Susan M. Murphy

Reduced cortical inhibition in a mouse model of familial childhood absence epilepsy

Mutations in the GABAA receptor γ2 subunit are associated with childhood absence epilepsy and febrile seizures. To understand better the molecular basis of absence epilepsy in man, we developed a mouse model harboring a γ2 subunit point mutation (R43Q) found in a large Australian family. Mice heterozygous for the mutation demonstrated behavioral arrest associated with 6-to 7-Hz spike-and-wave discharges, which are blocked by ethosuximide, a first-line treatment for absence epilepsy in man. Seizures in the mouse showed an abrupt onset at around age 20 days corresponding to the childhood nature of this disease. Reduced cell surface expression of γ2(R43Q) was seen in heterozygous mice in the absence of any change in α1 subunit surface expression, ruling out a dominant-negative effect. GABAA-mediated synaptic currents recorded from cortical pyramidal neurons revealed a small but significant reduction that was not seen in the reticular or ventrobasal thalamic nuclei. We hypothesize that a subtle reduction in cortical inhibition underlies childhood absence epilepsy seen in humans harboring the R43Q mutation.

Confocal microscopic estimation of GABAergic nerve terminals in the central nervous system.

We describe a method for estimating the average proportion of GABAergic terminal area relative to total nerve terminal area with confocal microscopy. Nerve terminal regions were identified with dual colour immunofluorescence on Vibratome sections with an antibody to synaptophysin (SYN), and GABAergic processes, including axon terminals, were identified with an antibody to glutamic acid decarboxylase (GAD). Sections were viewed in an Olympus AX70 microscope attached to a Biorad 1024 MRC scanning confocal system. Images were collected with a 100 x objective from the same tissue locations and imported into the NIH-Image program, where black and white binary images were obtained for co-localisation and quantitation. Measurements were made separately of areas of SYN/GAD (GABAergic terminals) and SYN labelling (all terminals). The relative proportion of GABAergic terminal areas in visual cortex (6.1+/-1%; mean +/- SE), CA1 hippocampus (2.6+/-0.5%) and deep cerebellar nuclei (46.6+/-3%) are consistent with what is known of the relative levels of inhibitory input to these structures. The assumptions that SYN labelling is restricted to axon terminals, and that SYN labels all axon terminals was tested by ultrastructural localisation of SYN in the three brain regions examined. Only 7.4+/-0.4% of SYN-labelled profiles could not be positively identified as synaptic vesicle-containing axon terminals, and between 93.4 and 99.2% of vesiculated axon profiles were SYN-positive. These results suggest that SYN is a very reliable marker for axon terminals, and validates the confocal analytical approach. The confocal method allows rapid sampling of many brain regions and would be suitable for examining terminals containing any neurotransmitter that can be detected immunocytochemically.

Relationships between nitric oxide synthase, vasoactive intestinal peptide and substance P immunoreactivities in neurons of the amphibian intestine

Nitric oxide synthase (NOS)-containing neurons (localized using NADPH diaphorase histochemistry or NOS immunoreactivity) and vasoactive intestinal peptide-immunoreactive (VIP-IR) neurons were found in the myenteric plexus of the gastrointestinal tract of the amphibian, Bufo marinus. Only limited co-localization of the two substances was observed in nerve cell bodies, about 11% of the NOS-containing neurons were also labelled by VIP-IR and about 37% of VIP-IR nerve cell bodies contained NOS. The relationship between VIP, NOS and SP-IR in nerve fibres in the circular muscle was examined. There was partial co-localization of VIP and NOS, but no co-localization of NOS or VIP with substance P. Of fibres that were immunoreactive for VIP or NOS, fewer than 10% contained VIP alone.

