fear (Burman et al, 2006;Czerniawski et al, 2009;Yoon & Otto, 2007). Therefore, from a neuroanatomical perspective, VH, more so than DH, is likely to participate significantly in forming associations between explicit stimuli and aversive events (Bannerman et al, 2004) Evidence from previous studies suggests that infusions of muscimol in the volumes and concentration used in the present study can exert inhibitory effects up to 2mm from the site of infusion (Edeline et al, 2002;Martin, 1991). So, while it is likely that significant portions of ventral CA1, CA3 and DG were inactivated, there is some possibility that muscimol may have diffused as far as the amygdala, and thus that the behavioral deficits described here could reflect inactivation of the amygdala.…”