Targeting Neurotrophins to Specific Populations of Neurons: NGF, BDNF, and NT-3 and Their Relevance for Treatment of Spinal Cord Injury.

Neurotrophins are a family of proteins that regulate neuronal survival, synaptic function, and neurotransmitter release, and elicit the plasticity and growth of axons within the adult central and peripheral nervous system. Since the 1950s, these factors have been extensively studied in traumatic injury models. Here we review several members of the classical family of neurotrophins, the receptors they bind to, and their contribution to axonal regeneration and sprouting of sensory and motor pathways after spinal cord injury (SCI). We focus on nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3), and their effects on populations of neurons within diverse spinal tracts. Understanding the cellular targets of neurotrophins and the responsiveness of specific neuronal populations will allow for the most efficient treatment strategies in the injured spinal cord.

Novel C-1 substituted cocaine analogs unlike cocaine or benztropine.

Despite a wealth of information on cocaine-like compounds, there is no information on cocaine analogs with substitutions at C-1. Here, we report on (R)-(-)-cocaine analogs with various C-1 substituents: methyl (2), ethyl (3), n-propyl (4), n-pentyl (5), and phenyl (6). Analog 2 was equipotent to cocaine as an inhibitor of the dopamine transporter (DAT), whereas 3 and 6 were 3- and 10-fold more potent, respectively. None of the analogs, however, stimulated mouse locomotor activity, in contrast to cocaine. Pharmacokinetic assays showed compound 2 occupied mouse brain rapidly, as cocaine itself; moreover, 2 and 6 were behaviorally active in mice in the forced-swim test model of depression and the conditioned place preference test. Analog 2 was a weaker inhibitor of voltage-dependent Na+ channels than cocaine, although 6 was more potent than cocaine, highlighting the need to assay future C-1 analogs for this activity. Receptorome screening indicated few significant binding targets other than the monoamine transporters. Benztropine-like "atypical" DAT inhibitors are known to display reduced cocaine-like locomotor stimulation, presumably by their propensity to interact with an inward-facing transporter conformation. However, 2 and 6, like cocaine, but unlike benztropine, exhibited preferential interaction with an outward-facing conformation upon docking in our DAT homology model. In summary, C-1 cocaine analogs are not cocaine-like in that they are not stimulatory in vivo. However, they are not benztropine-like in binding mechanism and seem to interact with the DAT similarly to cocaine. The present data warrant further consideration of these novel cocaine analogs for antidepressant or cocaine substitution potential.

Viral expression of constitutively active AKT3 induces CST axonal sprouting and regeneration, but also promotes seizures.

Increasing the intrinsic growth potential of neurons after injury has repeatedly been shown to promote some level of axonal regeneration in rodent models. One of the most studied pathways involves the activation of the PI3K/AKT/mTOR pathways, primarily by reducing the levels of PTEN, a negative regulator of PI3K. Likewise, activation of signal transducer and activator of transcription 3 (STAT3) has previously been shown to boost axonal regeneration and sprouting within the injured nervous system. Here, we examined the regeneration of the corticospinal tract (CST) after cortical expression of constitutively active (ca) Akt3 and STAT3, both separately and in combination. Overexpression of caAkt3 induced regeneration of CST axons past the injury site independent of caSTAT3 overexpression. STAT3 demonstrated improved axon sprouting compared to controls and contributed to a synergistic improvement in effects when combined with Akt3 but failed to promote axonal regeneration as an individual therapy. Despite showing impressive axonal regeneration, animals expressing Akt3 failed to show any functional improvement and deteriorated with time. During this period, we observed progressive Akt3 dose-dependent increase in behavioral seizures. Histology revealed increased phosphorylation of ribosomal S6 protein within the unilateral cortex, increased neuronal size, microglia activation and hemispheric enlargement (hemimegalencephaly).

Direct Injection of a Lentiviral Vector Highlights Multiple Motor Pathways in the Rat Spinal Cord.

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. Injection directly into spinal cord tissue bypasses these barriers, providing access to cell bodies or synapses where molecules can be incorporated. Combining viral vector technology with this method allows for introduction of target genes into nervous tissue for the purpose of gene therapy or tract tracing. Here a virus engineered for highly efficient retrograde transport (HiRet) is introduced at the synapses of propriospinal interneurons (PNs) to encourage specific transport to neurons in the spinal cord and brainstem nuclei. Targeting PNs takes advantage of the numerous connections they receive from motor pathways such as the rubrospinal and reticulospinal tracts, as well as their interconnection with each other throughout spinal cord segments. Representative tracing using the HiRet vector with constitutively active green fluorescent protein (GFP) shows high fidelity details of cell bodies, axons and dendritic arbors in thoracic PNs and in reticulospinal neurons in the pontine reticular formation. HiRet incorporates well into brainstem pathways and PNs but shows age dependent integration into corticospinal tract neurons. In summary, spinal cord injection using viral vectors is a suitable method for introduction of proteins of interest into neurons of targeted tracts.