Synaptic organisation of neuropeptide‐containing preganglionic boutons in lumbar sympathetic ganglia of guinea pigs

Within the lumbar sympathetic ganglia of guinea pigs, the endings of different populations of neuropeptide‐containing preganglionic neurons form well‐defined pericellular baskets of boutons around target neurons in specific functional pathways. We have used multiple‐labelling immunofluorescence, confocal microscopy, and ultrastructural immunocytochemistry to investigate synaptic organisation within pericellular baskets labelled for immunoreactivity to calcitonin gene‐related peptide (CGRP), substance P (SP), or the pro‐enkephalin‐derived peptide, met‐enkephalin‐arg‐gly‐leu (MERGL) in relation to their target neurons. Different functional populations of neurons, identified by their neurochemical profile, showed a significant degree of spatial clustering and predicted well the distribution of specific classes of pericellular baskets. Most of the boutons in a basket were completely surrounded by Schwann cell processes and did not form synapses. The synapses that were present were made mostly onto dendrites enclosed by the Schwann cell sheath surrounding the neuron within the basket. These dendrites probably originated from neurochemically similar neighbouring neurons. Nevertheless, some of the boutons in the baskets did form synapses with the cell body or proximal dendrites of the neuron they surrounded. Occasionally, cell bodies received a relatively high number of synapses and close appositions from boutons in a pericellular basket. Synaptic convergence of two immunohistochemically distinct types of preganglionic inputs was found in baskets of SP‐immunoreactive or MERGL‐immunoreactive, but not CGRP‐immunoreactive, boutons. Taken together, our results show that the appearance of pericellular baskets is primarily due to the packing of the target neurons. The grouping of functionally similar classes of neurons with their pathway‐specific projections of peptide‐containing preganglionic neurons suggests that peptides could exert their effects in relatively well‐defined zones within the ganglia. J. Comp. Neurol. 398:551–567, 1998.

Synaptic organisation of lumbar sympathetic ganglia of guinea pigs: serial section ultrastructural analysis of dye-filled sympathetic final motor neurons.

The authors serially sectioned seven dye‐filled neuronal somata and more than 1.6 mm of their dendrites from the lumbar sympathetic ganglia of guinea pigs and examined them ultrastructurally to determine the distribution of preganglionic synaptic inputs to their dendrites and cell bodies. Most of the surface of the neurons was covered with Schwann cells. Apposing boutons were rare, with an average density of one axosomatic bouton per 125 μm2 of somatic membrane and one axodendritic bouton per 25 μm of dendrite. Many dendritic segments that were more than 50 μm long completely lacked any apposing boutons. Although the average density of apposing boutons was low, local densities could be high, so that clusters of up to four adjacent boutons occurred on cell bodies and dendrites alike. The spatial arrangement of the apposing boutons for each of the cells examined here was not significantly different from a random distribution. Consequently, the number of apposing boutons observed for any neuron was simply proportional to the amount of neuronal surface sampled in the serial section run. About 50% of boutons directly apposing the neurons lacked any detectable presynaptic specialisations. When they were present, the presynaptic densities had a mean length of about 220 nm, with no difference between boutons that made axosomatic or axodendritic appositions. By applying these data to complete reconstructions of the dendritic trees of dye‐filled sympathetic neurons at the light microscopic level, the authors estimated that few neurons in the lumbar sympathetic chain of guinea pigs would receive more than 200 synapses or apposing boutons and that many of them would receive less than 100 synapses. Up to 50% of these boutons would be predicted to make axosomatic contacts. These new observations provide a strong morphological framework for a better understanding of how sympathetic final motor neurons process their preganglionic synaptic inputs. J. Comp. Neurol. 402:285–302, 1998.

