GABAergic Cell Types Research Articles

Single-cell Profiling Uncovers Evolutionary Divergence of Hypocretin/Orexin Neuronal Subpopulations.

Brain nuclei are traditionally defined by their anatomy, activity, and expression of specific markers. The hypothalamus contains discrete neuronal populations that coordinate fundamental behavioral functions, including sleep and wakefulness, in all vertebrates. Particularly, the diverse roles of hypocretin/orexin (Hcrt)-releasing neurons suggest functional heterogeneity among Hcrt neurons. Using single-cell RNA sequencing (scRNA-seq) and high-resolution imaging of the adult male and female zebrafish hypothalamic periventricular zone (PVZ), we identified 21 glutamatergic and 28 GABAergic cell types. Integration of zebrafish and mouse scRNA-seq revealed evolutionary conserved and divergent hypothalamic cell types. The expression of specific genes, including npvf, which encodes a sleep-regulating neuropeptide, was enriched in subsets of glutamatergic Hcrt neurons in both larval and adult zebrafish. The genetic profile, activity, and neurite processing of the neuronal subpopulation that co-expresses both Hcrt and Npvf (Hcrt+Npvf+) differ from other Hcrt neurons. These inter-species findings provide a unified annotation of hypothalamic cell types, and suggest that the heterogeneity of Hcrt neurons enables multi-functionality, such as consolidation of both wake and sleep by the Hcrt- and Npvf-releasing neuronal subpopulation.Significance Statement The study reveals the intricate heterogeneity within the hypothalamic periventricular zone (PVZ) of zebrafish, identifying 21 glutamatergic and 28 GABAergic cell types through single-cell RNA sequencing (scRNA-seq) and high-resolution imaging. Comparative analysis with mouse scRNA-seq data revealed conserved and divergent cell types, transcriptional regulatory mechanisms, and neuropeptide localization. Notably, we identified a unique neuronal subpopulation co-expressing both hypocretin/orexin (Hcrt) and neuropeptide VF (Npvf) neuropeptides in zebrafish. The distinct genetic profiles, activity patterns, and neurite processing of this subpopulation suggest a role in regulating both sleep and wakefulness.

The transcriptomic and spatial organization of telencephalic GABAergic neuronal types.

The telencephalon of the mammalian brain comprises multiple regions and circuit pathways that play adaptive and integrative roles in a variety of brain functions. There is a wide array of GABAergic neurons in the telencephalon; they play a multitude of circuit functions, and dysfunction of these neurons has been implicated in diverse brain disorders. In this study, we conducted a systematic and in-depth analysis of the transcriptomic and spatial organization of GABAergic neuronal types in all regions of the mouse telencephalon and their developmental origins. This was accomplished by utilizing 611,423 single-cell transcriptomes from the comprehensive and high-resolution transcriptomic and spatial cell type atlas for the adult whole mouse brain we have generated, supplemented with an additional single-cell RNA-sequencing dataset containing 99,438 high-quality single-cell transcriptomes collected from the pre- and postnatal developing mouse brain. We present a hierarchically organized adult telencephalic GABAergic neuronal cell type taxonomy of 7 classes, 52 subclasses, 284 supertypes, and 1,051 clusters, as well as a corresponding developmental taxonomy of 450 clusters across different ages. Detailed charting efforts reveal extraordinary complexity where relationships among cell types reflect both spatial locations and developmental origins. Transcriptomically and developmentally related cell types can often be found in distant and diverse brain regions indicating that long-distance migration and dispersion is a common characteristic of nearly all classes of telencephalic GABAergic neurons. Additionally, we find various spatial dimensions of both discrete and continuous variations among related cell types that are correlated with gene expression gradients. Lastly, we find that cortical, striatal and some pallidal GABAergic neurons undergo extensive postnatal diversification, whereas septal and most pallidal GABAergic neuronal types emerge simultaneously during the embryonic stage with limited postnatal diversification. Overall, the telencephalic GABAergic cell type taxonomy can serve as a foundational reference for molecular, structural and functional studies of cell types and circuits by the entire community.

