Rosemary C. Spike

Neurokinin-1 receptors on lumbar spinothalamic neurons in the rat

In order to determine whether spinothalamic neurons in the lumbar spinal cord of the rat process neurokinin-1 (substance P) receptors, we injected cholera toxin B subunit into the thalamus and carried out dual-labelling immunocytochemistry to search for neurons that were immunoreactive with antibodies to cholera toxin and neurokinin-1 receptor. We examined 356 spinothalamic neurons in transverse sections and found that 35% of these were neurokinin-1 receptor-immunoreactive. Double-labelled cells made up the majority of the spinothalamic population in lamina I and the lateral spinal nucleus, and were also present in laminae III-V and the area around the central canal. On the side contralateral to the injection site, 77% of spinothalamic neurons in lamina I also showed neurokinin-1 receptor immunoreactivity, while 33% of those in laminae III-V and 14% of the ventromedial group possessed the receptor. Several of the double-labelled neurons with cell bodies in laminae III and IV had dendrites which could be followed dorsally into the superficial dorsal horn. These results indicate that substance P released from nociceptive primary afferents into the superficial dorsal horn is likely to act on spinothalamic tract neurons in lamina I, and also on those with cells bodies in laminae III-IV and long dorsal dendrites.

The types of neuron in spinal dorsal horn which possess neurokinin-1 receptors

In order to provide further information about the types of spinal neuron which possess neurokinin-1 receptors, we have carried out pre-embedding immunocytochemistry on sections of rat lumbar spinal cord with an antiserum raised against a synthetic peptide corresponding to part of the sequence of the receptor, and combined this with post-embedding immunocytochemistry to detect GABA and glycine. Numerous neuronal cell bodies showing neurokinin-1 receptor-immunoreactivity were seen in lamina I, laminae III-VI, the lateral spinal nucleus and the area around the central canal. Most of the cells observed in lamina III were small and had relatively restricted dendritic trees which could often not be followed into lamina II, however some larger cells in laminae III and IV had dendrites which extended through lamina II and into lamina I. Cells of the latter type are likely to represent a major target of substance P released from small-diameter primary afferents in the superficial dorsal horn. The great majority (255 out of 283) of spinal neurons which possessed neurokinin-1 receptor-immunoreactivity, including all of those in lamina I, were not GABA- or glycine-immunoreactive, however a few cells in the deep part of the dorsal horn and the lateral spinal nucleus and several cells near the central canal were GABA-immunoreactive, and some of these were also glycine-immunoreactive. These results suggest that substance P acts through neurokinin-1 receptors mainly on excitatory neurons within the spinal cord.

A quantitative and morphological study of projection neurons in lamina I of the rat lumbar spinal cord

In the rat lumbar spinal cord the major supraspinal targets for lamina I projection neurons are the caudal ventrolateral medulla (CVLM), lateral parabrachial area (LPb) and periaqueductal grey matter (PAG). In this study we have estimated the number of lamina I neurons retrogradely labelled from each of these sites in the L4 segment, as well as the proportion that can be labelled by injecting different tracers into two separate sites. Our results suggest that this segment contains approximately 400 lamina I projection neurons on each side, and that approximately 85% of these can be labelled from either the CVLM or the LPb on the contralateral side. Around 120 lamina I cells in L4 project to the PAG, and over 90% of these cells can also be labelled from the CVLM or LPb. Most lamina I neurons projecting to CVLM or LPb are located in the contralateral dorsal horn, but in each case some cells were found to have bilateral projections. We also examined horizontal sections to investigate morphology and the expression of the neurokinin 1 (NK1) receptor in cells labelled from CVLM, LPb or PAG. There were no consistent morphological differences between these groups, however, while cells with strong or moderate NK1 receptor‐immunostaining were labelled from LPb or CVLM, they seldom projected to the PAG. These results suggest that many lamina I cells project to more than one site in the brain and that those projecting to PAG may represent a distinct subclass of lamina I projection neuron.

A quantitative study of neurons which express neurokinin-1 or somatostatin sst2a receptor in rat spinal dorsal horn.

