Marcia G. Honig

Dil and DiO: versatile fluorescent dyes for neuronal labelling and pathway tracing

The fluorescent carbocyanine dyes dil and diO have an extensive history of use in cell biology, but their use as neuronal tracers is relatively recent. We found in 1985 that these molecules were excellent retrograde and anterograde tracers in the developing nervous system. We went on to show that these dyes were retained in neurons placed in culture, that they initially labelled the processes as well as the cell bodies of cultured neurons, and that they were seemingly non-toxic. We suggested that the major mechanism of translocation for these molecules was lateral diffusion in the membrane, rather than fast axonal transport. This suggestion was recently confirmed in a striking manner by Godement et al., when they showed that these dyes can be used to label axonal projections in fixed tissues. Labelling with carbocyanine dyes has already allowed several exciting advances in developmental neurobiology. In this article we review the properties of carbocyanine dyes and point out some of their uses and advantages.

Biotinylated dextran amine as an anterograde tracer for single- and double-labeling studies.

Fluorescent dextran amines have recently been reported to be useful for anterograde pathway tracing. However, fluorescent markers are not always ideal for detailed mapping studies. We therefore evaluated the efficacy of a biotinylated dextran amine (BDA) for anterograde labeling in several different preparations. BDA was visualized with an avidin-biotinylated HRP (ABC) procedure followed by a standard or metal-enhanced diaminobenzidine (DAB) reaction. After iontophoretic injections of BDA into neocortex-like telencephalic regions in pigeons or into visual or somatosensory cortex in rats, there was excellent and abundant labeling of axons and terminals in forebrain, midbrain and hindbrain target areas with 1-week survival times. Large pressure injections of BDA into the avian telencephalon were also found to result in extensive anterograde labeling. We then carried out a series of studies using 2-color DAB double-labeling to determine effective approaches for combining BDA labeling with other labeling methods. Using an isolated embryonic chick spinal cord-hindlimb preparation, we combined BDA labeling with another anterograde labeling method to differentially label two sets of projections. In these studies, sensory neuron and motoneuron projections into the limb from the same segmental level, or motoneuron projections into the limb from two separate segments were differentially labeled by using HRP (visualized first with a blue/black metal-DAB reaction) and BDA (visualized second with a brown DAB reaction). In other double-labeling studies, we combined BDA labeling of axons and terminals with immunohistochemical labeling of neurons. In these experiments, telencephalic neurons in pigeons or rats were labeled immunohistochemically for parvalbumin or substance P (using a brown DAB reaction) and BDA-labeled axons were labeled blue/black (using a metal-intensified DAB reaction). Double-labeling was successful regardless of whether the entire immunohistochemical labeling procedure preceded or followed the BDA labeling procedure. Together, these studies show that BDA is effective for anterograde pathway tracing and can be used in double-label studies with other labeling methods.

Pathway tracing using biotinylated dextran amines

Biotinylated dextran amines (BDA) are highly sensitive tools for anterograde and retrograde pathway tracing studies of the nervous system. BDA can be reliably delivered into the nervous system by iontophoretic or pressure injection and visualized with an avidin-biotinylated HRP (ABC) procedure, followed by a standard or metal-enhanced diaminobenzidine (DAB) reaction. High molecular weight BDA (10 k) yields sensitive and exquisitely detailed labeling of axons and terminals, while low molecular weight BDA (3 k) yields sensitive and detailed retrograde labeling of neuronal cell bodies. The detail of neuronal cell body labeling can be Golgi-like. BDA tolerates EM fixation and processing well and can, therefore, be readily used in ultrastructural studies. Additionally, BDA can be combined with other anterograde or retrograde tracers (e.g. PHA-L or cholera toxin B fragment) and visualized either by multi-color DAB multiple-labeling - if permanent labels are desired, or by using multiple simultaneous immunofluorescence - if fluorescence viewing is desired. In the same manner, BDA pathway tracing and neurotransmitter immunolabeling can be combined. Note that BDA pathway tracing can also be combined with anterograde or retrograde labeling with fluorescent dextran amines, if one wishes to exclusively use tracers with the favorable transport properties and sensitivities of dextran amines. In this case, the BDA can be visualized together with the fluorescent dextran amines using fluorescence labeling for the BDA, or the fluorescent dextran amines can be visualized together with the BDA by multicolor DAB labeling via immunolabeling of the fluorescent dextran amines using anti-fluorophore antisera. BDA is, thus, a flexible and valuable pathway tracing tool that has gained widespread popularity in recent years.

