THE JOURNAL OF COMPARATIVE NEUROLOGY 294:633-646 (1990)

Immunohistochemical and Biochemical Evidence for the Putative Inhibitory Neurotransmitters Histamine and GABA in Lobster Olfactory Lobes EDWARD ORONA, BARBARA-ANNE BATI'ELLE, AND BARRY W. ACHE Whitney Laboratory and Departments of Zoology and Neuroscience, University of Florida, St. Augustine, Florida 32086

ABSTRACT As an initial effort to investigate possible inhibitory interactions in the olfactory system of the spiny lobster, studies were conducted to identify and localize the putative inhibitory neurotransmitters histamine and GABA in the olfactory lobe. Biochemical studies demonstrated that olfactory lobe tissue was capable of synthesizing histamine from radioactive histidine and GABA from glutamic acid. Immunohistochemistry was used to localize histamine and GABA in brain sections, by using either avidin-biotin conjugated peroxidase or fluorescein conjugated secondary antibody. Specific histamine-like and GABA-like immunoreactivity was found in soma clusters of olfactory interneurons, adjacent to the olfactory lobe. Small, putative glial cells displaying intense histamine-like immunoreactivity were found interspersed among the glomeruli of the lobe. The accessory lobe exhibited moderate immunostaining for both histamine and GABA. Positive immunostaining for histamine and GABA was also found in the olfactory lobes, with a predominance of staining in the outer caps of the glomeruli, which are thought to be the regions where the primary afferent terminals contact the processes of second-order olfactory neurons. These findings collectively implicate inhibition a t the first synaptic level of the olfactory pathway in the spiny lobster. Key words: olfaction, mixture suppression, synaptic inhibition, biogenic amines, crustaceans

Inhibition is a fundamental process in sensory biology that broadens the range for coding beyond that available from excitation alone. In olfaction, inhibition is a major component of information processing a t the earliest synaptic levels of the olfactory pathway of vertebrates (Halasz and Shepherd, '83; Mori, '87) and insects (Matsumoto and Hildebrand, '81; Waldrop et al., '87). Central inhibitory interactions have been implicated, for instance, in mixture suppression, a phenomenon in which the response intensity to an odor mixture is less than the summed responses to its individual components (see Laing et al., '89). Physiological correlates of mixture suppression occur in the olfactory system of the spiny lobster (Ache and Derby, '85; Ache et al., '88; Ache, '89). The persistence of this phenomenon when stimulatory and suppressive odorants are presented to separate parts of the olfactory receptor field (Derby et al., '85) implicates central inhibitory mechanisms in mixture suppression. In crustaceans, the axons of the primary receptor cells synapse with second-order neurons in the glomerular neuropil of the olfactory lobe (OL). o 1 9 9 0 WILEY-LISS, INC.

This is the first level of the olfactory pathway where central mechanisms of mixture suppression could be manifested. In order to study inhibitory interactions within the OL, we first attempted to identify and localize potential inhibitory neurotransmitters there. There is considerable evidence in many vertebrate and invertebrate systems that gamma-aminobutyric acid (GABA) can function as an inhibitory neurotransmitter (Usherwood, '78; Enna?'83; Bormann, '88). In particular, GABA has been implicated as a central inhibitory transmitter in olfaction. In the vertebrate olfactory bulb, GABA is present in periglomerular and tufted cells, but is predominantly located in the granule cells which are the major inhibitory interneurons (reviews: Mori, '87; Halasz and Shepherd, '83). GABA has been shown to mediate synaptic inhibition in the olfactory bulb of the rat (Halasz and Shepherd, '83),rabbit (Nicoll, '71), and turtle (Nowycky et al., '81). Evidence Accepted December 5,1989.

