Developing electrophysiological recordings of brain neuronal activity and their analysis provide a basis for exploring the structure of brain function and nervous system investigation. The recorded signals are typically a combination of action potentials (spikes) and noise. High amounts of background noise and possibility of electric signaling recording from several neurons adjacent to the recording site have led scientists to develop neuronal signal processing tools such as spike sorting to facilitate brain data analysis. Spike sorting plays a pivotal role in understanding the electrophysiological activity of neuronal networks. This process prepares recorded data for meaningful interpretations of interactions among neurons, investigating the connectivity between different brain regions, and ultimately understanding the overall structure of brain functions. Spike sorting consists of three steps: spike detection, feature extraction, and spike clustering. There are several methods to implement each of spike sorting steps. This paper provides a systematic comparison of various spike sorting sub-techniques applied to real extracellularly recorded data from a rat brain basolateral amygdala. An efficient sorted data resulted from careful choice of spike sorting sub-methods leads to better interpretation of the brain structures connectivity under different conditions, which is a very sensitive concept in diagnosis and treatment of neurological disorders. Here, spike detection is performed by appropriate choice of threshold level via three different approaches. Feature extraction is done through PCA and Kernel PCA methods, where Kernel PCA outperforms. We have applied four different algorithms for spike clustering including K-means, Fuzzy C-means, Bayesian and Fuzzy maximum likelihood estimation. As one requirement of most clustering algorithms, optimal number of clusters is achieved through validity indices for each method. Finally, the sorting results are evaluated using inter-spike interval histograms. Rat brain real data used in simulations was recorded in neuroscience research center, Shahid Beheshti University of Medical Sciences, Tehran, Iran.