The neurokinin-1 and somatostatin sst2a receptors have both been identified on spinal cord neurons. In this study we have estimated the proportions of neurons in different parts of the spinal cord which express these receptors, by using a monoclonal antibody against a neuronal nuclear protein named NeuN and combining the optical disector method with confocal microscopy. The NeuN antibody was initially tested on over 3200 neurons identified with antisera against a variety of compounds, including neuropeptides, enzymes and receptors, and also on astrocytes and oligodendrocytes. All of the neurons, but none of the glial cells that were examined possessed NeuN-immunoreactivity, which suggests that NeuN is a reliable marker for all spinal cord neurons. We found that approximately 45% of neurons in lamina I, 23-29% of those in laminae IV-VI and 18% in lamina X possessed the neurokinin-1 receptor, while the receptor was present on a smaller proportion of neurons in laminae II and III (6% and 11%, respectively). Thirteen percent of lamina I neurons and 15% of those in lamina II expressed the sst2a receptor. To provide further information about the types of neuron which possess the sst2a receptor, we searched for possible co-existence with the neurokinin-1 receptor as well as with GABA and glycine. sst2a and neurokinin-1 receptors were not co-localized on neurons in laminae I and II. All of the sst2a-immunoreactive neurons examined were also GABA-immunoreactive, and 83.5% were glycine-immunoreactive, indicating that the receptor is located on inhibitory neurons in the superficial dorsal horn. These results demonstrate the proportions of neurons in each region of the spinal cord which can be directly activated by substance P or somatostatin acting through these receptors. Levels of receptors can change in pathological states, and this method could be used to determine whether or not these changes involve alterations in the number of neurons which express receptors. In addition, the method can be used to estimate the sizes of neurochemically-defined populations of spinal cord neurons.

An immunohistochemical study of androgen, oestrogen and progesterone receptors in the vulva and vagina.

Objective Tomap potential sites of sex steroid action in the human vulva.

The Relationship Between Glycine and Gephyrin in Synapses of the Rat Spinal Cord

In order to examine the relationship between gephyrin (the peripheral membrane protein associated with glycine receptors) and glycinergic boutons, we have carried out a post‐embedding immunogold study of glycine‐like immunoreactivity on sections of rat lumbar spinal cord which had previously been reacted with monoclonal antibody to gephyrin. In all three areas examined (laminae I and II, lamina III and lamina IX) the majority of profiles which were presynaptic at gephyrin‐immunoreactive synapses were enriched with glycine‐like immunoreactivity. It was estimated that at least 83% of profiles presynaptic to gephyrin‐immunoreactive synapses in the superficial dorsal horn (laminae I and II) were glycine‐immunoreactive, while for lamina III and the ventral horn (lamina IX) the proportions were at least 91% and 98% respectively. This provides strong evidence that glycine is a transmitter at those synapses where gephyrin‐ and glycine‐like immunoreactivities are both present, but suggests that gephyrin may sometimes be expressed at non‐glycinergic synapses and indicates the need for caution in using gephyrin‐immunoreactivity as a marker for glycinergic synapses within the spinal cord. By reacting serial sections of dorsal horn with antisera to glycine and GABA, we have shown that many boutons in laminae I‐III of the dorsal horn show both types of immunoreactivity and are therefore likely to use both amino acids as inhibitory transmitters. Many of the boutons which were presynaptic at axoaxonic synapses in the ventral part of lamina II and in lamina III were glycine‐ and GABA‐immunoreactive and in many cases the postsynaptic element was the central axon of a type II synaptic glomerulus. Taken together with pharmacological evidence, this suggests that inhibitory intemeurons in the dorsal horn which use both GABA and glycine may be important in controlling the flow of information from hair follicle afferents to other spinal neurons.

Evidence that neuropeptide Y is present in GABAergic neurons in the superficial dorsal horn of the rat spinal cord

In order to determine whether or not neuropeptide Y coexists with GABA or glycine in rat dorsal horn, we have examined 84 neuropeptide Y-immunoreactive neurons in laminae I-III with a combined pre- and postembedding immunocytochemical method. All of the neuropeptide Y-immuno-reactive neurons were also GABA-immunoreactive, but they were either non-immunoreactive or weakly immunoreactive with the glycine antiserum. In addition, a double-label immunofluorescence method was used to search for co-localization of neuropeptide Y and [Met]enkephalin in spinal cord. Although the two types of peptide immunoreactivity often coexisted in varicosities around the central canal and in the ventral horn, such coexistence was not seen in the superficial dorsal horn. These results suggest that neuropeptide Y is present in GABAergic neurons in laminae I-III of rat dorsal horn, but that it is largely or completely restricted to those neurons which do not contain glycine. In addition, the cells that contain GABA and neuropeptide Y appear to form a different population from those that contain GABA and [Met]enkephalin. Neuropeptide Y administered by intrathecal injection causes analgesia, and there is evidence that this may involve a presynaptic mechanism. The results of the present study suggest that neuropeptide Y may act in conjunction with GABA to produce presynaptic inhibition of nociceptive primary afferents.