E. ORONA ET AL.

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Core Fibers VPMLC cells VPPLC cells Olfactory lobe -Accessory lobe tract Olfactory-Globular tract (to medulla terminalis)

Fig. 1. A schematic diagram of the lobster brain showing the olfactory lobes (OLs) and the principal synaptic connections. A: T h e main olfactory organs are tufts of aesthetasc sensilla found on the lateral filaments of the antennule (see Grunert and Ache, '88). T h e primary receptor cells (about 320 cells per sensilla) have ipsilateral, monosynaptic projections to t h e OL. T h e cell bodies of second-order olfactory neurons are found in the adjacent soma clusters, VPPLC and VPMLC (ventral paired posterolateral and posteromedial clusters, respectively). The accessory lobes and medulla terminales (in the eyestalks) are centers thought to be involved in higher-order olfactory processing. All interneurons eventually synapse with cells exiting the circumesophageal connectives. T h e brain is shown in a ventral view, with the anterior end of t h e brain oriented to the right. B T h e synaptology within the OL is shown here. Primary afferents encircle the rim of t h e OL, occasionally looping down into the glomeruli. Synapses are exclusively localized in

the glomeruli, with the majority of synapses between the primary afferents and the processes of second-order cells are primarily in the cap regions (Sandeman and Luff, '73). The glomerulus is a focal unit of olfactory neuropil, with synaptic interactions between primary afferent terminals and olfactory interneurons. A hypothetical neuron is shown in the VPPLC soma cluster with a neurite extending into the cap regions of the glomeruli, where they contact the primary afferents. There is a 5 0 0 1 convergence ratio of primary receptor cells to a glomerulus (Blaustein e t al., '88), and a n estimated 120,000 synapses in a single glomerulus in crayfish OLs (Sandeman and Luff, '73). T h e components of the fibers located a t the periphery of the glomeruli and with the core regions of the OL are indicated. Relatively little is known about the branching patterns of individual primary axon8 or about the dendritic morphologies of t h e second-order neurons (cf. Arbas e t al., '88; Derby and Blaustein, '88).

indicates that GABA is synthesized and localized in the antenna1 (olfactory) lobes of an arthropod, Manduca sexta (Maxwell et al., '78; Hoskins, et al., '86), where it is also believed to function as an inhibitory transmitter in olfactory interneurons (Waldrop et al., '87).

Histamine (HA) is another important neuroactive substance in vertebrates and invertebrates (Haas, '85; Prell and Green, '86; Schwartz et al., '86). In arthropod photoreceptors (Hardie, '88; Nassel et al., '88; Pirvola et al., '88), HA has recently emerged as a candidate neurotransmitter whose

HISTAMINE AND GABA IN LOBSTER OLFACTORY LOBES

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Fig. 2. HA synthesis. A radiochromatogram of OL tissue that was incubated with 10 pCi/ml of 'H-histidine (HIS), extracted in acid, and subjected to high-voltage paper electrophoresis at pH 1.9. A peak of radioactivity co-migrates with the HA standard (shown in brackets). The other peaks represent unidentified HIS metabolites.

action mimicks that of natural visual stimulation by hyperpolarizing the second-order neurons (Simmons and Hardie, '88; Callaway and Stuart, '89; Schlemermeyer et al., '89). HA has also been implicated in the olfactory CNS. For instance, HA-like immunoreactivity (HA-IR) is present in the cockroach antennal lobe (Pirvola et al., '88), and in Manduca sexta, the antennal lobe tissue is capable of synthesizing HA (Maxwell et al., '78). In addition, HA-IR fibers are present a t the glomerular layer of the rat olfactory bulb (Panula et al., '89), and HA can produce physiological effects when applied to the bulb (Rhoades et al., '88). HA has been implicated as an inhibitory neurotransmitter in the lobster stomatogastric ganglion (Claiborne and Selverston, '84), and has recently been shown in our laboratory to gate a chloride channel in lobster olfactory receptor cells (McClintock and Ache, '89). Collectively, these diverse lines of evidence suggest that HA and GABA are candidate inhibitory neurotransmitters in the olfactory CNS of crustaceans. Therefore, immunohistochemical and biochemical techniques were used to attempt to localize HA and GABA within the OLs of the spiny lobster. A preliminary report of this study has appeared elsewhere (Orona et al., '89).

MATERIALS AND METHODS Animal and tissue preparation Male and female specimens of the Caribbean spiny lobster, Panulirus argus, were obtained from the Florida Keys and maintained in the laboratory in flowing seawater a t ambient temperature. All were intermolt adult specimens with a carapace length of 50-70 mm. Animals were sub-

merged under ice for 15-20 minutes before their brain was quickly removed into Panulirus saline (Schmiedel-Jakob et al., '89).