Single-cell spatial transcriptome reveals cell-type organization in the macaque cortex

Elucidating the cellular organization of the cerebral cortex is critical for understanding brain structure and function. Using large-scale single-nucleus RNA sequencing and spatial transcriptomic analysis of 143 macaque cortical regions, we obtained a comprehensive atlas of 264 transcriptome-defined cortical cell types and mapped their spatial distribution across the entire cortex. We characterized the cortical layer and region preferences of glutamatergic, GABAergic, and non-neuronal cell types, as well as regional differences in cell-type composition and neighborhood complexity. Notably, we discovered a relationship between the regional distribution of various cell types and the region's hierarchical level in the visual and somatosensory systems. Cross-species comparison of transcriptomic data from human, macaque, and mouse cortices further revealed primate-specific cell types that are enriched in layer 4, with their marker genes expressed in a region-dependent manner. Our data provide a cellular and molecular basis for understanding the evolution, development, aging, and pathogenesis of the primate brain.

Marco Capogna, a pioneering neuroscientist and true European

Marco Capogna, a pioneering neuroscientist and true European

Transcriptional and cell type profiles of cortical brain regions showing ultradian cortisol rhythm dependent responses to emotional face stimulation

The characteristic endogenous circadian rhythm of plasma glucocorticoid concentrations is made up from an underlying ultradian pulsatile secretory pattern. Recent evidence has indicated that this ultradian cortisol pulsatility is crucial for normal emotional response in man. In this study, we investigate the anatomical transcriptional and cell type signature of brain regions sensitive to a loss of ultradian rhythmicity in the context of emotional processing. We combine human cell type and transcriptomic atlas data of high spatial resolution with functional magnetic resonance imaging (fMRI) data. We show that the loss of cortisol ultradian rhythm alters emotional processing response in cortical brain areas that are characterized by transcriptional and cellular profiles of GABAergic function. We find that two previously identified key components of rapid non-genomic GC signaling – the ANXA1 gene and retrograde endocannabinoid signaling – show most significant differential expression (q = 3.99e−10) and enrichment (fold enrichment = 5.56, q = 9.09e−4). Our results further indicate that specific cell types, including a specific NPY-expressing GABAergic neuronal cell type, and specific G protein signaling cascades underly the cerebral effects of a loss of ultradian cortisol rhythm. Our results provide a biological mechanistic underpinning of our fMRI findings, indicating specific cell types and cascades as a target for manipulation in future experimental studies.

Integrative cross-species analysis of GABAergic neuron cell types and their functions in Alzheimer’s disease

Understanding the phenotypic and functional diversity of cerebral cortical GABAergic neurons requires a comprehensive analysis of key transcriptional signatures and neuronal subtype identity. However, the diversity and conservation of GABAergic neurons across multiple mammals remain unclear. Here, we collected the single-nucleus RNA sequencing (snRNA-seq) datasets of cerebral cortex from human, macaque, mouse, and pig to identify the conserved neuronal cell types across species. After systematic analysis of the heterogeneity of GABAergic neurons, we defined four major conserved GABAergic neuron subclasses (Inc SST, Inc LAMP5, Inc PVALB, and Inc VIP) across species. We characterized the species-enriched subclasses of GABAergic neurons from four mammals, such as Inc Meis2 in mouse. Then, we depicted the genetic regulatory network (GRNs) of GABAergic neuron subclasses, which showed the conserved and species-specific GRNs for GABAergic neuron cell types. Finally, we investigated the GABAergic neuron subclass-specific expression modules of Alzheimer’s disease (AD)-related genes in GABAergic neuron cell types. Overall, our study reveals the conserved and divergent GABAergic neuron subclasses and GRNs across multiple species and unravels the gene expression modules of AD-risk genes in GABAergic neuron subclasses, facilitating the GABAergic neurons research and clinical treatment.

Differential effects of group III metabotropic glutamate receptors on spontaneous inhibitory synaptic currents in spine-innervating double bouquet and parvalbumin-expressing dendrite-targeting GABAergic interneurons in human neocortex.