Double-labeling of tissue containing the carbocyanine dye DiI for immunocytochemistry

The fluorescent carbocyanine dye DiI can be used for retrograde and anterograde labeling of neuronal pathways. To investigate the possible neurochemical identity of DiI-labeled neuronal cell bodies and terminals, we used a procedure for double-labeling of the same tissue with antisera to specific neuroactive substances. This procedure involves visualizing the immunohistochemical label with an FITC-conjugated secondary antiserum. Both labels can be viewed in the same tissue by fluorescence microscopy, and individual cell bodies and processes double-labeled with DiI and antiserum can be identified by switching between filter sets appropriate for rhodamine (to see the DiI labeling) and for fluorescein (to see the immunhistochemical labeling). The method has been used with primary antisera to excitatory and inhibitory amino acid neurotransmitters, as well as to neuropeptides, and is likely to be useful with antibodies against a wide variety of substances. Several other immunocytochemical methods were found to be incompatible with DiI labeling.

A Novel Closed-Head Model of Mild Traumatic Brain Injury Caused by Primary Overpressure Blast to the Cranium Produces Sustained Emotional Deficits in Mice

Emotional disorders are a common outcome from mild traumatic brain injury (TBI) in humans, but their pathophysiological basis is poorly understood. We have developed a mouse model of closed-head blast injury using an air pressure wave delivered to a small area on one side of the cranium, to create mild TBI. We found that 20-psi blasts in 3-month-old C57BL/6 male mice yielded no obvious behavioral or histological evidence of brain injury, while 25–40 psi blasts produced transient anxiety in an open field arena but little histological evidence of brain damage. By contrast, 50–60 psi blasts resulted in anxiety-like behavior in an open field arena that became more evident with time after blast. In additional behavioral tests conducted 2–8 weeks after blast, 50–60 psi mice also demonstrated increased acoustic startle, perseverance of learned fear, and enhanced contextual fear, as well as depression-like behavior and diminished prepulse inhibition. We found no evident cerebral pathology, but did observe scattered axonal degeneration in brain sections from 50 to 60 psi mice 3–8 weeks after blast. Thus, the TBI caused by single 50–60 psi blasts in mice exhibits the minimal neuronal loss coupled to “diffuse” axonal injury characteristic of human mild TBI. A reduction in the abundance of a subpopulation of excitatory projection neurons in basolateral amygdala enriched in Thy1 was, however, observed. The reported link of this neuronal population to fear suppression suggests their damage by mild TBI may contribute to the heightened anxiety and fearfulness observed after blast in our mice. Our overpressure air blast model of concussion in mice will enable further studies of the mechanisms underlying the diverse emotional deficits seen after mild TBI.

Motor, Visual and Emotional Deficits in Mice after Closed-Head Mild Traumatic Brain Injury Are Alleviated by the Novel CB2 Inverse Agonist SMM-189

