Nicholas C. Brecha

A cDNA that suppresses MPP+ toxicity encodes a vesicular amine transporter

Classical neurotransmitters are transported into synaptic vesicles so that their release can be regulated by neural activity. In addition, the vesicular transport of biogenic amines modulates susceptibility to N-methyl-4-phenylpyridinium (MPP+), the active metabolite of the neurotoxin N-methyl-1,2,3,6-tetrahydropyridine that produces a model of Parkinsons disease. Taking advantage of selection in MPP+, we have used gene transfer followed by plasmid rescue to identify a cDNA clone that encodes a vesicular amine transporter. The sequence predicts a novel mammalian protein with 12 transmembrane domains and homology to a class of bacterial drug resistance transporters. We have detected messenger RNA transcripts for this transporter only in the adrenal gland. Monoamine cell populations in the brain stem express a distinct but highly related protein.

A novel α subunit in rat brain GABAA receptors

Abstract Two cDNAs (α1 and α4) from rat brain cDNA libraries encode isoforms of the a subunit of the GABA/benzodiazepine receptor, which differ at 30% of their amino acid residues. Northern blot analysis and in situ hybridization histochemistry show that α1 and α4 mRNAs have distinct sizes and distinct regional and cellular distributions in rat brain: both mRNAs are found in the cortex and hippocampus; however, only the α1 mRNA is detected in the cerebellum. We injected RNA transcribed from αa1 and α4 cDNAs into Xenopus oocytes, together with an RNA for a rat β subunit. We obtained GABA-dependent inward currents that were reversibly blocked by picrotoxin. Picrotoxin alone, applied to oocytes producing the α and β polypeptides, elicited an outward current. We suggest that these polypeptides together produce GABA-gated ion channels that can also open spontaneously.

The morphology and distribution of peptide-containing neurons in the adult and developing visual cortex of the rat. I. Somatostatin

SummaryUsing conventional immunocytochemical techniques, we have examined the morphology and distribution of somatostatin-like immunoreactive neurons in the visual cortex of albino rats between the first postnatal day and maturity. In the adult, somatostatin-immunoreactive neurons were observed in layers II to VI but were concentrated in layers II and III. These cells displayed morphological features characteristic of the multipolar and bitufted varieties of cortical non-pyramidal neurons as described in Golgi preparations of rat visual cortex.On the first postnatal day and in the subsequent few days, immunoreactivity was confined to immature bipolar and multipolar neurons concentrated in layers V and VI. Labelled cells first appeared in the more superficial layers at the beginning of the second postnatal week and attained a distribution similar to that observed in adult animals at the end of this week. At this time they closely resembled their adult counterparts from which they appeared indistinguishable by the end of the third postnatal week. The late appearance of labelled cells in the superficial layers, where they are predominantly located in adult animals, suggests that the somatostatin immunoreactivity exhibited by most of these neurons develops several days after they have completed their migration and assumed their positions in the visual cortex.

The nob2 mouse, a null mutation in Cacna1f : Anatomical and functional abnormalities in the outer retina and their consequences on ganglion cell visual responses

Glutamate release from photoreceptor terminals is controlled by voltage-dependent calcium channels (VDCCs). In humans, mutations in the Cacna1f gene, encoding the alpha1F subunit of VDCCs, underlie the incomplete form of X-linked congenital stationary night blindness (CSNB2). These mutations impair synaptic transmission from rod and cone photoreceptors to bipolar cells. Here, we report anatomical and functional characterizations of the retina in the nob2 (no b-wave 2) mouse, a naturally occurring mutant caused by a null mutation in Cacna1f. Not surprisingly, the b-waves of both the light- and dark-adapted electroretinogram are abnormal in nob2 mice. The outer plexiform layer (OPL) is disorganized, with extension of ectopic neurites through the outer nuclear layer that originate from rod bipolar and horizontal cells, but not from hyperpolarizing bipolar cells. These ectopic neurites continue to express mGluR6, which is frequently associated with profiles that label with the presynaptic marker Ribeye, indicating potential points of ectopic synapse formation. However, the morphology of the presynaptic Ribeye-positive profiles is abnormal. While cone pedicles are present their morphology also appears compromised. Characterizations of visual responses in retinal ganglion cells in vivo, under photopic conditions, demonstrate that ON-center cells have a reduced dynamic range, although their basic center-surround organization is retained; no alteration in the responses of OFF-center cells was evident. These results indicate that nob2 mice are a valuable model in which to explore the pathophysiological mechanisms associated with Cacna1f mutations causing CSNB2, and the subsequent effects on visual information processing. Further, the nob2 mouse represents a model system in which to define the signals that guide synapse formation and/or maintenance in the OPL.