Diverse neocortical GABAergic neurons specialize in synaptic targeting and their effects are modulated by presynaptic metabotropic glutamate receptors (mGluRs) suppressing neurotransmitter release in rodents, but their effects in human neocortex are unknown. We tested whether activation of group III mGluRs by L-AP4 changes GABAA receptor-mediated spontaneous inhibitory postsynaptic currents (sIPSCs) in 2 distinct dendritic spine-innervating GABAergic interneurons recorded in vitro in human neocortex. Calbindin-positive double bouquet cells (DBCs) had columnar "horsetail" axons descending through layers II-V innervating dendritic spines (48%) and shafts, but not somata of pyramidal and nonpyramidal neurons. Parvalbumin-expressing dendrite-targeting cell (PV-DTC) axons extended in all directions innervating dendritic spines (22%), shafts (65%), and somata (13%). As measured, 20% of GABAergic neuropil synapses innervate spines, hence DBCs, but not PV-DTCs, preferentially select spine targets. Group III mGluR activation paradoxically increased the frequency of sIPSCs in DBCs (to median 137% of baseline) but suppressed it in PV-DTCs (median 92%), leaving the amplitude unchanged. The facilitation of sIPSCs in DBCs may result from their unique GABAergic input being disinhibited via network effect. We conclude that dendritic spines receive specialized, diverse GABAergic inputs, and group III mGluRs differentially regulate GABAergic synaptic transmission to distinct GABAergic cell types in human cortex.

Neuronal and Non‐neuronal Cell Type Mapping in the Mouse Brain

The brain contains multiple neuronal and non‐neuronal cell types with distinct spatial relationship that serve as a basic building block in the brain. I previously developed high resolution mapping methods to visualize and to quantify these individual brain cell types across the entire mouse brain. In this talk, I will present my lab’s effort to understand spatial arrangement of several major cell types in the mouse brain across lifetime. I previously applied my mapping methods to examine GABAergic neuronal cell types, c‐Fos‐labeled cells representing functional network activation, and long‐range anatomical connectivity of oxytocin neurons. Moreover, I used the method to trace the complete network of cerebrovasculature and pericytes that showed striking correlation with cortical parvalbumin and neuronal nitric oxide synthase (nNOS) positive neurons. Recently, my lab established a new online anatomical atlas for the adult mouse brain that combines the segmentation of the Allen Mouse Brian Atlas and Paxinos Mouse Brain Atlas, and developed new atlas framework for early postnatal brains. Using these resources, I have mapped developmental trajectories of oxytocin receptor‐expressing neurons during early postnatal development, unveiling spatial temporal patterns of the brain cell type. In summary, high resolution anatomical mapping of cell types helps to understand overall anatomical and functional organization of the brain.

Optoception: Perception of Optogenetic Brain Perturbations.

How do animals experience brain manipulations? Optogenetics has allowed us to manipulate selectively and interrogate neural circuits underlying brain function in health and disease. However, little is known about whether mice can detect and learn from arbitrary optogenetic perturbations from a wide range of brain regions to guide behavior. To address this issue, mice were trained to report optogenetic brain perturbations to obtain rewards and avoid punishments. Here, we found that mice can perceive optogenetic manipulations regardless of the perturbed brain area, rewarding effects, or the stimulation of glutamatergic, GABAergic, and dopaminergic cell types. We named this phenomenon optoception, a perceptible signal internally generated from perturbing the brain, as occurs with interoception. Using optoception, mice can learn to execute two different sets of instructions based on the laser frequency. Importantly, optoception can occur either activating or silencing a single cell type. Moreover, stimulation of two brain regions in a single mouse uncovered that the optoception induced by one brain region does not necessarily transfer to a second not previously stimulated area, suggesting a different sensation is experienced from each site. After learning, they can indistinctly use randomly interleaved perturbations from both brain regions to guide behavior. Collectively taken, our findings revealed that mice’s brains could “monitor” perturbations of their self-activity, albeit indirectly, perhaps via interoception or as a discriminative stimulus, opening a new way to introduce information to the brain and control brain-computer interfaces.

Development of the mammalian main olfactory bulb.

ABSTRACTThe mammalian main olfactory bulb is a crucial processing centre for the sense of smell. The olfactory bulb forms early during development and is functional from birth. However, the olfactory system continues to mature and change throughout life as a target of constitutive adult neurogenesis. Our Review synthesises current knowledge of prenatal, postnatal and adult olfactory bulb development, focusing on the maturation, morphology, functions and interactions of its diverse constituent glutamatergic and GABAergic cell types. We highlight not only the great advances in the understanding of olfactory bulb development made in recent years, but also the gaps in our present knowledge that most urgently require addressing.

