R. Maciewicz

Behavioral evidence of trigeminal neuropathic pain following chronic constriction injury to the rat's infraorbital nerve

Video recordings of free behavior and responses to mechanical facial stimulation were analyzed to assess whether chronic constriction injury (CCI) to the rats infraorbital nerve (IoN) results in behavioral alterations indicative of neuropathic pain. A unilateral CCI was produced by placing loose chromic gut ligatures around the IoN. After CCI to the IoN, rats exhibited changes in both non-evoked and evoked behavior. Behavioral changes developed in two phases. Early after CCI (postoperative days 1–15), rats showed increased face-grooming activity with face-wash strokes directed to the injured nerve territory, while the responsiveness to stimulation of this area was decreased. Later after CCI (postoperative days 15–130), the prevalence of asymmetric face grooming was reduced but remained significantly increased compared to control rats. The early hyporesponsiveness was abruptly replaced by an extreme hyperresponsiveness: all stimulus intensities applied to the injured nerve territory evoked the “maximal” response (brisk head withdrawal, avoidance behavior plus directed face grooming). This response was never observed in control rats. Concurrently, IoN ligation rats showed a limited increase in the responsiveness to stimulation of the contralateral IoN territory, and around postoperative days 30–40 the responsiveness to stimulation of facial areas outside the IoN territories also increased. The hyperresponsiveness to stimulation of the ligated IoN territory slightly decreased from 60 d postoperative. Throughout the study, IoN ligation rats showed decreased exploratory behavior, displayed more freezing-like behavior, had a slower body weight gain, and a higher defecation rate, compared to control rats. The behavioral alterations observed after CCI to the IoN are indicative of severe sensory disturbances within the territory of the injured nerve: mechanical allodynia develops after a period of relative hypo- /anesthesia during which behavioral signs of recurrent spontaneous, aversive (possibly painful) sensations (paresthesias/dysesthesias) are maximal.

MK-801 blocks the development of thermal hyperalgesia in a rat model of experimental painful neuropathy

Loose ligation of the sciatic nerve in the rat can produce behavioral signs of hyperalgesia in the hindpaw. This study examined the effect of an NMDA (N-methyl-D-aspartate) receptor antagonist (MK-801) on the development of hyperalgesia in this model. Rats received i.p. injections of saline or MK-801 (1.0 mg/kg) prior to and then for 7 days after a unilateral sciatic nerve ligation. Testing of each hindpaw for latency to withdrawal from a standardized thermal stimulus was performed prior to ligation and then at 10, 12, 17, 27, and 37 days postoperatively. Hyperalgesia of the operated hindpaw developed in saline-treated animals as measured by a decrease in withdrawal latency. Hyperalgesia did not develop in animals treated with MK-801. MK-801 may therefore prevent the development of hyperalgesia following experimental nerve injury, possibly through an NMDA receptor-mediated effect.

Edinger-Westphal nucleus: cholecystokinin immunocytochemistry and projections to spinal cord and trigeminal nucleus in the cat

Immunocytochemical methods were used to determine the distribution of cells with cholecystokinin-like immunoreactivity (CCK-LI) in the cat Edinger-Westphal complex (EW). Numerous cells with CCK-LI are found throughout the length of EW. The distribution and frequency of such cells are similar to the pattern of EW neurons that show substance P-like immunoreactivity (SP-LI). Companion retrograde transport experiments reveal that EW neurons which project to spinal cord or the region of the caudal trigeminal nucleus are found throughout the length of EW, and that some EW neurons which project to spinal cord also show CCK-LI.

The distribution of substance P-containing neurons in the cat Edinger-Westphal nucleus: relationship to efferent projection systems

The light microscopic localization of substance P-like immunoreactivity (SPLI) was examined in the cat Edinger--Westphal complex using the peroxidase--antiperoxidase method. A high density of cell bodies and processes staining for SPLI were found in the caudal part of the Edinger--Westphal complex (EWc) capping the somatic divisions of the oculomotor nucleus. This distribution continued rostrally into the anteromedian nucleus (AM). Cells labeled with SPLI were also found arranged in a thin layer dorsally capping the oculomotor nucleus, and scattered cells were found in the periaqueductal gray region at the same level. This distribution of SPLI-positive cells was then compared with the distribution of cells in EWc and AM that are retrogradely labeled by horseradish peroxidase or Nuclear Yellow injections into spinal cord, cerebellum, or ciliary ganglion. Injections of horseradish peroxidase into both cervical and lumbar cord labeled a large number of cells throughout the length of EWc and the more rostral AM. A similar pattern of labeling was seen following injections of Nuclear Yellow into the deep cerebellar nuclei. In contrast, cells innervating the ciliary ganglion were found predominantly outside of the Edinger--Westphal complex in AM, the rostral periaqueductal region, and the tegmentum ventral to the oculomotor complex. The distribution of cells projecting to spinal cord or cerebellum and the pattern of SPLI staining was found to closely overlap, evidence that substance P may be contained in cells that give rise to the central projections of the Edinger--Westphal complex.