Localization of calcitonin gene-related peptide-like immunoreactivity in neurons of the rat gastrointestinal tract

Calcitonin gene-related peptide (CGRP)-like immunoreactivity was localized in neuronal processes and somata of the rat gastrointestinal tract. Varicose processes were observed in the myenteric and submucosal plexuses, smooth muscles, submucosa, mucosa and around blood vessels. Immunoreactive somata were visualized in the myenteric and submucosal ganglia of the intestine in colchicine-treated rats. These observations, together with previous neuroanatomical and pharmacological studies, suggest that CGRP may be involved in regulatory functions of the gastrointestinal tract.

The morphology and distribution of peptide-containing neurons in the adult and developing visual cortex of the rat. II. Vasoactive intestinal polypeptide

SummaryUsing immunocytochemistry, we have examined the morphology and distribution of vasoactive intestinal polypeptide-like immunoreactive neurons in the visual cortex of albino rats whose ages were closely spaced in time between the first postnatal day and adulthood. In the adult, immunoreactive neurons were located in layers II to VI but were concentrated in layers II and III. All labelled neurons had the morphological characteristics of cortical non-pyramidal cells with the majority being of the bipolar variety as described in Golgi preparations. Some multipolar forms were also present.Vasoactive intestinal polypeptide-immunoreactivity appeared to develop in postnatal life. Labelled cells were first seen in layers V and VI at day 4. During the subsequent few days, some labelled cells were observed in the more superficial layers and by day 8 they were predominantly present in layers II and III. Although the distribution of immunoreactive cells at this time resembled that of adult animals, their morphology displayed immature features. The size and extent of their dendritic branching appeared to increase considerably during the second and third weeks and their morphological maturation was attained by the middle of the fourth postnatal week.

Expression of the proto-oncogene, trk, receptors in the developing rat retina.

The neurotrophins, including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and NT-4/5 are important in a variety of developmental processes in the peripheral and central nervous systems. These molecules bind to a low-affinity receptor and to distinct high-affinity receptors. The high-affinity receptor for NGF is the proto-oncogene product, p140trkA(trkA). Isoforms of p140trkA, p145trkB(trkB), and p140trkC(trkC), are the primary high-affinity receptors for BDNF and NT-3, respectively. We evaluated the developmental regulation of the high-affinity neurotrophin receptors in the rat retina using polyclonal antibodies directed to a highly conserved region of the C-terminus of the p140trkA isoforms (pantrk) and antibodies directed to unique amino-acid sequences of p140trkA, p145trkB, and p140trkC. Immunoreactivities for trkA and trkB, as well as pantrk, were detected in the developing retina and showed similar distributions. At similar antibody concentrations, trkC immunoreactivity was not detected. In the embryo, immunoreactivties were present in cells located throughout the neuroblastic retina, especially in the inner retinal layers, and in fibers in the nerve fiber layer and optic nerve. In the newborn retina, immunoreactivities for these two receptor isoforms were localized to numerous somata in the inner nuclear layer (INL), as well as to cells in the ganglion cell layer (GCL) and axons in the nerve fiber layer and optic nerve. A similar pattern of immunostaining persisted throughout the first postnatal week. By postnatal day-10, immunostaining was confined to large-diameter cells in the GCL, both heavily stained and lightly stained cells in the INL and a plexus of processes in the inner plexiform layer (IPL).(ABSTRACT TRUNCATED AT 250 WORDS)

Multiple γ‐aminobutyric acid plasma membrane transporters GAT‐1, GAT‐2, GAT‐3 in the rat retina