Parvalbumin expressing interneurons control spike-phase coupling of hippocampal cells to theta oscillations

Encoding of information by hippocampal neurons is defined by the number and the timing of action potentials generated relative to ongoing network oscillations in the theta (5–14 Hz), gamma (30–80 Hz) and ripple frequency range (150–200 Hz). The exact mechanisms underlying the temporal coupling of action potentials of hippocampal cells to the phase of rhythmic network activity are not fully understood. One critical determinant of action potential timing is synaptic inhibition provided by a complex network of Gamma-amino-hydroxy-butyric acid releasing (GABAergic) interneurons. Among the various GABAergic cell types, particularly Parvalbumin-expressing cells are powerful regulators of neuronal activity. Here we silenced Parvalbumin-expressing interneurons in hippocampal areas CA1 and the dentate gyrus in freely moving mice using the optogenetic silencing tool eNpHR to determine their influence on spike timing in principal cells. During optogenetic inhibition of Parvalbumin-expressing cells, local field potential recordings revealed no change in power or frequency of CA1 or dentate gyrus network oscillations. However, CA1 pyramidal neurons exhibited significantly reduced spike-phase coupling to CA1 theta, but not gamma or ripple oscillations. These data suggest that hippocampal Parvalbumin-expressing interneurons are particularly important for an intact theta-based temporal coding scheme of hippocampal principal cell populations.

Vision-dependent specification of cell types and function in the developing cortex

The role of postnatal experience in sculpting cortical circuitry, while long appreciated, is poorly understood at the level of cell types. We explore this in the mouse primary visual cortex (V1) using single-nucleus RNA sequencing, visual deprivation, genetics, and functional imaging. We find that vision selectively drives the specification of glutamatergic cell types in upper layers (L) (L2/3/4), while deeper-layer glutamatergic, GABAergic, and non-neuronal cell types are established prior to eye opening. L2/3 cell types form an experience-dependent spatial continuum defined by the graded expression of ∼200 genes, including regulators of cell adhesion and synapse formation. One of these genes, Igsf9b, a vision-dependent gene encoding an inhibitory synaptic cell adhesion molecule, is required for the normal development of binocular responses in L2/3. In summary, vision preferentially regulates the development of upper-layer glutamatergic cell types through the regulation of cell-type-specific gene expression programs.

Cholinergic boutons are closely associated with excitatory cells and four subtypes of inhibitory cells in the inferior colliculus

Acetylcholine (ACh) is a neuromodulator that has been implicated in multiple roles across the brain, including the central auditory system, where it sets neuronal excitability and gain and affects plasticity. In the cerebral cortex, subtypes of GABAergic interneurons are modulated by ACh in a subtype-specific manner. Subtypes of GABAergic neurons have also begun to be described in the inferior colliculus (IC), a midbrain hub of the auditory system. Here, we used male and female mice (Mus musculus) that express fluorescent protein in cholinergic cells, axons, and boutons to look at the association between ACh and four subtypes of GABAergic IC cells that differ in their associations with extracellular markers, their soma sizes, and their distribution within the IC. We found that most IC cells, including excitatory and inhibitory cells, have cholinergic boutons closely associated with their somas and proximal dendrites. We also found that similar proportions of each of four subtypes of GABAergic cells are closely associated with cholinergic boutons. Whether the different types of GABAergic cells in the IC are differentially regulated remains unclear, as the response of cells to ACh is dependent on which types of ACh receptors are present. Additionally, this study confirms the presence of these four subtypes of GABAergic cells in the mouse IC, as they had previously been identified only in guinea pigs. These results suggest that cholinergic projections to the IC modulate auditory processing via direct effects on a multitude of inhibitory circuits.

Interneuron Types and Their Circuits in the Basolateral Amygdala.

The basolateral amygdala (BLA) is a cortical structure based on its cell types, connectivity features, and developmental characteristics. This part of the amygdala is considered to be the main entry site of processed and multisensory information delivered via cortical and thalamic afferents. Although GABAergic inhibitory cells in the BLA comprise only 20% of the entire neuronal population, they provide essential control over proper network operation. Previous studies have uncovered that GABAergic cells in the basolateral amygdala are as diverse as those present in other cortical regions, including the hippocampus and neocortex. To understand the role of inhibitory cells in various amygdala functions, we need to reveal the connectivity and input-output features of the different types of GABAergic cells. Here, I review the recent achievements in uncovering the diversity of GABAergic cells in the basolateral amygdala with a specific focus on the microcircuit organization of these inhibitory cells.

