Somatostatin (SOM) is a peptide encoded by the SST gene. Its isoform somatostatin-14 is expressed in a major subset of GABAergic neurons [1, 2] and is involved in the modulation of cortical microcircuits, which is essential for regulating higher cognitive functions [3–9]. SOM+ neurons play an important role in spatial working memory by selectively modulating pyramidal neurons . In addition, due to their involvement in synaptic plasticity, they represent an important component in the processes of learning .
Reduced levels of SOM expression have been reported in several neuropsychiatric and neurodegenerative disorders, such as major depressive disorder, schizophrenia, bipolar disorder and Alzheimer’s disease[10, 11] and SOM+ neurons appear to play an important role in the pathogenesis these disorders. Nevertheless, the exact causes of reduced SOM expression in these disorders remain largely unknown. Most of these disorders affect the prefrontal cortex (PFC), which plays a key role in the delicate integration of relevant information from other regions of the brain. It is possible that even smaller alterations to a particular subpopulation of SOM+ neurons could potentially lead to serious dysfunction of the PFC. Therefore, providing a detailed understanding the molecular organization of SOM+ neurons in the human PFC is of great importance and would enable determining the intricate physiological roles of SOM+ neurons. Furthermore, SOM and its analogs may have potential as future pharmacological treatments for brain disorders characterized by reduced SOM expression, which gives even more incentive to focus research on SOM+ neurons [10, 11]. Nevertheless, data on the molecular characterization of SOM+ neurons in the human PFC is severely lacking.
In general, the calcium-binding proteins parvalbumin (PV), calbindin (CB) and calretinin (CR) are the most common markers used to define the major populations of GABAergic neurons , though other molecular, morphological and electrophysiological classification criteria are also described [5, 13–15]. In most classifications, SOM immunolabelling is considered to visualize the vast majority of CB+ neurons as well as additional GABAergic neurons that are not visualized by CB immunolabelling. This is why SOM is typically considered a more eligible marker that encompasses a larger subset of cortical GABAergic neurons than CB  – in essence CB+ neurons are increasingly being regarded as merely a subpopulation of SOM+ neurons and are rarely included in newer interneuron classifications, especially in rodents . However, it is important to accentuate that this classification has never been explicitly confirmed in the primate brain and especially not in humans. This means that inferences on the GABAergic cortical network are often done by attempting to extrapolate data from rodents, i.e. descriptions of cortical network connectivity (and conclusions on its role in the pathogenesis of human diseases) are heavily reliant on whether the rodent model is correct for the human brain.
Based on their morphology, SOM+ neurons are typically classified into two major subpopulations – Martinotti and non-Martinotti cells [16–19]. Martinotti cells have an ascending axon with synaptic targets located in layer I of the cerebral cortex, where they form significant axonal plexuses [20, 21]. There, the axons of Martinotti cells establish synapses with the terminal branches of the apical dendrites of principal cells. Unlike Martinotti cells that have a distinct axonal morphology with clearly defined synaptic targets, non-Martinotti cells are simply defined as all SOM+ cells that lack significant axonal plexuses in layer I . Besides their intriguing morphology, studies in rodents revealed that a portion of SOM + neurons co-express (to varying degrees) different molecular markers , including CR , CB [23, 24], neuropeptide Y (NPY) , cholecystokinin (CCK) , nNOS [16, 17, 24, 25], and substance P receptor (SPR) [18, 26].
In most species, SOM+ cell bodies are present in all cortical layers, except layer I. In primates, SOM+ cells have been described as less densely packed than in the rat cortex and as being located in superficial and deep bands in the cerebral cortex . In the PFC of the cynomolgus monkey (Macaca fascicularis), SOM+ cell bodies were found in layers II – III and V – VI . In the human brain, their laminar distribution has been described as uneven, with some studies describing them only in layers IV – VI , others describing them as predominantly present in layers II and III , and yet others describing them in all cortical layers, including in layer I [31, 32]. In general, previous studies on SOM+ neurons in humans used different methodology (either only immunohistochemistry or only RNA in situ hybridization), analyzed different cortical regions (though none of them focused on the PFC) and assessed varying pathologies, while coming to somewhat conflicting findings. In order to resolve these discrepancies in literature, it is necessary to conduct systematic studies that first assess SOM+ neurons in non-pathological tissue utilizing multiple complementary histological methods.
The role of SOM+ neurons in the human brain is still understudied and their laminar distribution as well as morphological and molecular features in the human brain are not resolved. In particular, no studies so far addressed the molecular expression, morphological features and laminar distribution of SOM+ neurons in the human PFC. Therefore, the aim of this paper is to give a comprehensive molecular and morphological characterization of SOM+ neurons in phylogenetically and functionally entirely different cortical areas (Brodmann areas 9 and 14r) of the human PFC using novel histological methodology combining immunofluorescence and RNAscope in situ hybridization.