How many types of cholinergic sympathetic neuron are there in the rat stellate ganglion

Sympathetic cholinergic postganglionic neurons are present in many sympathetic ganglia. Three classes of sympathetic cholinergic neuron have been reported in mammals; sudomotor neurons, vasodilator neurons and neurons innervating the periosteum. We have examined thoracic sympathetic ganglia in rats to determine if any other classes of cholinergic neurons exist. We could identify cholinergic sudomotor neurons and neurons innervating the rib periosteum, but confirmed that cholinergic sympathetic vasodilator neurons are absent in this species. Sudomotor neurons contained vasoactive intestinal peptide (VIP) and calcitonin gene-related peptide (CGRP) and always lacked calbindin. Cholinergic neurons innervating the periosteum contained VIP and sometimes calbindin, but always lacked CGRP. Cholinergic neurons innervating the periosteum were usually surrounded by terminals immunoreactive for CGRP. We conclude that if any undiscovered populations of cholinergic neurons exist in the rat thoracic sympathetic chain, then they are indistinguishable in size, neurochemistry and inputs from sudomotor or cholinergic neurons innervating the periosteum. It may be that the latter two populations account for all cholinergic neurons in the rat thoracic sympathetic chain ganglia.

Chemically distinct preganglionic inputs to iris-projecting postganglionic neurons in the rat: A light and electron microscopic study

Individual autonomic postganglionic neurons are surrounded by pericellular baskets of preganglionic terminals that are easily identifiable with the light microscope. It has been assumed that the target cell of a pericellular basket of preganglionic terminals is the neuron at the centre of the basket. This assumption has enabled the connectivity of preganglionic neurons to be determined at the light microscopic level. However, if the preganglionic terminals in a pericellular basket make synapses with the dendrites of nearby, but functionally different, postganglionic neurons, then the conclusions of light microscopic studies are far less certain. We have used a serial section ultrastructural study to determine the target of the preganglionic pericellular basket in a situation where the apparent target cell is surrounded by neurons of dissimilar function. In the rat superior cervical ganglion, postganglionic neurons projecting to the iris were identified, using retrograde tracers, as single neurons (i.e., not in clusters). We have used immunohistochemistry to show that iris‐projecting neurons are surrounded by preganglionic nerve terminals containing calcitonin gene‐related peptide (CGRP). We have demonstrated that the pericellular basket of CGRP‐immunoreactive preganglionic terminals provides inputs only to the soma at the centre of the basket and not to the dendrites of surrounding neurons. This suggests that, in autonomic ganglia, light microscopic identification of the preganglionic terminal baskets is likely to be a reliable method for identifying the targets of subclasses of preganglionic neurons. J. Comp. Neurol. 412:606–616, 1999.

Chemical coding of sympathetic neurons controlling the tarsal muscle of the rat

Sympathetic axons in the upper eyelid and in tissues in the superior retro-orbital space were examined for NPY immunoreactivity. Sympathetic nerve terminals containing co-localised NPY were associated with blood vessels, the conjunctiva and the Meibomian glands. The acini of the Harderian gland completely lacked sympathetic innervation. Sympathetic axons lacking NPY were only found in the tarsal muscle. In addition, a minority of terminals, located in the more proximal part of the tarsal muscle, contained weak immunoreactivity to NPY. Injections of the retrograde tracer, Fast Blue, into the eyelid or retro-orbital space labelled postganglionic somata in the superior cervical ganglion. While many retrogradely labelled somata were immunoreactive for NPY, around half lacked NPY immunoreactivity and so are likely to project to the tarsal muscle. Most of the retrogradely labelled postganglionic somata lacking NPY were surrounded by terminals immunoreactive for met-enkephalin, leu-enkephalin and met-enkephalin arg-gly-leu which were all found to be present in the same nerve terminals. Sectioning the cervico-sympathetic trunk eliminated all enkephalin-immunoreactive pericellular baskets. Many enkephalin-immunoreactive pericellular terminals contained co-localised VAChT, calretinin and calbindin immunoreactivity, but completely lacked nitric oxide synthase immunoreactivity. A second population of nerve terminals that were immunoreactive for nitric oxide synthase also surrounded tarsal muscle-projecting neurons, but these terminals lacked immunoreactivity to enkephalin. Thus, postganglionic neurons projecting to the tarsal muscle are of at least two chemical phenotypes (with or without NPY) and they receive convergent input from at least two populations of preganglionic neurons with distinctive chemical phenotypes.