Single-cell co-expression analysis reveals that transcriptional modules are shared across cell types in the brain.

Gene-gene relationships are commonly measured via the co-variation of gene expression across samples, also known as gene co-expression. Because shared expression patterns are thought to reflect shared function, co-expression networks describe functional relationships between genes, including co-regulation. However, the heterogeneity of cell types in bulk RNA-seq samples creates connections in co-expression networks that potentially obscure co-regulatory modules. The brain initiative cell census network (BICCN) single-cell RNA sequencing (scRNA-seq) datasets provide an unparalleled opportunity to understand how gene-gene relationships shape cell identity. Comparison of the BICCN data (500,000 cells/nuclei across 7 BICCN datasets) with that of bulk RNA-seq networks (2,000 mouse brain samples across 52 studies) reveals a consistent topology reflecting a shared co-regulatory signal. Differential signals between broad cell classes persist in driving variation at finer levels, indicating that convergent regulatory processes affect cell phenotype at multiple scales.

Total Number and Ratio of GABAergic Neuron Types in the Mouse Lateral and Basal Amygdala.

GABAergic neurons are key circuit elements in cortical networks. Despite growing evidence showing that inhibitory cells play a critical role in the lateral (LA) and basal (BA) amygdala functions, neither the number of GABAergic neurons nor the ratio of their distinct types has been determined in these amygdalar nuclei. Using unbiased stereology, we found that the ratio of GABAergic neurons in the BA (22%) is significantly higher than in the LA (16%) in both male and female mice. No difference was observed between the right and left hemispheres in either sex. In addition, we assessed the ratio of the major inhibitory cell types in both amygdalar nuclei. Using transgenic mice and a viral strategy for visualizing inhibitory cells combined with immunocytochemistry, we estimated that the following cell types together compose the vast majority of GABAergic cells in the LA and BA: axo-axonic cells (5.5%-6%), basket cells expressing parvalbumin (17%-20%) or cholecystokinin (7%-9%), dendrite-targeting inhibitory cells expressing somatostatin (10%-16%), NPY-containing neurogliaform cells (14%-15%), VIP and/or calretinin-expressing interneuron-selective interneurons (29%-38%), and GABAergic projection neurons expressing somatostatin and neuronal nitric oxide synthase (5.5%-8%). Our results show that these amygdalar nuclei contain all major GABAergic neuron types as found in other cortical regions. Furthermore, our data offer an essential reference for future studies aiming to reveal changes in GABAergic cell number and in inhibitory cell types typically observed under different pathologic conditions, and to model functioning amygdalar networks in health and disease.SIGNIFICANCE STATEMENT GABAergic cells in cortical structures, as in the lateral and basal nucleus of the amygdala, have a determinant role in controlling circuit operation. In this study, we provide the first estimate for the total number of inhibitory cells in these two amygdalar nuclei. In addition, our study is the first to define the ratio of the major GABAergic cell types present in these cortical networks. Taking into account that hyperexcitability in the amygdala, arising from the imbalance between excitation and inhibition typifies many altered brain functions, including anxiety, post-traumatic stress disorder, schizophrenia, and autism, uncovering the number and ratio of distinct amygdalar inhibitory cell types offers a solid base for comparing the changes in inhibition in pathologic brain states.

Sleep down state-active ID2/Nkx2.1 interneurons in the neocortex.

Pyramidal cells and GABAergic interneurons fire together in balanced cortical networks. In contrast to this general rule, we describe a distinct neuron type in mice and rats whose spiking activity is anti-correlated with all principal cells and interneurons in all brain states but, most prevalently, during the down state of non-REM (NREM) sleep. We identify these down state-active (DSA) neurons as deep-layer neocortical neurogliaform cells that express ID2 and Nkx2.1 and are weakly immunoreactive to neuronal nitric oxide synthase. DSA neurons are weakly excited by deep-layer pyramidal cells and strongly inhibited by several other GABAergic cell types. Spiking of DSA neurons modified the sequential firing order of other neurons at down-up transitions. Optogenetic activation of ID2+Nkx2.1+ interneurons in the posterior parietal cortex during NREM sleep, but not during waking, interfered with consolidation of cue discrimination memory. Despite their sparsity, DSA neurons perform critical physiological functions.