The vestibulothalamic pathway: Contribution of the ascending tract of deiters

Axoplasmic transport techniques were used to determine the contribution of the ascending tract of Deiters (ATD) to the vestibulothalamic projection in cats. Large injections of HRP into the thalamus centered on the border region between the ventrobasal complex and the caudal ventrolateral nucleus resulted in bilateral retrograde labeling of cells in the vestibular nuclear complex and the nucleus prepositus hypoglossi (PH). Similar thalamic injections were also made in animals with extensive bilateral lesions of the medial longitudinal fasciculus (MLF) and the brachium conjunctivum (BC). HRP-positive neurons in these cases were localized principally to the ventral lateral vestibular nucleus and adjacent superior vestibular nucleus ipsilateral to the thalamic injection, evidence that vestibulothalamic neurons in these nuclei may project to the thalamus over the unlesioned ATD. Injections of [35S]methionine into the rostral vestibular nuclear complex in animals with MLF and BC lesions confirmed these findings, demonstrating orthograde transport of radiolabel in the ATD with termination in thalamus. These experiments document a contribution of the ATD to the ipsilateral vestibulothalamic projection; other sources of the vestibulothalamic pathway (PH, Y group) likely travel through projection systems destroyed in the lesions made in the present study.

Edinger-Westphal neurons that project to spinal cord contain substance P

Combined retrograde transport and immunocytochemical methods were used to determine whether Edinger-Westphal neurons projecting to spinal cord also demonstrate substance P-like immunoreactivity (SPLI). Large injections of horseradish peroxidase (HRP) into cervical and lumbar enlargements retrogradely labeled cells throughout the length of the Edinger-Westphal complex (EW). Nearly all HRP-labeled EW neurons also stained for SPLI, evidence that EW is the origin of a direct substance P pathway linking rostral mesencephalon with spinal cord.

The oculomotor internuclear pathway: a double retrograde labeling study

To determine whether oculomotor internuclear neurons (INOs) establish unilateral or bilateral connections with the abducens nucleus (nVI), horseradish peroxidase (HRP) and either DAPI or Nuclear Yellow (NY) were injected into nVI and the oculomotor complex (nIII) examined for retrogradely labeled cells. Such injections resulted in retrograde labeling of a large number of INOs within and just dorsal to nIII in the ventral periaqueductal gray; approximately 10% of HRP-positive cells were also labeled with a fluorescent marker. Within the conditions of the experiment, this frequency of double-labeling was largely independent of the amount of HRP or fluorescent label injected or the degree of spread of label at the injections sites. It was also independent of which fluorochrome was used as a second label. Individual double-labeled cells were scattered within and immediately dorsal to nIII within the distribution of single-labeled INOs. These findings provide strong evidence for a subset of nIII INOs that have bilateral projections to nVI; the low frequency of double-labeling, however, suggests that the population of INOs projecting to both nVI may be small.

Serotonin immunocytochemistry of physiologically characterized raphe magnus neurons

SummaryTo determine if PAG stimulation activates serotonin containing neurons in RM, intracellular techniques were used to identify raphe magnus (RM) cells that are excited by antinociceptive periaqueductal gray (PAG) stimulation in the cat. RM neurons that received a monosynaptic EPSP from PAG shock were intracellularly labeled with ethidium bromide. Intracellularly stained RM cells were large, medium and small sized neurons. To determine if these cells contain serotonin, sections containing intracellularly labeled RM neurons were processed for serotonin immunocytochemistry. None of the RM cells (N = 32) which were intracellularly stained with ethidium bromide contained immunoreactive serotonin. These results are evidence that non-serotonergic RM cells are activated by antinociceptive PAG stimulation; these cells may be important in the relay of PAG induced antinociception.

Effect of midbrain raphe and lateral mesencephalic stimulation on spontaneous and evoked activity in the lateral geniculate of the cat.

SummarySingle shock stimulation of the midbrain raphe and lateral reticular formation altered the spontaneous and evoked activity of single cells in the dorsal lateral geniculate of the cat. The effect of stimulation was to produce a facilitation of 64 units with a latency of 15 msec or greater and an inhibition of 30 units with a latency of 7 to 10 msec. These two effects were jointly confirmed by computation of post-stimulus time histograms and by conditioning-test procedures employing stimulation of optic tract and midbrain.

Choline acetyltransferase immunocytochemistry of Edinger-Westphal and ciliary ganglion afferent neurons in the cat

The distribution of cholinergic neurons in the region of the cat Edinger-Westphal nucleus (EW) was determined by immunocytochemical localization of the acetylcholine-synthesizing enzyme choline acetyltransferase (ChAT). Neurons containing ChAT-like immunoreactivity (ChAT-LI) were densely distributed within EW, the anteromedian nucleus (AM), and the oculomotor nucleus (III), and were also present in immediately adjacent regions of the periaqueductal gray and ventral tegmental region. The majority of labelled neurons in EW and AM showed a markedly lower intensity of ChAT-LI than the labelled neurons in III and adjacent regions. To determine the relationship of cells with ChAT-LI to the distribution of ciliary ganglion afferent neurons, a double labelling immunocytochemistry/retrograde transport technique was also used. These experiments showed that many of the cells located outside of III that stained intensely for ChAT-LI project to ciliary ganglion. Very few ciliary ganglion afferent neurons were found in EW or AM itself; instead, the distribution of lightly labelled ChAT-LI-positive neurons in EW and AM more closely matched the known distribution of peptide-containing cells that have descending, central projections.