We have developed a focal blast model of closed-head mild traumatic brain injury (TBI) in mice. As true for individuals that have experienced mild TBI, mice subjected to 50–60 psi blast show motor, visual and emotional deficits, diffuse axonal injury and microglial activation, but no overt neuron loss. Because microglial activation can worsen brain damage after a concussive event and because microglia can be modulated by their cannabinoid type 2 receptors (CB2), we evaluated the effectiveness of the novel CB2 receptor inverse agonist SMM-189 in altering microglial activation and mitigating deficits after mild TBI. In vitro analysis indicated that SMM-189 converted human microglia from the pro-inflammatory M1 phenotype to the pro-healing M2 phenotype. Studies in mice showed that daily administration of SMM-189 for two weeks beginning shortly after blast greatly reduced the motor, visual, and emotional deficits otherwise evident after 50–60 psi blasts, and prevented brain injury that may contribute to these deficits. Our results suggest that treatment with the CB2 inverse agonist SMM-189 after a mild TBI event can reduce its adverse consequences by beneficially modulating microglial activation. These findings recommend further evaluation of CB2 inverse agonists as a novel therapeutic approach for treating mild TBI.

Dextran Amines: Versatile Tools for Anterograde and Retrograde Studies of Nervous System Connectivity

Dextran amines are versatile and sensitive tools for anterograde and retrograde investigation of neural connectivity. Because of their tolerance of diverse fixatives, they are ideal for various light and electron microscopic studies. They can be iontophoretically or pressure-injected, and then depending on the type of dextran amine used and the type of detection method, visualized by transmitted light microscopy, fluorescence microscopy, or electron microscopy. High-molecular-weight biotinylated dextran amines (BDAs; 10 kDa) yield sensitive and exquisitely detailed labeling of axons and terminals, while low-molecular-weight BDAs (3 kDa) yield sensitive and detailed Golgi-like retrograde labeling of neurons. Labeling with the BDAs can be visualized with an avidin-biotinylated horseradish peroxidase (ABC) procedure followed by a standard or a metal-enhanced diaminobenzidine (DAB) reaction, or with any of several fluorescent probes that bind to biotin. Fluorescent dextran amines can be directly visualized by fluorescence microscopy or rendered suitable for transmitted light or electron microscopic viewing by immunohistochemical detection of the given fluorophore. The variety of dextran amines and the methods for their visualization make them well-suited for multiple-label studies. The dextran amines can also be combined with other anterograde or retrograde tracers, or intracellular labeling, and the disparate markers separately visualized either by multicolor DAB or by DAB-VIP® labeling, or by multiple fluorescence viewing. In the same manner, pathway tracing with dextran amines and immunohistochemical labeling can be combined. The dextran amines are thus flexible and valuable pathway-tracing tools that have gained widespread popularity since being introduced.

Mild Traumatic Brain Injury Produces Neuron Loss That Can Be Rescued by Modulating Microglial Activation Using a CB2 Receptor Inverse Agonist

We have previously reported that mild TBI created by focal left-side cranial blast in mice produces widespread axonal injury, microglial activation, and a variety of functional deficits. We have also shown that these functional deficits are reduced by targeting microglia through their cannabinoid type-2 (CB2) receptors using 2-week daily administration of the CB2 inverse agonist SMM-189. CB2 inverse agonists stabilize the G-protein coupled CB2 receptor in an inactive conformation, leading to increased phosphorylation and nuclear translocation of the cAMP response element binding protein (CREB), and thus bias activated microglia from a pro-inflammatory M1 to a pro-healing M2 state. In the present study, we showed that SMM-189 boosts nuclear pCREB levels in microglia in several brain regions by 3 days after TBI, by using pCREB/CD68 double immunofluorescent labeling. Next, to better understand the basis of motor deficits and increased fearfulness after TBI, we used unbiased stereological methods to characterize neuronal loss in cortex, striatum, and basolateral amygdala (BLA) and assessed how neuronal loss was affected by SMM-189 treatment. Our stereological neuron counts revealed a 20% reduction in cortical and 30% reduction in striatal neurons bilaterally at 2–3 months post blast, with SMM-189 yielding about 50% rescue. Loss of BLA neurons was restricted to the blast side, with 33% of Thy1+ fear-suppressing pyramidal neurons and 47% of fear-suppressing parvalbuminergic (PARV) interneurons lost, and Thy1-negative fear-promoting pyramidal neurons not significantly affected. SMM-189 yielded 50–60% rescue of Thy1+ and PARV neuron loss in BLA. Thus, fearfulness after mild TBI may result from the loss of fear-suppressing neuron types in BLA, and SMM-189 may reduce fearfulness by their rescue. Overall, our findings indicate that SMM-189 rescues damaged neurons and thereby alleviates functional deficits resulting from TBI, apparently by selectively modulating microglia to the beneficial M2 state. CB2 inverse agonists thus represent a promising therapeutic approach for mitigating neuroinflammation and neurodegeneration.