Synthesis of HA and GABA from radioactive precursors Brains were dissected to obtain samples consisting of 80-90% olfactory tissue (OL tissue), which included the paired OLs, accessory lobes, and adjacent soma clusters of olfactory interneurons (as in Fig. 1A). OL tissue from a single animal was incubated overnight in 2 ml of saline containing 10 yCi/ml of radiolabelled precursor (3Hhistidine [HIS], 51.0 Ci/mmol, or 3H-glutamic acid, 54.7 Ci/mmol obtained from DuPont/NEN, Wilmington, DE). A total of 8 animals was used for these experiments; tissue from 4 animals was assayed for HA synthesis and tissue from a separate set of 4 animals was assayed for GABA synthesis. After the incubations, the OL tissues were extracted with formic acid in acetone. To separate the radiola-

TABLE 1. Synthesis of HA and GABA in Lobster Olfactory Lobes'

DPM x lo" ["HI HA

DPM

No. 1113 No.2 70 No.3 94 N0.4 21 i = 74.6 x lo3 SEM = 23 x I d

No. 5 128

103 r

3 ~ GABA 1

No. 6 223 No. 7 112 No. 8 210 = 168 x 108 SEM = 32 = 103

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'Diaintegrationaper minute (DPME)per mg protein, above hackgroundco-migrating with the HA and GABA atandarda.T L u e ~ a m ~from l e ~four differentanimals were used in each set of analyses. The mean (3and standard error of the mean (SEMI are indicated.

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Distance from Origin ( c m ) Fig. 3. GABA synthesis. A portion of a radiochromatogram of OL tissue that was incubated with 10 &/ml of ‘H-glutamic acid, extracted in acid, and subjected to high-voltage paper electrophoresis a t pH 1.9. A peak of radioactivity co-migrates with the GABA standard (shown in brackets).

belled precursor from its products, the extracts were subjected to high-voltage paper electrophoresis at pH 1.9 together with appropriate standards. The positions of the standards were visualized with diazotized sulfanilic acid for HA or ninhydrin for GABA (Hildebrand et al., ’71). The electrophoretograms were cut into 1 cm strips, and the radioactivity, which co-migrated with the standards, was measured in a liquid scintillation counter. To confirm the identities of the radioactive products of interest, electrophoresis was also followed by ascending paper chromatography. One track was processed for scintillation counting and the other chromatographed in a second dimension, orthogonal to the direction of the electrophoresis. The chromatography was in butano1:pyridine:water (1:l:l) for HA, or in butano1:pyridine:water (2:l:l) for GABA. The percentage of counts in the first dimension, which migrated with the marker compound in the second dimension, was measured for both HA and GABA. The amount of protein content in the OL tissue was determined according to the method of Lowry (Lowry et al., ’51).

For HA immunohistochemistry, whole brains were partially desheathed and fixed overnight in 4% paraformaldehyde made in 0.1 M Sorensen’s buffer. The brains were then rinsed in Sorensen’s buffer and stored overnight in 30% sucrose. Cryostat sections were taken at 15 pm and dried for a t least 30 minutes prior to staining. The sections were first rinsed in Tris-buffered saline (TBS), then incubated in normal goat serum and Triton (0.1-0.3 % ) to block nonspecific staining. A polyclonal antibody (Cat. No. AB134, lot No. 051188M) raised against HA was obtained from Chemicon International (El Segundo, CA). The sections were incubated in the primary antibody, diluted 1500 to 1:1,000 in goat serum and Triton, overnight at 4°C. The antigen-antibody complex was visualized with either avidin-biotin conjugated peroxidase or a fluorescein isothiocyanate (FITC) conjugated secondary antibody. An AvidinBiotin (ABC) Vectastain Kit (Vector Labs) was used for the peroxidase reaction. The biotinylated secondary antibody (goat anti-rabbit IgG) was diluted per the kit directions (1 drop/5 ml TBS; or a dilution of about -1925) and applied to the sections for 2 hours. The ABC reagent complex was also applied to the sections for 2 hours. The peroxidase reaction incorporated 3,3’-diaminohenzidine tetrahydrochloride (DAB) as the chromagen. The sections were rinsed in TBS, dehydrated, and coverslipped with Permount. For immunafluorescent visualization, the FITC secondary antibody was applied to separate sections at a dilution of 1:lOO to 1:200 for 2 hours. Tissue was then rinsed in phosphate-buffered saline and Triton, then mounted in 50% glycerol with p-phenylenediamine for fluorescence microscopy.

