Novel Striatal GABAergic Interneuron Populations Labeled in the 5HT3a(EGFP) Mouse.

Histological and morphological studies indicate that approximately 5% of striatal neurons are cholinergic or γ-aminobutyric acidergic (GABAergic) interneurons (gINs). However, the number of striatal neurons expressing known interneuron markers is too small to account for the entire interneuron population. We therefore studied the serotonin (5HT) receptor 3a-enhanced green fluorescent protein (5HT3a(EGFP)) mouse, in which we found that a large number of striatal gINs are labeled. Roughly 20% of 5HT3a(EGFP)-positive cells co-express parvalbumin and exhibit fast-spiking (FS) electrophysiological properties. However, the majority of labeled neurons do not overlap with known molecular interneuron markers. Intrinsic electrical properties reveal at least 2 distinct novel subtypes: a late-spiking (LS) neuropeptide-Y (NPY)-negative neurogliaform (NGF) interneuron, and a large heterogeneous population with several features resembling low-threshold-spiking (LTS) interneurons that do not express somatostatin, NPY, or neuronal nitric oxide synthase. Although the 5HT3a(EGFP) NGF and LTS-like interneurons have electrophysiological properties similar to previously described populations, they are pharmacologically distinct. In direct contrast to previously described NPY(+) LTS and NGF cells, LTS-like 5HT3a(EGFP) cells show robust responses to nicotine administration, while the 5HT3a(EGFP) NGF cell type shows little or no response. By constructing a molecular map of the overlap between these novel populations and existing interneuron populations, we are able to reconcile the morphological and molecular estimates of striatal interneuron numbers.

Matrix metalloprotease-9 release from monocytes increases as a function of differentiation: implications for neuroinflammation and neurodegeneration.

Naïve monocytes extravasate in response to monocyte chemoattractant-1 (MCP-1) and subsequently, following differentiation within tissue, carry out effector functions. Consistent with this concept, expression of the MCP-1 receptor CCR2 decreases with monocyte differentiation, as production of cytokines increases (Fantuzzi et al., 1999). Because matrix metalloproteases (MMPs) may also play an important role in the ability of monocytes to migrate into tissues and/or to promote pathogen clearance/tissue injury, we have examined production of matrix metalloprotease-9 as a function of both monocyte differentiation in vitro and expression of CCR2. Increased time in culture, which is linked to monocyte differentiation, resulted in enhanced production of MMP-9, assessed by gelatin substrate zymography. Further, CCR2-negative monocytes produced greater quantities of MMP-9 than did naïve CCR2-positive cells. Our results indicate that MMP-9 release increases during monocyte differentiation, consistent with a prominent role in effector functions. Because extracellular matrix proteins are important to cell structure and survival (Wee Yong et al., 1998), increased expression of MMP-9 could contribute to tissue damage following monocyte differentiation.

Cytotoxicity by matrix metalloprotease-1 in organotypic spinal cord and dissociated neuronal cultures.

Extracellular matrix (ECM) proteins, including collagens and laminins, are critical to the structure of the neuronal synapse and may also be involved in cell survival. In the present study, we therefore examined the possibility that select ECM degrading proteins might be toxic to organotypic spinal cord and dissociated neuronal cultures. Of those proteins tested, including MMP-1, -7, and -9, we observed that MMP-1 was toxic to spinal cord cultures as determined by release of lactic acid dehydrogenase as well as uptake of propidium iodide. Pretreatment of cell cultures with 50 microM alpha-tocopherol partially reversed these effects. We also observed that MMP-1 was toxic to human neurons grown in dissociated cultures and that increased amounts of MMP-1 were released by astrocytes following their stimulation with IL-1beta. These results suggest that further studies may be warranted to determine whether MMP-1 contributes to neurodegenerative conditions in which activated astrocytes may play a role.

Cerebrospinal fluid levels of MMP-2, 7, and 9 are elevated in association with human immunodeficiency virus dementia.

Pathological evidence suggests that alterations of the blood-brain barrier (BBB) may occur in association with human immunodeficiency virus (HIV) dementia (HIVD). Increased BBB permeability could contribute to the development of dementia by facilitating the entry of activated and infected monocytes, as well as potentially toxic serum proteins, into the central nervous system. One mechanism by which BBB permeability may be altered is through increased activity of select matrix metalloproteinases (MMPs). In the present study, we examined the possibility that MMPs that target critical BBB proteins, including laminin, entactin, and collagen type IV, are elevated in the cerebrospinal fluid (CSF) of patients with HIVD. We also examined the possibility that such MMPs could be produced by brain-derived cells, and that MMP production by these cells might be increased by tumor necrosis factor-alpha, an inflammatory cytokine that is produced by HIV-infected monocytes/microglia and is elevated in HIVD. By using western blot and enzyme-linked immunosorbent assay, we observed that CSF levels of pro-MMP-2 and pro-MMP-7 were increased in association with HIVD. In addition, through the use of gelatin substrate zymography, a sensitive functional assay for MMP-2 and MMP-9, we observed that MMP-2 or pro-MMP-9 activity was more frequently detectable in the CSF of individuals with HIV dementia (9/16) than in the CSF from either nondemented seropositive (2/11) or seronegative (0/11) controls. Although the presence of MMPs in the serum could contribute to elevated levels in the CSF, we also show that brain-derived cells release MMP-2, 7, and 9, and that such release is increased after their stimulation with tumor necrosis factor-alpha. Together, these results suggest that elevated CSF levels of select MMPs may reflect immune activation within the central nervous system. They also suggest that further studies may be warranted to determine whether these proteins may play a role in the development of symptomatic neurological disease.