Richard E. Coggeshall

Methods for determining numbers of cells and synapses: a case for more uniform standards of review.

Neuron and synapse numbers are important assays in neuroscience. These numbers are estimated by one of four methods: 1) profile counts, 2) assumption‐based methods, 3) serial reconstructions, and 4) stereological methods. The criteria for these methods are diverse. This creates a disparity in that some reviewers accept estimates from any of these methods, while others accept only specific methods. An equally important issue is the diversity of sampling strategies, since unbiased estimates of neuronal or synaptic numbers are contingent upon both counting and sampling techniques. The purpose of this commentary is to institute a dialog that will lead to a better understanding of the strengths and weaknesses of the above methods, and to propose guidelines that should lead to more uniform and thus fairer judging of the studies that provide estimates of neuron or synapse numbers. In addition, adoption of more uniform standards for obtaining unbiased numerical estimates should result in the generation of an unbiased database that will be of considerable use in future studies.

A consideration of neural counting methods

It is often necessary to obtain unbiased estimates of neuronal or synaptic numbers. In the past, estimates were almost always done by counting profiles of these structures in single histological sections. Assumptions were then made and calculations were done to determine particle numbers or ratios. To the extent that the assumptions deviated from reality, the conclusions will be biased. That these biases are, in fact, serious has recently become apparent. To obtain unbiased particle counts, the presently available methods are serial-section reconstructions (which are accurate but cumbersome), and the recently developed disector method. The disector method, because it is unbiased and easy to use, is becoming the method of choice. The goals of this paper are to show why previous methods are biased and to describe the rationale behind the disector method so that neuroscientists can consider its appropriateness for their work.

Localization and activation of glutamate receptors in unmyelinated axons of rat glabrous skin

Immunohistochemical staining for the glutamate receptor subtypes N-methyl-D-aspartate (NMDA), kainate, and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) results in a significant number of labeled unmyelinated axons in the glabrous skin of the rat hindpaw. Injection of glutamate into the rat hindpaw results in behavioral changes interpreted as mechanical allodynia and mechanical hyperalgesia. The anatomical findings provide a reasonable explanation for the action of the exogenous peripheral glutamate, namely that activation of these receptors leads to increased primary afferent activity in unmyelinated axons and thus to pain behaviors. AMPA receptors are frequently associated with small clear vesicles in the axoplasm of the unmyelinated axons, many of which have been previously shown to contain high concentrations of glutamate. This finding indicates that these might be autoreceptors and so glutamate itself might regulate certain types of peripheral impulse traffic. The presence of peripheral glutamate receptors associated with unmyelinated axons suggests the possibility that glutamate antagonists applied peripherally might prevent or attenuate some pain-related behaviors.

Reactive oxygen species (ROS) play an important role in a rat model of neuropathic pain

&NA; Reactive oxygen species (ROS) are free radicals produced in biological systems that are involved in various degenerative brain diseases. The present study tests the hypothesis that ROS also play an important role in neuropathic pain. In the rat spinal nerve ligation (SNL) model of neuropathic pain, mechanical allodynia develops fully 3 days after nerve ligation and persists for many weeks. Systemic injection of a ROS scavenger, phenyl‐N‐tert‐butylnitrone (PBN), relieves SNL‐induced mechanical allodynia in a dose‐dependent manner. Repeated injections cause no development of tolerance or no loss of potency. Preemptive treatment with PBN is also effective in preventing full development of neuropathic pain behavior. Systemic injection was mimicked by intrathecal injection with a little less efficacy, while intracerebroventricular administration produced a much smaller effect. These data suggest that PBN exerts its anti‐allodynic action mainly by spinal mechanisms. Systemic treatment with other spin‐trap reagents, 5,5‐dimethylpyrroline‐N‐oxide and nitrosobenzene, showed similar analgesic effects, suggesting that ROS are critically involved in the development and maintenance of neuropathic pain. Thus this study suggests that systemic administration of non‐toxic doses of free radical scavengers could be useful for treatment of neuropathic pain.

Blocking Caspase Activity Prevents Transsynaptic Neuronal Apoptosis and the Loss of Inhibition in Lamina II of the Dorsal Horn after Peripheral Nerve Injury

We show that transsynaptic apoptosis is induced in the superficial dorsal horn (laminas I-III) of the spinal cord by three distinct partial peripheral nerve lesions: spared nerve injury, chronic constriction, and spinal nerve ligation. Ongoing activity in primary afferents of the injured nerve and glutamatergic transmission cause a caspase-dependent degeneration of dorsal horn neurons that is slow in onset and persists for several weeks. Four weeks after spared nerve injury, the cumulative loss of dorsal horn neurons, determined by stereological analysis, is >20%. GABAergic inhibitory interneurons are among the neurons lost, and a marked decrease in inhibitory postsynaptic currents of lamina II neurons coincides with the induction of apoptosis. Blocking apoptosis with the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD) prevents the loss of GABAergic interneurons and the reduction of inhibitory currents. Partial peripheral nerve injury results in pain-like behavioral changes characterized by hypersensitivity to tactile or cold stimuli. Treatment with zVAD, which has no intrinsic analgesic properties, attenuates this neuropathic pain-like syndrome. Preventing nerve injury-induced apoptosis of dorsal horn neurons by blocking caspase activity maintains inhibitory transmission in lamina II and reduces pain hypersensitivity.