Some inhibitory neurons in the spinal cord develop c-fos-immunoreactivity after noxious stimulation

In order to determine which types of spinal neuron produce c-fos in response to noxious stimulation, we have combined pre-embedding detection of c-fos-like immunoreactivity with post-embedding immunocytochemistry using antibodies against GABA and glycine, 2 h after subcutaneous injection of formalin into a hindpaw of anaesthetized rats. Throughout the spinal cord, the majority of c-fos-immunoreactive neurons (72-81%) did not possess GABA- or glycine-like immunoreactivity, while the remaining cells contained one or both types of immunoreactivity. In the superficial dorsal horn (laminae I and II) and dorsal white matter, between 14 and 20% of c-fos-immunoreactive neurons were GABA-immunoreactive, and some of these were also glycine-immunoreactive. A single neuron in lamina I in one animal was glycine- but not GABA-immunoreactive. In the remainder of the spinal cord, between 21 and 35% of the c-fos-immunoreactive cells were GABA- or glycine-immunoreactive, and the majority of these neurons contained both types of immunoreactivity. These results suggest that some inhibitory neurons in both the superficial and deep parts of the dorsal horn are activated by noxious stimuli. It is known that some of the cells which produce c-fos in response to noxious stimulation are projection neurons, with axons ascending to the brainstem or thalamus, however, because of the large number of c-fos-immunoreactive cells in the dorsal horn, it is likely that many are interneurons, and some of these are probably excitatory cells which use glutamate as a transmitter. It therefore appears that after noxious stimulation c-fos is produced in several types of spinal neuron, including projection cells and both excitatory and inhibitory interneurons.

Immunohistochemical evidence that Met-enkephalin and GABA coexist in some neurones in rat dorsal horn

A pre-embedding immunohistochemical method to detect Met-enkephalin was combined with postembedding immunohistochemistry with GABA and glycine antisera, in order to determine whether or not Met-enkephalin coexisted with either of these inhibitory transmitters in neuronal cell bodies within the superficial dorsal horn of the rat. The distribution of immunostaining with the three antisera was similar to that which has been described previously. Of 74 enkephalin-immunoreactive neurones in laminae II and III, 51 were immunoreactive with the GABA antiserum and 23 were not. All of the neurones which were not GABA-immunoreactive were located in lamina II. None of the enkephalin-immunoreactive cells showed glycine-like immunoreactivity. These results suggest that enkephalin is present both in GABAergic neurones and in neurones which do not contain GABA within the rat superficial dorsal horn. It is likely that the population of neurones immunoreactive with both enkephalin and GABA antisera includes lamina II islet cells and that the population which were enkephalin-immunoreactive but not GABA-immunoreactive includes stalked cells. In addition, this latter group may correspond to those cells which possess both enkephalin- and substance P-like immunoreactivity and which have been described previously in this area.

An ultrastructural study of the glycine transporter GLYT2 and its association with glycine in the superficial laminae of the rat spinal dorsal horn

The glycine transporter GLYT2 is present in axonal boutons throughout the spinal cord, and its laminar distribution matches that of glycine-enriched axons, which are presumed to be glycinergic. In order to determine whether boutons which possess GLYT2 are glycine-enriched, we have carried out pre-embedding immunocytochemistry with antibody raised against GLYT2, and combined this with post-embedding detection of glycine, in the rat. GLYT2 immunoreactivity was present in boutons which formed symmetrical axodendritic, axosomatic or axoaxonic synapses, and was often seen in peripheral axons of type II synaptic glomeruli. One hundred and fifty GLYT2-immunoreactive boutons were analysed quantitatively, and in 142 (94.6%) of these the density of gold particles representing glycine-like immunoreactivity exceeded the background level (over presumed glutamatergic boutons) by at least a factor of two. Within immunoreactive boutons, the GLYT2 reaction product was associated with the plasma membrane, but often appeared as discrete clumps and was generally excluded from the region of the active sites of synapses. These results confirm that GLYT2 is associated with glycine-enriched axonal boutons in the superficial dorsal horn. They also suggest that GLYT2 is unevenly distributed on the plasma membrane of these boutons, and raise the possibility that it may be excluded from synaptic clefts.