GABA immunohistochemistry For the GABA immunohistochemistry, the fixative for the brains was either 1% ‘ paraformaldehyde + 1.25% glutaraldehyde, or a mixture of 1%)paraformaldehyde, 0.5% glutaraldehyde, and 15% picric acid. All fixatives were made in 0.1 M Sorensen’s buffer. The immunohistochemistry was performed on frozen sections (as above), as well as paraffin sections. After fixation and rinsing, brains were dehydrated through an ethanol and xylene series, and embedded in Paraplast. Brain sections (15 pm) were obtained with a rotary microtome, dried overnight, then deparaffinized, rehydrated, and stained (below). The GABA immunostaining protocol employed a commercially available antiserum (Chemicon, Cat. No. AB131) and the “affinity purified” GABA antiserum (Chemicon, Cat. No. AB141), conjugated to keyhole-limpet hemocyanin prepared in glutaraldehyde. The protocol for GABA was generally similar to the method used for HA (above), except that the peroxidase antiperoxidase method was also used. The sections were blocked with nonspecific sheep serum, then incubated in the GABA antiserum at a final dilution of 1:500 to 1:1,000 overnight. Immunoperoxidase staining employed the sheep antirabbit secondary antibody a t a dilution of 1:lOO-1:500 for 2 hours.

Controls and specificity of staining To control for nonspecific staining, the procedures (above) were repeated on equivalent sections, except that the primary antibody was omitted or it was preabsorbed with the

HISTAMINE A N D GABA I N LOBSTER OLFACTORY LOBES

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Fig. 4. A HA-like immunoreactivity (HA-IR) demonstrated in a cryostat section processed with avidin-biotin conjugated peroxidase. In this superficial section of the OL, glomeruli (G) tend to look more spherical than columnar. All glomeruli appeared to exhibit positive

immunoreactivity. B: Lack of staining in glomeruli of a control section (preabsorbed overnight with 100 pg of HA per ml of diluted antisera). Calibration bar: 100 fim for A, 50 Wm for B.

antigen (preabsorbed overnight with 100 pg of antigen per ml of diluted antisera). Information about the specificity of the antibodies was provided by the supplier. The HA antibody does not cross-react with noradrenaline, glucagon, and histidine. The cross-reactivity of the GABA antisera

(#AB131 and “affinity purified” AB141) was determined by either radioimmunoassay or enzyme-linked immunosorbent assays. As certified by the supplier, affinity purification reduces cross-reactivity to other neurotransmitters, such as glutamate, aspartate, glycine, and taurine.

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Figure 5

HISTAMINE AND GABA IN LOBSTER OLFACTORY LOBES

RESULTS HA and GABA synthesis The OL tissues (as shown in Fig. 1A) contain enzymes for synthesizing HA and GABA from their respective precursors. The OL tissues synthesized a product from radiolabelled HIS that co-migrated with standard HA in highvoltage paper electrophoresis (Fig. 2). The average amount of 3H-HA synthesized in four separate experiments was 75 x103 DPM (SEM 23) or 666 fmoles (SEM * 207) of 3H-HA synthesized per mg protein (Table 1).Greater than 82 % of this radioactivity co-migrated with the HA standard when electrophoresis was followed by ascending paper chromatography. A portion of a radiochromatogram of OL tissue, incubated with 'H-glutamic acid, shows a peak of radioactivity that co-migrates with the GABA standard (Fig. 3). The average amount of 'H-GABA synthesized in four separate experiments was 168 x lo3 DPM (SEM i 32) or 1.4 pmol (SEM 2 0.3) per mg protein (Table 1).Greater than 95% of this radioactivity co-migrated with GABA in the second dimension. The high proportion of radioactivity co-migrating with GABA and HA in the second dimension corroborates the likelihood that these neuroactive substances are synthesized and stored in the OL tissues of the lobster brain. Synthesis of GABA or HA, however, was not demonstrated in samples of peripheral olfactory tissue (unpublished data).