Receptor localization in the mammalian dorsal horn and primary afferent neurons

The dorsal horn of the spinal cord is a primary receiving area for somatosensory input and contains high concentrations of a large variety of receptors. These receptors tend to congregate in lamina II, which is a major receiving center for fine, presumably nociceptive, somatosensory input. There are rapid reorganizations of many of these receptors in response to various stimuli or pathological situations. These receptor localizations in the normal and their changes after various pertubations modify present concepts about the wiring diagram of the nervous system. Accordingly, the present work reviews the receptor localizations and relates them to classic organizational patterns in the mammalian dorsal horn.

Ultrastructural analysis of NMDA, AMPA, and kainate receptors on unmyelinated and myelinated axons in the periphery

The present study determines the proportions of unmyelinated cutaneous axons at the dermal–epidermal junction in glabrous skin and of myelinated and unmyelinated axons in the sural and medial plantar nerves that immunostain for subunits of the ionotropic glutamate receptors. Approximately 20% of the unmyelinated cutaneous axon profiles at the dermal–epidermal junction immunostain for either N‐methyl‐D‐aspartate (NMDA), α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA), or kainate receptor subunits. These findings are consistent with previous observations that NMDA and non‐NMDA antagonists ameliorate nociceptive behaviors that result from noxious peripheral stimulation. In the sural nerve, where the large majority of myelinated fibers are sensory, approximately half of the myelinated axon profiles immunostain for the NMDA receptor 1 (R1) subunit, 28% immunostain for the glutamate receptor 1 (GluR1) AMPA subunit, and 11% for the GluR5,6,7 kainate subunits. Even higher proportions immunostain for these receptors in the medial plantar nerve, a mixed sensory and motor nerve. In the sural nerve, 20% of the unmyelinated axon profiles immunostain for NMDAR1 and only 7% label for GluR1 or GluR5,6,7. Because the sural nerve innervates hairy skin, these data suggest that glutamate will activate a higher proportion of unmyelinated axons in glabrous skin than in hairy skin. Measurements of fiber diameters indicate that all sizes of myelinated axon profiles, including Aδ and Aβ, are positively labeled for the ionotropic receptors. The presence of glutamate receptors on large‐diameter myelinated axons suggests that these mechanosensitive receptors, presumably transducing touch and pressure, may also respond to local glutamate and thus be chemosensitive. J. Comp. Neurol. 391: 78–86, 1998.

Region-specific generation of cholinergic neurons from fetal human neural stem cells grafted in adult rat

Pluripotent or multipotent stem cells isolated from human embryos or adult central nervous system (CNS) may provide new neurons to ameliorate neural disorders. A major obstacle, however, is that the majority of such cells do not differentiate into neurons when grafted into non-neurogenic areas of the adult CNS. Here we report a new in vitro priming procedure that generates a nearly pure population of neurons from fetal human neural stem cells (hNSCs) transplanted into adult rat CNS. Furthermore, the grafted cells differentiated by acquiring a cholinergic phenotype in a region-specific manner. This technology may advance stem cell–based therapy to replace lost neurons in neural injury or neurodegenerative disorders.

Peripheral NMDA and non-NMDA glutamate receptors contribute to nociceptive behaviors in the rat formalin test

THE present study demonstrates that local cutaneous administration of either the N-methyl-D-aspartate (NMDA) glutamate receptor antagonist MK-801 or the non-NMDA glutamate receptor antagonist 6-cyano7-nitroquinoxaline-2,3-dione (CNQX) significantly attenuates formalin-induced nociceptive behaviors. Specifically, pretreatment with either drug reduced the magnitude and time course of lifting and licking behavior in the late phase of formalin pain; however, flinching behavior was not affected. In contrast, posttreatment of formalin pain with either antagonist did not affect lifting and licking behavior, although flinching behavior was mildly attenuated. We hypothesize that these actions result from blocking of peripheral glutamate receptors located on unmyelinated axons at the dermal–epidermal junction. These data suggest that peripheral glutamate receptors on cutaneous axons can be manipulated to reduce certain aspects of pain of peripheral origin. This route of administration offers the advantage of avoiding the side effects of systemic administration.

Opioid receptors on peripheral sensory axons

Opioid receptors have been demonstrated by light microscopic techniques in fine cutaneous nerves in naive animals. The present study extends these findings by showing that 29 and 38% of unmyelinated cutaneous sensory axons can be immunostained for mu- or delta-opioid receptors respectively. Local cutaneous injection of DAMGO, a mu-opioid ligand, ameliorates the nociceptive behaviors caused by local cutaneous injection of glutamate, a purely nociceptive chemical stimulus showing that the mu-receptors are functional. By contrast the delta-opioid ligand [2-D-penicillamine, 5-D-penicillamine]enkephalin (DPDPE) had no effect on these behaviors. These findings indicate a wider function for opioid receptors in naive animals than previously envisioned.