Elsevier

Neuroscience

Volume 146, Issue 3, 25 May 2007, Pages 931-945
Neuroscience

Cellular neuroscience
Electrophysiological and morphological heterogeneity of slow firing neurons in medial septal/diagonal band complex as revealed by cluster analysis

https://doi.org/10.1016/j.neuroscience.2007.02.047Get rights and content

Abstract

Slow firing septal neurons modulate hippocampal and neocortical functions. Electrophysiologically, it is unclear whether slow firing neurons belong to a homogeneous neuronal population. To address this issue, whole-cell patch recordings and neuronal reconstructions were performed on rat brain slices containing the medial septum/diagonal band complex (MS/DB). Slow firing neurons were identified by their low firing rate at threshold (<5 Hz) and lack of time-dependent inward rectification (Ih). Unsupervised cluster analysis was used to investigate whether slow firing neurons could be further classified into different subtypes. The parameters used for the cluster analysis included latency for first spike, slow afterhyperpolarizing potential, maximal frequency and action potential (AP) decay slope. Neurons were grouped into three major subtypes. The majority of neurons (55%) were grouped as cluster I. Cluster II (17% of neurons) exhibited longer latency for generation of the first action potential (246.5±20.1 ms). Cluster III (28% of neurons) exhibited higher maximal firing frequency (25.3±1.7 Hz) when compared with cluster I (12.3±0.9 Hz) and cluster II (11.8±1.1 Hz) neurons. Additionally, cluster III neurons exhibited faster action potentials at suprathreshold. Interestingly, cluster II neurons were frequently located in the medial septum whereas neurons in cluster I and III appeared scattered throughout all MS/DB regions. Sholl’s analysis revealed a more complex dendritic arborization in cluster III neurons. Cluster I and II neurons exhibited characteristics of “true” slow firing neurons whereas cluster III neurons exhibited higher frequency firing patterns. Several neurons were labeled with a cholinergic marker, Cy3-conjugated 192 IgG (p75NTR), and cholinergic neurons were found to be distributed among the three clusters. Our findings indicate that slow firing medial septal neurons are heterogeneous and that soma location is an important determinant of their electrophysiological properties. Thus, slow firing neurons from different septal regions have distinct functional properties, most likely related to their diverse connectivity.

Section snippets

Animals and medial septum/diagonal band slices

Experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) and experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23). Every effort was made to minimize the number of animals used and their suffering. Briefly, slices containing MS/DB were prepared from 15 to 25 day old male Sprague–Dawley rats. Animals were deeply anesthetized with ketamine (10 mg/kg

Electrophysiological variability of intrinsic membrane properties of MS/DB neurons

Neurons were first visually classified as “slow firing neurons” according to described criteria and then electrophysiological variables were analyzed for development of non-supervised cluster analysis. Normal probability plots and Shapiro-Wilcox test for normality revealed that the distribution of several electrophysiological variables deviated from normality (Table 1). Property variability was evident in Table 1 and Fig. 2. The AP half-width was not normally distributed (P=0.048). The AP

Discussion

Unsupervised cluster analysis was used to determine whether the population of MS/DB neurons is composed of highly heterogeneous subgroups of neuronal subtypes or discrete groups of neuronal subtypes displaying a continuum of electrophysiological properties for each firing phenotype (range of individual variations). These methods have recently been used to more objectively characterize neuronal populations in several brain areas such as neocortex and suprachiasmatic nucleus (Azouz et al 1997,

Acknowledgments

We thank Dr. Brian H. Bland (University of Calgary) for graciously reviewing the manuscript and providing us with valuable insights. Grant sponsor: NIH grants: # NS4271 (L. V. Colom); # NSG M068855 (L. V. Colom and E. R. Garrido-Sanabria); # P20MD001091 and # P20MD000161 funded by NIH/NCMHD (RIMI) and also by NIH/NCMHD (EXPORT) (L. V. Colom and E. R. Garrido-Sanabria) as well as MBRS-RISE grant #1R25Gm06592501A1.

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