Diverse GABAergic neurons organize into subtype-specific sublaminae in the ventral lateral geniculate nucleus.

In the visual system, retinal axons convey visual information from the outside world to dozens of distinct retinorecipient brain regions and organize that information at several levels, including either at the level of retinal afferents, cytoarchitecture of intrinsic retinorecipient neurons, or a combination of the two. Two major retinorecipient nuclei which are densely innervated by retinal axons are the dorsal lateral geniculate nucleus, which is important for classical image‐forming vision, and ventral LGN (vLGN), which is associated with non‐image‐forming vision. The neurochemistry, cytoarchitecture, and retinothalamic connectivity in vLGN remain unresolved, raising fundamental questions of how it receives and processes visual information. To shed light on these important questions, used in situ hybridization, immunohistochemistry, and genetic reporter lines to identify and characterize novel neuronal cell types in mouse vLGN. Not only were a high percentage of these cells GABAergic, we discovered transcriptomically distinct GABAergic cell types reside in the two major laminae of vLGN, the retinorecipient, external vLGN (vLGNe) and the non‐retinorecipient, internal vLGN (vLGNi). Furthermore, within vLGNe, we identified transcriptionally distinct subtypes of GABAergic cells that are distributed into four adjacent sublaminae. Using trans‐synaptic viral tracing and in vitro electrophysiology, we found cells in each these vLGNe sublaminae receive monosynaptic inputs from retina. These results not only identify novel subtypes of GABAergic cells in vLGN, they suggest the subtype‐specific laminar distribution of retinorecipient cells in vLGNe may be important for receiving, processing, and transmitting light‐derived signals in parallel channels of the subcortical visual system.

Developmental loss of MeCP2 from VIP interneurons impairs cortical function and behavior.

Rett Syndrome is a devastating neurodevelopmental disorder resulting from mutations in the gene MECP2. Mutations of Mecp2 that are restricted to GABAergic cell types largely replicate the behavioral phenotypes associated with mouse models of Rett Syndrome, suggesting a pathophysiological role for inhibitory interneurons. Recent work has suggested that vasoactive intestinal peptide-expressing (VIP) interneurons may play a critical role in the proper development and function of cortical circuits, making them a potential key point of vulnerability in neurodevelopmental disorders. However, little is known about the role of VIP interneurons in Rett Syndrome. Here we find that loss of MeCP2 specifically from VIP interneurons replicates key neural and behavioral phenotypes observed following global Mecp2 loss of function.

GABAergic cell types in the superficial layers of the mouse superior colliculus.

To begin to unravel the complexities of GABAergic circuits in the superior colliculus (SC), we utilized mouse lines that express green fluorescent protein (GFP) in cells that contain the 67 kDa isoform of glutamic acid decarboxylase (GAD67-GFP), or Cre-recombinase in cells that contain glutamic acid decarboxylase (GAD; GAD2-cre). We used Cre-dependent virus injections in GAD2-Cre mice and tracer injections in GAD67-GFP mice, as well as immunocytochemical staining for gamma amino butyric acid (GABA) and parvalbumin (PV) to characterize GABAergic cells that project to the pretectum (PT), ventral lateral geniculate nucleus (vLGN) or parabigeminal nucleus (PBG), and interneurons in the stratum griseum superficiale (SGS) that do not project outside the SC. We found that approximately 30% of SGS neurons in the mouse are GABAergic. Of these GABAergic neurons, we identified three categories of potential interneurons in the GAD67-GFP line (GABA+GFP ~45%, GABA+GFP + PV ~15%, and GABA+PV ~10%). GABAergic cells that did not contain GFP or PV were identified as potential projection neurons (GABA only ~30%). We found that GABAergic neurons that project to the PBG are primarily located in the SGS and exhibit narrow field vertical, stellate, and horizontal dendritic morphologies, while GABAergic neurons that project to the PT and vLGN are primarily located in layers ventral to the SGS. In addition, we examined GABA and GAD67-containing elements of the mouse SGS using electron microscopy to further delineate the relationship between GABAergic circuits and retinotectal input. Approximately 30% of retinotectal synaptic targets are the presynaptic dendrites of GABAergic interneurons, and GAD67-GFP interneurons are a source of these presynaptic dendrites.