HA-like immunoreactivity (HA-IR) Specific HA-IR was observed in the glomeruli of the OL (Figs. 4A, 5A), and nearly all glomeruli were positively stained. This was best demonstrated in superficial sections through the OL, in which most of the glomeruli can be observed (Fig. 4A). No specific HA-IR was obtained when the primary antibody was omitted or when the antiserum was preabsorbed with HA (Fig. 4B). The staining pattern tended to demarcate individual glomeruli in the lobe, giving them the characteristic columnar appearance that is typical of the crustacean OL (Fig. 5A,B). There was a predominance of staining in the outer caps of the glomeruli (Fig. 5A-C), which are thought to be the principal regions of synaptic contacts between the primary afferent terminals and secondorder olfactory neurons (see Fig. 1A,B; and Sandeman and Luff, '73). The specific HA-IR in the glomerular caps does not appear to represent positive staining of the axonal terminals of the primary receptor cells for two reasons. The somata of the primary receptor cells are not HA positive (,unpublished). In addition, the primary afferent fiber layer of the OL was typically not stained (Fig. 5B), although at some

Fig. 5. Demonstration of the predominance of HA-IR in the outer cap regions of the glomeruli. A Low magnification of the HA-IR in the OL and the adjacent soma cluster VPMLC (arrow). Note the intense peroxidase reaction product in the outer areas (arrowhead) of the glomeruli, B Higher magnification (of the boxed area in A) showing the predominance of staining in the outer glomerular caps. Note the typical columnar organization of the olfactory glomerulus ( G ) . The separate regions, including the primary d e r e n t s (PA), cap, and base of the glomeruli are indicated. C In different sections through the OL, the primary afferent layer was not always entirely distinguishable from the cap regions of the glomeruli. Nonetheless, this outer region of the glomeruli (arrowheads) exhibits intense HA-IR. Calibration bar: 250 p m for A, 50 pm for B, 100 pm for C.

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levels (or planes of section) of the OL, the primary afferent layer was not clearly delineated from the cap region of the glomeruli (Fig. 5C). Therefore, we interpret this staining to result from HA-IR processes of olfactory interneurons (Fig. 6A,B), with somata in the clusters, VPMLC and VPPLC (as in Fig. 1).Many, but not all, neurons in these two clusters stained for HA (Fig. 6B). These HA-positive somata were generally uniform in size, with a diameter of about 10-20 km. Interneuronal fibers from the soma clusters generally enter through the core of the OL, although some processes from the VPMLC cluster enter via the rim of the lobe (see Fig. 1B; Blaustein et al., '88). In certain sections, HApositive interneuronal fibers from the somata could be observed in the core of the OL (Fig. 6C). Moderate HA-IR was also present in the accessory lobe (Fig. 6D). Here the HA-positive staining revealed glomeruli which were more spherical in shape than those of the OL; spherical glomeruli are typical for the accessory lobe. Again, we tentatively attribute the staining of the glomeruli in the accessory lobe to olfactory interneurons in the VPMLC and VPPLC clusters, since these cells are known to innervate both olfactory and accessory lobes (Arbas et al., '88; Blaustein et al., '88). Other cells which displayed intense HA-IR were found interspersed between the OL glomeruli. These cells are presumably glia, since neuronal somata are not present in the OL itself, but in the adjacent cell body clusters (Blaustein et al., '88; Sandeman and Luff, '73). These cells were concentrated near the outer caps of glomeruli, adjacent to the first synaptic region within the OL (Fig. 7A). The somata of these putative glial cells were located on the outer rim of the OL, sending long processes between the glomeruli (Fig. 7B). These cells seemed to surround or encapsulate the glomeruli, with their processes appearing to enter the columns on only rare occasion (Fig. 7C). The morphology of these putative glial cells is similar to that of glia previously described in crustacean OLs (Sandeman and Luff, '73), in being confined to the areas between the glomeruli without innervating the glomerular neuropil to any great extent. Hence, it is not likely that the extensive HA-like immunostaining throughout the many glomeruli of the OL can be attributed to the processes of these cells.