γ‐Aminobutyric acid (GABA) plasma membrane transporters (GATs) influence synaptic neurotransmission by high‐affinity uptake and release of GABA. The distribution and cellular localization of GAT‐1, GAT‐2, and GAT‐3 in the rat retina have been evaluated by using affinity‐purified polyclonal antibodies directed to the C terminus of each of these GAT subtypes. Small GAT‐1‐immunoreactive cell bodies were located in the proximal inner nuclear layer (INL) and ganglion cell layer (GCL), and processes were distributed to all laminae of the interplexiform layer (IPL). Varicose processes were in the optic fiber layer (OFL) and the outer plexiform layer (OPL). Weak GAT‐1 immunostaining surrounded cells in the INL and GCL, and it was found in the OFL and OPL and in numerous processes in the outer nuclear layer (ONL) that ended at the outer limiting membrane. GAT‐1 is therefore strongly expressed by amacrine, displaced amacrine, and interplexiform cells and weakly expressed by Müller cells. GAT‐2 immunostaining was observed in the retina pigment epithelium and the nonpigmented ciliary epithelium. GAT‐3 immunoreactivity was distributed to the OFL, to all laminae of the IPL, GCL and INL, and to processes in the ONL that ended at the outer limiting membrane. Small GAT‐3‐immunoreactive cell bodies were in the proximal INL and GCL. GAT‐3 is therefore strongly expressed by Müller cells, and by some amacrine and displaced amacrine cells. Together, these observations demonstrate a heterologous distribution of GATs in the retina. These transporters are likely to take up GABA from, and perhaps release GABA into, the synaptic cleft and extracellular space. This suggests that GATs regulate GABA levels in these areas and thus influence synaptic neurotransmission.

Neuronal and glial localization of GAT-1, a high-affinity gamma-aminobutyric acid plasma membrane transporter, in human cerebral cortex: with a note on its distribution in monkey cortex

High‐affinity γ‐aminobutyric (GABA) plasma membrane transporters (GATs) influence the action of GABA, the main inhibitory neurotransmitter in the human cerebral cortex. In this study, the cellular expression of GAT‐1, the main cortical GABA transporter, was investigated in the human cerebral cortex by using immunocytochemistry with affinity‐purified polyclonal antibodies directed to the C‐terminus of rat GAT‐1.

Somatostatin 2A receptor is expressed by enteric neurons, and by interstitial cells of Cajal and enterochromaffin-like cells of the gastrointestinal tract.

Somatostatin exerts multiple effects by activating distinct G protein‐coupled receptors. Here we report the cellular sites of expression of the somatostatin subtype 2A (sst2A) receptor in the rat enteric nervous system by using a C‐terminus‐specific, affinity‐purified antiserum and immunohistochemistry. Antibody specificity was confirmed by the cell surface staining of human embryonic kidney 293 cells expressing the sst2A receptor, the lack of staining of cells expressing the somatostatin subtype 2B receptor, and the abolition of staining by preincubating the antiserum with the C‐terminus peptide used for immunization, sst2A(361‐369). The sst2A receptor antibody recognized a broad 80 kDa band on Western blots of membranes prepared from cells transfected with sst2A receptor cDNA; following receptor membrane deglycosylation, the antibody detected an additional 40 kDa band. In the enteric nervous system, the sst2A antibody primarily stained neurons of the myenteric and submucosal plexuses, and abundant fibers distributed to the muscle, mucosa, and vasculature. Immunoreactive staining was also observed in non‐neuronal cells, including presumed interstitial cells of Cajal of the intestine and enterochromaffin‐like cells of the stomach. Fibers expressing sst2A receptor immunoreactivity were often in close proximity to D cells of the gastric and intestinal mucosa. Colocalization of somatostatin and sst2A receptor immunoreactivities was not observed in endocrine cells nor in enteric neurons. Double‐label immunohistochemistry revealed colocalization of sst2A and vasoactive intestinal peptide immunoreactivities in enteric neurons. The multiple types of cells expressing the sst2A receptor, including enteric neurons and non‐neuronal structures, in addition to the relationship between somatostatin and sst2A receptor elements, provide evidence that the sst2A receptor mediates somatostatin effects in the gastrointestinal tract via neuronal and paracrine pathways. J. Comp. Neurol. 386:396‐408, 1997.