Interneuron Markers

Pan-GABAergic Interneuron Markers

Glutamate decarboxylase (GAD) is an enzyme responsible for synthesizing the inhibitory neurotransmitter GABA released by inhibitory interneurons.^5,6 GAD67 (encoded by Gad1) and GAD65 (encoded by Gad2) serve as pan-interneuron markers to characterize inhibitory interneurons across different nervous system regions. Although both isoforms are expressed in inhibitory neurons, GAD67 is constitutively active and widely distributed in soma and dendrites,⁷ whereas GAD65 is expressed at synaptic terminals and dynamically regulated during neurotransmission.⁸ Their broad expression makes them essential tools for delineating GABAergic interneurons from excitatory glutamatergic neurons in immunohistochemistry (IHC), Western blot (WB), and flow cytometry. For a more comprehensive overview of pan-GABAergic interneuron markers, please refer to the GABAergic Neuron Markers page.

Figure 1: IHC of rat brain tissue stained with Anti-GAD65 Antibody (A12729) in red. Nuclei were stained by DAPI in blue.

Figure 2: IHC of rat eye cells stained with Anti-GAD67 Antibody (A14262) in red. Nuclei were stained by DAPI in blue.

Parvalbumin (PV)

PV is a calcium-binding protein that serves as a key molecular marker for a major interneuron class that makes up approximately 40% of interneurons in the cortex1. PV-expressing (PV+) interneurons are functionally diverse and include subclasses such as basket, chandelier, and translaminar interneurons, each targeting distinct compartments of principal neurons and playing critical roles in modulating network oscillations,⁹ synaptic plasticity¹⁰ and excitatory-inhibitory balance.^11,12 While most PV interneurons are classically defined as fast-spiking interneurons, there are also non-fast-spiking PV interneuron subclasses such as a layer 4 PV interneuron subclass.¹³ IHC of PV helps to distinguish PV interneurons from other inhibitory neuron classes, locate their layer distribution and study their connectivity and co-expression with other markers.

Figure 3: IHC of rat hippocampal tissue stained with Anti-Parvalbumin Antibody [3C9] (A85317) in red and Anti-Calretinin Antibody (A104312) in green. Nuclei were stained by DAPI in blue.

Figure 4: IHC of rat eye cells stained with Anti-Parvalbumin Antibody (A85316) in green and Anti-GFAP Antibody (A85422) in red. Nuclei were stained by DAPI in blue.

Somatostatin (SST)

SST is a neuropeptide widely expressed in the nervous system and acts as both neurotransmitter and neuromodulator to regulate network activity.¹⁴ In the cortex, SST marks a major interneuron class, known as SST-expressing (SST+) interneurons or SST interneurons, that constitutes 30% of the cortical interneuron population.

Figure 5: IHC of rat brain tissue stained with Anti-Somatostatin Antibody [ARC50670] (A308885).

Figure 6: IHC of rat pancreas cells stained with Anti-Somatostatin Antibody (A16303) in orange. Nuclei were stained by DAPI in blue.

SST interneurons, which encompass the well-characterized subclasses Martinotti, non-Matinotti and long-projecting neurons, predominantly target the distal dendrites of pyramidal neurons, thereby modulating excitatory input,¹⁵ shaping sensory processing,¹⁶ and regulating cortical network activity.^17,18 SST interneurons are also essential for excitation-to-inhibition balance, synaptic plasticity, learning,¹⁹ and memory.²⁰ IHC of SST can be applied in combination with calretinin (CR) and calbindin (CB) to characterize Martinotti interneurons (SST+/CR+/CB+); and with nitric oxide synthase (NOS) to identify long-projecting SST interneurons (SST+/NOS+).

Figure 7: IHC of rat cerebellum stained with Anti-Calretinin Antibody [3G9] (A85367) in green and Anti-Calbindin Antibody (A85359) in red.

Figure 8: IHC of rat cerebellum stained with Anti-Calbindin Antibody [5A9] (A85362) in red and Anti-GFAP Antibody (A85419) in green. Nuclei were stained by DAPI in blue.