GABA-like immunoreactivity (GABA-IR) Specific GABA-IR was also present in somata of many olfactory interneurons in both VPPLC and VPMLC (Fig 8A). However, the positive staining for GABA resembled the pattern of HA-IR in only some respects. The size of these neurons resembled the small globuli cells that had also stained for HA. Positive staining was not observed in control

Fig. 6 (appears on overleaf'). The positive HA-IR in the glomeruli of the OL is attributed to staining of the processes of second-order olfactory neurons. A: Staining was present in many interneurons in both soma clusters. Cells in the VPMLC cluster (small arrow) are shown. Both the small globuli cells and somewhat larger neurons (arrowhead) were stained. Though specific staining was present in the interneurons, more intense HA-IR was exhibited by the putative glial cells (large arrows) in the OL (see text and Fig. 7). B Higher magnification of somata in the VPMLC cluster, showing both positively stained neurons (arrowhead) and nonstained neurons (asterisk). C Fibers from interneurons in the core region (arrowhead) of the area below the glomeruli of the OL. D: Moderate HA-IR was also present in the spherically organized glmieruli of the accessory lobe (AL). Calibration bar: 100 pm for A and C, 25 p m for B, 50 pm for D.

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HISTAMINE AND GABA IN LOBSTER OLFACTORY LOBES

Figure 7 (legend appears on overleafi

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sections incubated with normal serum (Fig. 8B). Hence, both GABA- and HA-IR were present in many of the uniformly small cells in the VPPLC and VPMLC, rather than the large diameter cells which have been found to display serotonin-like immunoreactivity (Sandeman and Sandeman, '87). Although many interneurons of the VPPLC and VPMLC clusters stained for GABA and HA, no quantitative analysis was performed to compare directly the numbers of neurons that were stained. However, it appeared that relatively more GABA-IR cells were present in VPPLC, whereas more HA-IR cells were present in VPMLC. While there may be some degree of co-localization of GABA- and HA-IR, this idea was not directly tested in double-labelling experiments. Unlike the HA staining, the GABA-IR in the OL did not strictly demarcate the glomeruli. Instead, the specific GABAIR was confined to only the outer regions of the glomeruli (Fig. 9A,B). There was also an indication of a separate band of fibers just below the cap region of glomeruli (Fig. 9B). As with the HA-IR, however, the GABA-IR in the glomeruli is attributed to the positive staining of olfactory interneurons with somata in the two clusters. This immunostaining also does not appear to reflect specific GABA immunostaining of the primary receptor cells, since neither the somata of the receptor cells (unpublished) nor the afferent terminal layer in the OL demonstrated positive staining. Peripheral olfactory tissue does not exhibit the capacity to synthesize GABA from its precursor in either the American (Barker et al., '72) or the spiny lobster (unpublished). Within the OL, there were no putative glial cells near the glomeruli which stained for GABA, as they had for HA. Within the accessory lobe, the glomeruli also displayed GABA-IR (Fig. 8A). Almost all glomeruli exhibited moderately intense GABA-like irnrnunoreactivity. This staining, like that in the OL, may again be attributed to the processes of second-order neurons with cell bodies in the VPPLC and VPMLC clusters.

DISCUSSION Our results indicate that HA and GABA are present within the olfactory CNS of the lobster, where they may function as neurotransmitters or modulators. That HA and GABA can he synthesized in olfactory brain tissue from their precursors supports the interpretation that endogenous HA and GABA are present in the OL. The olfactory receptor cells of arthropods are believed to use acetylcholine as their principal neurotransmitter (Sanes and Hildebrand, '76; Buchner et al., '86). It has been demonstrated that acetylcholine can be synthesized in peripheral olfactory tissue of both American (Barker et al., '72) and spiny lobsters (unpublished data). Our finding that

Fig. 7 (located on previous page). HA-IR was also present in the putative glial cells in the OL, demonstrated by using peroxidase- (A) and FITC conjugated secondary antibody (B,C) on cryostat sections. A: Putative glial cells with cell bodies (arrows) located near the outer cap regions of the glomeruli ( G )of the OL also exhibited HA-IR. These glial cells were intensely stained and were distributed near the first synaptic zone (glomerular caps) of the OL. B FITC staining of these putative glial cells. These cells had long finger-like processes (arrow) that were present between and around the glomeruli. C: Processes (arrows) of these glial cells appeared to enter the glomerular columns on only rare occasion. Calibration bar: 100 p m for A, 50 p m for B, 25 ptn for C.