Projections of nitric oxide synthase- and peptide-containing neurons in the small and large intestines of the toad (Bufo marinus)

The projections of galanin (GAL)- and vasoactive intestinal peptide (VIP)-immunoreactive (IR) and nitric oxide synthase (NOS)-containing neurons in the small and large intestines of the amphibian Bufo marinus were investigated by their reactions to surgical interruption (myotomy). In the small intestine, myotomy caused accumulation of GAL- and VIP-IR and of NADPH diaphorase reaction product (revealing NOS) in cut axons on the oral side of the operation site. On the anal side there was loss of GAL-IR axons from the circular muscle and myenteric plexus and long, anally directed processes could be traced from GAL-IR nerve cell bodies. There was no significant loss of VIP-IR or NADPH diaphorase from nerve fibres in the myenteric plexus or circular muscle layer, although anally-directed axons could be traced from nerve cell bodies on the anal side of the operation sites. In the large intestine, myotomy caused accumulation of VIP-IR and of NADPH diaphorase reaction product in cut axons on the oral side of the operation site. Anal to the cut, although there was no significant loss of these fibres from the muscle or myenteric plexus, anally directed axons could be traced from nerve cell bodies. GAL-IR fibres in the large intestine are of two types: a few contain GAL-IR alone and are thought to arise from enteric neurons; many contain both GAL- and SOM-IR and are thought to arise from nerve cell bodies in the hindgut. Myotomy caused an accumulation of GAL/SOM-IR material in fibres on the anal side of the cut and a substantial decrease in the number of fibres on the oral side. There was no detectable effect of myotomy on the GAL-IR fibres, although an abnormally high density of GAL-IR nerve cell bodies was found oral to the cut. These results indicate that VIP-IR and NOS-containing enteric neurons project in an oral to anal direction in the toad small and large intestines. Some of the neurons have short anal projections to the circular muscle. GAL-IR enteric neurons have similar projections in the small intestine, but their projections could not be determined in the large intestine. GAL/SOM-IR axons in the large intestine project from anal to oral. Myotomy in the large intestine appears to induce an increased or de novo expression of GAL-IR in enteric neurons oral to the cut.

Pre-embedding staining for GAD67 versus postembedding staining for GABA as markers for central GABAergic terminals.

Pre-embedding immunocytochemistry for the active form of glutamate decarboxylase (GAD67) and postembedding staining for γ-aminobutyric acid (GABA) were compared as markers for central GABAergic terminals in the phrenic motor nucleus, in which phrenic motor neurons had been retrogradely labeled with cholera toxin B-horseradish peroxidase. Nerve terminals with or without GAD67 immunoreactivity were identified in one ultrathin section. GABA was localized with immunogold in an adjacent section after etching and bleaching. GABA labeling density was assessed over 519 GAD67-positive and GAD67-negative nerve terminals in the phrenic motor nucleus. Frequency histograms showed that statistically higher densities of gold particles occurred over most GAD67-positive terminals. However, some GAD67-negative terminals also showed high densities of gold particles, and some GAD67-positive terminals showed low densities. Preabsorption of the anti-GABA antibody with a GABA-protein conjugate, but not with other amino acid-protein conjugates, significantly reduced gold labeling over both GAD67-positive and GAD67-negative terminals. These results show that the presence of GAD67 immunoreactivity correlates strongly with high densities of immunogold labeling for GABA in nerve terminals in the phrenic motor nucleus. Preabsorption controls indicate that authentic GABA was localized in the postembedding labeling procedure. Only a small proportion of intensely GABAimmunoreactive terminals lack GAD67, suggesting that both GAD67 and GABA are reliable markers of GABAergic nerve terminals.