Identification of Cerebellin2 in chick and its preferential expression by subsets of developing sensory neurons and their targets in the dorsal horn

The cerebellins are a family of four secreted proteins, two of which, Cbln1 and Cbln3, play an important role in the formation and maintenance of parallel fiber‐Purkinje cell synapses. We have identified the chicken homologue of Cbln2 and, through the use of in situ hybridization, shown that it is expressed by specific subsets of neurons in the dorsal root ganglia (DRGs) and spinal cord starting shortly after those neurons are generated. In the developing spinal cord, Cbln2 is highly expressed by dI1, dI3, dI5, and dILB dorsal interneurons and to a lesser extent by dI2, dI4, dI6, and dILA dorsal interneurons, but not by ventral (v0–v3) interneurons. After the spinal cord has matured and neurons have migrated to their final destinations, Cbln2 is abundant in the dorsal horn. In the DRGs, Cbln2 is expressed by TrkB+ and TrkC+ sensory neurons, but not by TrkA+ sensory neurons. Interestingly, regions of the spinal cord where TrkB+ and TrkC+ afferents terminate (i.e., laminae II, III, IV, and VI) exhibit the highest levels of Cbln2 expression. Cbln2 is also expressed by preganglionic sympathetic neurons and their targets in the sympathetic chain ganglia. Thus, the results show that Cbln2 is frequently expressed by synaptically connected neuronal populations. This, in turn, raises the possibility that if Cbln2, like Cbln1, plays a role in the formation and maintenance of synapses, it may somehow mediate bi‐directional communication between discrete populations of neurons and their appropriate neuronal targets. J. Comp. Neurol. 518:2818–2840, 2010.

Parcellation of cerebellins 1, 2, and 4 among different subpopulations of dorsal horn neurons in mouse spinal cord.

The cerebellins (Cblns) are a family of secreted proteins that are widely expressed throughout the nervous system, but whose functions have been studied only in the cerebellum and striatum. Two members of the family, Cbln1 and Cbln2, bind to neurexins on presynaptic terminals and to GluRδs postsynaptically, forming trans‐synaptic triads that promote synapse formation. Cbln1 has a higher binding affinity for GluRδs and exhibits greater synaptogenic activity than Cbln2. In contrast, Cbln4 does not form such triads and its function is unknown. The different properties of the three Cblns suggest that each plays a distinct role in synapse formation. To begin to elucidate Cbln function in other neuronal systems, we used in situ hybridization to examine Cbln expression in the mouse spinal cord. We find that neurons expressing Cblns 1, 2, and 4 tend to occupy different laminar positions within the dorsal spinal cord, and that Cbln expression is limited almost exclusively to excitatory neurons. Combined in situ hybridization and immunofluorescent staining shows that Cblns 1, 2, and 4 are expressed by largely distinct neuronal subpopulations, defined in part by sensory input, although there is some overlap and some individual neurons coexpress two Cblns. Our results suggest that differences in connectivity between subpopulations of dorsal spinal cord neurons may be influenced by which Cbln each subpopulation contains. Competitive interactions between axon terminals may determine the number of synapses each forms in any given region, and thereby contribute to the development of precise patterns of connectivity in the dorsal gray matter. J. Comp. Neurol. 522:479–497, 2014.