Withdrawal from chronic administration of cocaine decreases delta opioid receptor signaling and increases anxiety- and depression-like behaviors in the rat.

Chronic administration of cocaine has been shown to attenuate the functional capacity of delta opioid receptors to inhibit adenylyl cyclase activity. Abuse and withdrawal from cocaine in humans is associated with increases in anxiety and depression. Since recent research supports the role of delta opioid receptors in anxiety- and depression-like behaviors in rodents, we hypothesized that functional desensitization of delta opioid receptors contributes to anxiety- and depression-like behavioral phenotypes following short-term withdrawal from chronic administration of cocaine. To test this hypothesis, delta opioid receptor signaling and behaviors were evaluated 24h after 14days of binge-pattern cocaine administration (15mg/kg three times daily at 1h intervals) in male Sprague-Dawley rats. Results showed that the inhibition of adenylyl cyclase by delta opioid receptor agonists was attenuated in the frontal cortex, nucleus accumbens and caudate putamen 24h after cessation of cocaine administration. One day withdrawal from chronic administration of cocaine resulted in increased anxiety- and depression-like behaviors as measured by the elevated plus maze and the forced swim test respectively, and no change in locomotor activity. The anxiety- and depression-like behaviors were dose-dependently reduced by acute administration of the selective delta opioid receptor agonist, SNC80. These results demonstrate that early withdrawal from cocaine resulted in increased anxiety and depression, which accompanies the desensitization of delta opioid receptor function. Furthermore, cocaine-induced anxiety- and depression-like behaviors were reversible by the delta opioid receptor agonist SNC80.

Retrogradely Transportable Lentivirus Tracers for Mapping Spinal Cord Locomotor Circuits.

Retrograde tracing is a key facet of neuroanatomical studies involving long distance projection neurons. Previous groups have utilized a variety of tools ranging from classical chemical tracers to newer methods employing viruses for gene delivery. Here, we highlight the usage of a lentivirus that permits highly efficient retrograde transport (HiRet) from synaptic terminals within the cervical and lumbar enlargements of the spinal cord. By injecting HiRet, we can clearly identify supraspinal and propriospinal circuits innervating motor neuron pools relating to forelimb and hindlimb function. We observed robust labeling of propriospinal neurons, including high fidelity details of dendritic arbors and axon terminals seldom seen with chemical tracers. In addition, we examine changes in interneuronal circuits occurring after a thoracic contusion, highlighting populations that potentially contribute to spontaneous behavioral recovery in this lesion model. Our study demonstrates that the HiRet lentivirus is a unique tool for examining neuronal circuitry within the brain and spinal cord.

Ketamine reverses stress-induced depression-like behavior and increased GABA levels in the anterior cingulate: an 11.7 T 1H-MRS study in rats.

Gamma-aminobutyric acid (GABA) is the major inhibitory amino acid neurotransmitter in the brain and is primarily responsible for modulating excitatory tone. Clinical neuroimaging studies show decreased GABA levels in the anterior cingulate of patients with mood disorders, including major depressive disorder. Chronic unpredictable stress (CUS) is an animal model thought to mimic the stressful events that may precipitate clinical depression in humans. In this study male Sprague-Dawley rats were subjected to a modified CUS paradigm that used a random pattern of unpredictable stressors twice daily for 10 days to explore the early developmental stages of depression-like endophenotypes. Control rats were handled daily for 10 days. Some rats from each treatment group received an injection of ketamine (40 mg/kg) after the final stressor. One day following the final stressor rats were tested for behavioral effects in the forced swim test and then euthanized to collect trunk blood and anterior cingulate brain samples. GABA levels were measured in anterior cingulate samples ex vivo using proton magnetic resonance spectroscopy ((1)H-MRS) at 11.7 T. Animals subjected to CUS had lower body weights, higher levels of blood corticosterone, and increased immobility in the forced swim test; all of which suggest that the stress paradigm induced a depression-like phenotype. GABA levels in the anterior cingulate were significantly increased in the stressed animals compared to controls. Administration of ketamine on the last day of treatment blunted the depression-like behavior and increased GABA levels in the anterior cingulate following CUS. These data indicate that stress disrupts GABAergic signaling, which may over time lead to symptoms of depression and ultimately lower basal levels of cortical (1)H-MRS GABA that are seen in humans with depression. Furthermore, the data suggests that ketamine modulates cortical GABA levels as a mechanism of its antidepressant activity.