HA-IR is not observed at the central terminals of the primary receptor cells is consistent with this idea, as is the recent demonstration that HA-IR is present in the cockroach antennal lobe but not in the antennal nerve itself (Pirvola et al., '88). Thus, the specific HA-IR in the OL presumably reflects the positive staining of putative histaminergic olfactory interneurons with processes in the glomeruli. The occurrence of synapses between the primary olfactory afferents and second-order neurons are thought to be exclusively in the OL glomeruli, and principally in the outer cap regions (Sandeman and Luff, '73). HA release could occur, therefore, at either the axonal or dendritic arborizations of olfactory interneurons which terminate in this region (Arbas et al., '88; Blaustein et al., '88). There is precedence for inhibitory actions of HA in the CNS of arthropods. Increased chloride conductances have been associated with the actions of HA in the lobster stomatogastic ganglion (Claiborne and Selverston, '84) and in fly photoreceptors (Hardie, '89). Furthermore, we have recently identified a HA-gated chloride channel on the olfactory receptor cells of the lobster (McClintock and Ache, '89). Thus, histaminergic interneurons that arborize in the outer caps of the glomeruli could inhibit the primary afferents presynaptically. This possibility implies that there are inhibitory synapses from interneurons onto the primary afferent terminals, although such presynaptic contacts were not described in a previous electron-microscopic study of the crayfish OL (Sandeman and Luff, '73). Alternatively, inter-glomerular inhibition by olfactory interneurons could be homologous to the lateral inhibition proposed for the vertebrate olfactory bulb (reviews: Kauer, '87; Mori, '87; Scott and Harrison, '87), in which periglomerular cells synapse with the dendrites of the projection neurons (rnitral and tufted cells) and not onto the primary afferents (Pinching and Powell, '71). Based on our current results, it appears that a detailed examination of the glomerular synaptology of the lobster OLs is clearly warranted. The small, HA-positive cells juxtaposed t o the OL glomeruli are presumably glia, since the somata of olfactory interneurons are not believed to be present in the OL itself (Blaustein et al., '88; Tsvileneva and Titova, '85). Moreover, these cells bear a remarkable similarity to glial cells previously described in the crayfish OL that extend long fingerlike processes between glomeruli (Sandeman and Luff, '73). Cells with similar morphology at the same location in the lobster OLs also stain for glutamine synthetase (unpublished data), an acknowledged glial marker (Henn, '82), lending further support to the idea that these cells are glia. The presence of these cells near the terminals of the primary afferents suggests that they are involved with neurotransmitter uptake or inactivation of transmitter release (reviews: Henn, '82; Pentreath, '87). This role would not be surprising, as accumulation of HA by glial cells has been observed in the optic lobe of locusts (Elias and Evans, '84). The relative location of these cells is reminiscent of the glial sheaths around glomeruli in the vertebrate olfactory bulb (Pinching and Powell, '71) and of the glial cells which surround glomeruli in arthropods and are involved in their development and formation (Oland and Tolbert, '89; Tolbert and Oland, '89). The pattern of GABA-IR in the lobster OL resembled that of the HA-IR, insofar as olfactory interneurons and the outer caps of the glomeruli were stained. This apparent overlap of some GABA and HA staining leaves open the possibility that these neuroactive substances may coexist in

HISTAMINE AND GABA IN LOBSTER OLFACTORY LOBES

Fig. 8. GABA-like immunoreactivity (GABA-IR) was exhibited in peroxidase-stained cryostat sections. A Specific staining of neurons in the cluster VPPLC, and in the glomeruli of the accessory lobe (AL). Many of the interneurons in the soma clusters stained positively for

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GABA, and were generally uniform in morphology and size. B: Control sections, treated with normal serum, showed no specific staining of the soma clusters (arrow). Calibration bar: 100 pm for A, 50 pm for B.

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E.ORONA ET AL.