Serotonin receptor 3A (5HT3A)

5HT3A is an ionotropic serotonin receptor that directly controls ion channels for rapid synaptic transmission upon the binding of serotonin.²¹ 5HT3A is predominantly expressed by a major interneuron class derived from a transient embryonic brain region, the caudal ganglionic eminence,²² in contrast to the PV and SST interneuron classes generated from the medial ganglionic eminence.²³ 5HT3A interneurons constitute approximately 30% of cortical interneurons and are especially enriched in cortical layers 2/3.²¹ These interneurons regulate neural activities during cognitive and emotional brain processes, including spatial memory, fear and anxiety.²⁴ In addition, cortical 5HT3A interneurons modulate neurovasculature by inducing vasodilation mediated by nitric oxide (NO) and vasoconstriction mediated by neuropeptide Y (NPY).²⁵

5HT3A interneurons are functionally and molecularly heterogeneous and comprise several subclasses with distinct roles in cortical circuits. Key 5HT3A interneuron subclasses include:

• Vasoactive intestinal peptide (VIP)-expressing interneurons (5HT3A +/VIP+), which mediate disinhibition by inhibiting PV and SST interneurons.^4,26
• Cholecystokinin (CCK)-expressing (5HT3A +/VIP+/CCK+) or CCK-negative interneurons, subclasses of VIP interneurons.
• Neuropeptide Y (NPY)-expressing interneurons (5HT3A+/NPY+), which target pyramidal neuron dendrites and influence dendritic maturation.
• Reelin (RELN)-expressing neurogliaform cells (5HT3A+/RELN+/NPY+), which provide slow and diffuse inhibition through volumetric GABA transmission.²⁷
• MEIS2-expressing interstitial interneurons (5HT3A+/ MEIS2+), recently identified by single cell transcriptomics, which reside in the white matter and extend axons to deep layers of the cortex and striatum.²⁸

Figure 9: IHC of mouse brain tissue stained with Anti-5HT3A Receptor Antibody (A11858).

Figure 10: IHC of human brain tissue stained with Anti-NPY Antibody (A98310).

GLYT2 and VGAT

In the spinal cord, three principal glycinergic interneuron types have been described. Renshaw cells are located in the ventral horn and are well known for providing recurrent inhibition to motor neurons. This inhibition is mediated by large synaptic terminals expressing the membrane-bound glycine transporter 2 (GLYT2), which are a hallmark of glycinergic neurotransmission.³¹ Another marker, the vesicular GABA transporter (VGAT), also known as VIAAT and SLC32A1, which transports both GABA and glycine into synaptic vesicles, needs to be co-expressed with GLYT2 in a neuron to fulfil glycinergic transmission.³¹

Glycine receptors (GlyRs) and Gephyrin

At the postsynaptic level, glycinergic signaling is mediated by glycine receptors (GlyRs), which are pentameric ligand-gated chloride channels constituting α (α1-α4) and β subunits. The α1 subunit (GlyRα1) is the most abundant in adult spinal cord and brainstem neurons, making it a useful marker for glycinergic synapses.^32,33 Gephyrin (GPHN), a scaffolding protein that anchors GlyRs at inhibitory synapses, is another valuable marker.²⁹

All the aforementioned glycinergic markers can also be used to identify dorsal horn interneurons as well as ventral inhibitory commissural interneurons. Dorsal horn interneurons are primarily found in laminae I-III of the spinal cord, responsible for gating nociceptive (pain) and pruritic (itch) signals,³⁴ while ventral inhibitory commissural interneuron project glycinergic axons across the midline to regulate timing and synchronization of motor activity on both sides of the body.³⁵

mGluR2 and neurogranin

In the brain stem, 65% of Golgi cells located in the cerebellar granule layer are capable of co-releasing glycine and GABA. They can be identified by their co-expression of molecular markers such as mGluR2 and neurogranin.³⁶ These features allow Golgi cells to modulate granule cell activity and contribute to the fine-tuning of cerebellar processing. On the other hand, Lugaro and globular cells represent other cerebellar interneuron classes that are also glycinergic and GABAergic and can be distinguished by their lack of mGluR2 expression.³⁶ They play a role in regulating circuits within the molecular layer of the cerebellum, further highlighting the functional heterogeneity among glycinergic interneurons.^36,37

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