Fig. 9. GABA-IR demonstrated by using avidin-biotin conjugated peroxidase on a paraffin section. A: Specific GABA-IR is seen in the outer cap regions (arrow) of the OL. B: A higher magnification of A. T h e staining, like t h a t for HA, was also predominant in the outer cap regions of the glomeruli. I t did not appear, however to resemble the columnar

organization of the glomerular neuropil, but t h a t of bands or fiber bundles (arrows) in the outer parts of the glomeruli. The edge of the tissue (arrowhead) demonstrates the absence of staining in the primary afferent layer. Calibration bar: 250 Wm for A, 100 pm for B.

a subpopulation of second-order deurons. Other lines of evidence have indicated that G A B A and HA can be colocalized, or act through similar mechanisms. For example, it was recently reported (Klihler and Ericson, '88) that every histaminergic cell in the rat hypothalamus also contained

glutamic acid decarboxylase, the synthesizing enzyme for GABA.Interestingly, GABA and the HA metabolite, imidazoleacetic acid, produce nearly identical increases in the chloride conductance of lobster muscle fibers (Constanti and Quilliam, '74). Although these results were interpreted

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HISTAMINE AND GABA IN LOBSTER OLFACTORY LOBES as characteristic of two substances competing for common receptors, not enough is known a t present about GABA or HA receptors in arthropods to make generalizations about receptor mechanisms or subtypes (cf. Bormann, '88; Schwartz et al., '86). Nonetheless, there were two important differences in the pattern and location of GABA- and HA-IR, indicating that the two compounds are a t least partially expressed in different subpopulations of olfactory interneurons. First, it appeared as though more GABA-like immunoreactive cells were present in VPPLC, whereas relatively more HA-like cells may have been present in VPMLC. Second, the HA-IR clearly demarcated individual glomeruli in the OL, while the GABA-IR was indicative of labeled fibers traversing around the rim of the glomeruli. This distinction in the staining pattern in the OL suggests that some of the HA-and GABA-containing interneurons differentially innervate the glomeruli. Previous work has implicated GABA as an inhibitory neurotransmitter in the olfactory system of vertebrates and arthropods (see the introduction). GABA has been postulated to mediate presynaptic inhibition in the visual and statocyst systems of the crayfish (Glantz e t al., '85; WangBennett and Glantz, '87). The putative inhibition by GABA in the lobster may not be presynaptic to the afferent terminals like that proposed for HA, however, since primary olfactory receptor cells do not appear to be sensitive to GABA (Bayer et al., '89). Rather, it may be more plausible to expect that GABA mediates inhibition at a different locus in the lobster than does HA, perhaps at second- or higherorder olfactory interneurons. This interpretation would be consistent with the findings in the insect Manduca sexta, where GABA-IR was also localized in olfactory interneurons and all glomeruli of the antennal (olfactory) lobe (Hoskins et al., '86). It was hypothesized that the primary afferents excite GABA-containing local (amacrine) interneurons, which in turn inhibit postsynaptically the projection neurons of the antennal lobe (Waldrop et al., '87). Overall, our findings with GABA and HA implicate inhibition a t the first synaptic level of the lobster olfactory pathway, and suggest that the glomerulus may be a potential locus of central mixture suppression. HA- and GABA-IR were also present in the accessory lobe, presumably reflecting the labeling of crustacean olfactory interneurons known to arborize in both the OL and the accessory lobe (Arbas et al., '88; Blaustein et al., '88). Hence, GABA- and HA-mediated inhibition at other loci in the olfactory pathways are also potentially implicated in central mixture suppression. Our results, of course, must be interpreted in light of the potentially great neurochemical diversity in olfactory CNS of crustaceans, as suggested by the positive immunoreactivity for serotonin (Elofsson, '83; Sandeman and Sandeman, '87; Sandeman et al., '88) and the peptide FMRF-amide (Mangerich and Keller, '88). Combined pharmacological and physiological studies of identified interneurons and the microcircuits they form will be necessary to unravel how information is processed within the lobster olfactory system.

ACKNOWLEDGMENTS This research was supported by NSF grants 85-11256 and 88-10261. The authors thank Lynn Milstead for assistance with figures and graphs, James Netherton for photographic assistance, and Thomas Beyer for preliminary work on HA synthesis.

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